Enzymes As Additives Or Processing Aids in Food Biotechnology

Enzyme Research

Enzymes as Additives or Processing Aids in Food Biotechnology

Guest Editors: Raffaele Porta, Ashok Pandey, and Cristina M. Rosell Enzymes as Additives or Processing Aids in Food Biotechnology Enzyme Research

Enzymes as Additives or Processing Aids in Food Biotechnology

This is a special issue published in volume 2010 of “Enzyme Research.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Enzyme Research

Editorial Board

Sabbir Ahmed, UK D. M. G. Freire, Brazil Michael J. McLeish, USA Mario Amzel, USA Vilmos Fulop, UK Peter Moody, UK Vasu D. Appanna, Canada Giovanni Gadda, USA William David Nes, USA David Ballou, USA J. Guisan, Spain Toshihisa Ohshima, Japan Ulrich Baumann, Switzerland Munishwar Nath Gupta, India Michael Page, UK Fabrizio Briganti, Italy R. S. Gupta, Canada Jose Miguel Palomo, Spain Joaquim Cabral, Portugal Albert Jeltsch, Germany Robert Pike, Australia Gerald M. Carlson, USA Marilyn S. Jorns, USA Raﬀaele Porta, Italy Sunney I. Chan, USA Mari Kaartinen, Canada Alireza R. Rezaie, USA Christopher Davies, USA Eva Nordberg Karlsson, Sweden Ali Akbar Saboury, Iran Narasimha Rao Desirazu, India Leszek Kleczkowski, Sweden Engin Serpersu, USA John David Dignam, USA William Konigsberg, USA Assia Shisheva, USA Colin Dingwall, UK H. Kuhn, Germany R. D. Tanner, USA Jean-Marie Dupret, France David Lambeth, USA John J. Tanner, USA Paul Engel, Ireland A-Lien Lu-Chang, USA Gianluigi Veglia, USA Roberto Fernandez Lafuente, Spain Paul Malthouse, Ireland Qi-Zhuang Ye, USA Contents

Enzymes as Additives or Processing Aids in Food Biotechnology,Raﬀaele Porta, Ashok Pandey, and Cristina M. Rosell Volume 2010, Article ID 436859, 2 pages

Enzymes in Food Processing: A Condensed Overview on Strategies for Better Biocatalysts, Pedro Fernandes Volume 2010, Article ID 862537, 19 pages

Some Nutritional, Technological and Environmental Advances in the Use of Enzymes in Meat Products, Anne y Castro Marques, Mario´ Roberto Marostica´ Jr., and Glaucia´ Maria Pastore Volume 2010, Article ID 480923, 8 pages

Enzymatic Strategies to Detoxify Gluten: Implications for Celiac Disease, Ivana Caputo, Marilena Lepretti, Stefania Martucciello, and Carla Esposito Volume 2010, Article ID 174354, 9 pages

Uses of Laccases in the Food Industry, Johann F. Osma, Jose´ L. Toca-Herrera, and Susana Rodr´ıguez-Couto Volume 2010, Article ID 918761, 8 pages

Fungal Laccases: Production, Function, and Applications in Food Processing, Khushal Brijwani, Anne Rigdon, and Praveen V. Vadlani Volume 2010, Article ID 149748, 10 pages

Potential Applications of Immobilized β-Galactosidase in Food Processing Industries, Parmjit S. Panesar, Shweta Kumari, and Reeba Panesar Volume 2010, Article ID 473137, 16 pages

Screen-Printed Carbon Electrodes Modiﬁed by Rhodium Dioxide and Glucose Dehydrogenase, Vojtechˇ Polan, Jan Soukup, and Karel Vytrasˇ Volume 2010, Article ID 324184, 7 pages

Preparation of Antioxidant Enzymatic Hydrolysates from Honeybee-Collected Pollen Using Plant Enzymes, Margarita D. Marinova and Bozhidar P. Tchorbanov Volume 2010, Article ID 415949, 5 pages

Characterization of Activity of a Potential Food-Grade Leucine Aminopeptidase from Kiwifruit, A. A. A. Premarathne and David W. M. Leung Volume 2010, Article ID 517283, 5 pages SAGE-Hindawi Access to Research Enzyme Research Volume 2010, Article ID 436859, 2 pages doi:10.4061/2010/436859

Editorial Enzymes as Additives or Processing Aids in Food Biotechnology

Raffaele Porta,1 Ashok Pandey,2 and Cristina M. Rosell3

1 Department of Food Science, University of Naples Federico II, Portici, 80055 Napoli, Italy 2 Biotechnology Division, National Institute for Interdisciplinary Science and Technology, CSIR, Trivandrum, Kerala 695019, India 3 Institute of Agrochemistry and Food Technology (IATA-CSIC), 46980 Paterna, Valencia, Spain

Correspondence should be addressed to Raﬀaele Porta, raﬀ[email protected]

Received 31 December 2010; Accepted 31 December 2010

Copyright © 2010 Raﬀaele Porta et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Essential in the metabolism of all living organisms, the of natural products are altered to fit the nutritional or enzymes are increasingly used to drive chemical reactions technological needs changing. outside their natural localization. In particular, the use of the The economic benefit of using technical enzyme prepa- biocatalysts as food additives and in processing raw materials rations lies in lowered process costs, in the reduction of the has been practiced for a long time. In fact, enzymatic environmental impact by making use of renewable resources, preparations from the extracts of plants or animal tissues and often in increasing the quality of the products. Also, were used well before much was known about the nature and preservation makes a significant impact on the quality of properties of enzymes. food as well of beverages. It is well known, for example, Food industry is constantly seeking advanced technolo- that modern processes convert juices into concentrates that, gies to meet the demand of the consumers, and enzymes have except for aroma, can be stored for a long time without loss long been used by the industrial product makers as major in quality. Stabilizing flavor and color is also an example of tools to transform the raw materials into end-products. improved preservation. Finally, the advent of biotechnology Their clean label (GRAS, generally recognized as safe) has also allowed significant refinements in the methodologies consideration from the legal point of view has prompted offering unpredictable solutions to many persistent problems their extensive use in food technology. When purified and and opening up exciting new possibilities. Among these, added to food preparations, several enzymes are able to enzymes are proposed as exemplary agents of “green” improve their flavor, texture, digestibility, and nutritional technology since they can also be used either to treat the value. However, it was not until the mid of the past century biological wastes or to prevent their formation. Currently that the rapid development in protein technology occurred, used enzymes sometimes originate in animals and plants but and only in the last 30 years, the use of commercial enzymes most come from a range of beneficial microorganisms. Thus, has grown in the food industry, progressively becoming an numerous purified enzymes are now being widely used not important aspect of the manufacturing of meat, vegetables, only in food processing but also as food additives. In this fruit, baked goods, milk products, and both alcoholic and respect, it is noteworthy that the enzymes, like all proteins, nonalcoholic beverages. As a matter of fact, an increasing can cause reactions only when people have been sensitized number of articles, mostly describing the enhanced product through exposure to large quantities. Therefore, since their yields, have been published during the last ten years, both levels in the food are generally very low, the enzymes are in food and beverage manufacturing. Moreover, since it highly unlikely to cause allergies. is desirable in different branches of food technology to This special issue of Enzyme Research is devoted to change the physical and chemical properties of protein, many contribute to highlight some expanding fields of enzyme previously unexplored enzymes are currently employed to applications in food technology, mostly explaining how some produce a variety of foods in which the biocatalysts replace different biocatalysts bring advantages in some food proces- potentially carcinogenic or otherwise harmful chemicals. sing improvement and innovation. It comprises six review This includes also new methods in which the characteristics articles and three research articles. The first review article 2 Enzyme Research is a condensed and concise overview on the applications of enzymes in food and feed processing, outlining the development of better biocatalysts through microbial screening, protein engineering, and immobilization techniques. The second review article summarizes the nutritional, technological, and environmental advances in meat products and, in particular, the application of the proteolytic enzymes, phytases, and transglutaminase in the meat industry. Trans- glutaminase, as well as bacterial-derived endopeptidases, are the subject of the third review article which reports the most recent developments of the attempts to detoxify gluten. The fourth and the fifth review articles describe, respectively, the uses of laccases as additives in food and beverage processing and the production, function, and applications in food industry of fungal laccases. The last review article has been focused on the different types of techniques used for the immobilization of β-galactosidase and its potential applications in food and dairy processing industries. The three research articles describe (i) a new glucose biosensor based on a screen-printed carbon electrode modified by glucose dehydrogenase immobilized on its surface, (ii) the preparation of antioxidant hydrolysates of honeybee- collected pollen by using proteinase and aminopeptidases of plant origin, and (iii) the characterization of a potential food-grade leucine aminopeptidase extracted from kiwifruit. We sincerely hope that the present volume may represent only the first of a special issue series in which Enzyme Research will periodically stimulate authors to publish the highlights and original research articles reporting how enzymes bring new advantages in food preparation improvement and innovation. Raffaele Porta Ashok Pandey Cristina M. Rosell SAGE-Hindawi Access to Research Enzyme Research Volume 2010, Article ID 862537, 19 pages doi:10.4061/2010/862537

Review Article Enzymes in Food Processing: A Condensed Overview on Strategies for Better Biocatalysts

Pedro Fernandes

Institute for Biotechnology and Bioengineering (IBB), Centre for Biological and Chemical Engineering, Instituto Superior T´ecnico, Avenue Rovisco Pais, 1049-001 Lisboa, Portugal

Correspondence should be addressed to Pedro Fernandes, [email protected]

Received 7 July 2010; Accepted 1 September 2010

Academic Editor: Cristina M. Rosell

Copyright © 2010 Pedro Fernandes. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Food and feed is possibly the area where processing anchored in biological agents has the deepest roots. Despite this, process improvement or design and implementation of novel approaches has been consistently performed, and more so in recent years, where significant advances in enzyme engineering and biocatalyst design have fastened the pace of such developments. This paper aims to provide an updated and succinct overview on the applications of enzymes in the food sector, and of progresses made, namely, within the scope of tapping for more efficient biocatalysts, through screening, structural modification, and immobilization of enzymes. Targeted improvements aim at enzymes with enhanced thermal and operational stability, improved specific activity, modification of pH-activity profiles, and increased product specificity, among others. This has been mostly achieved through protein engineering and enzyme immobilization, along with improvements in screening. The latter has been considerably improved due to the implementation of high-throughput techniques, and due to developments in protein expression and microbial cell culture. Expanding screening to relatively unexplored environments (marine, temperature extreme environments) has also contributed to the identification and development of more efficient biocatalysts. Technological aspects are considered, but economic aspects are also briefly addressed.

1. Introduction the use of amylases and amyloglucosidases (glucoamylases), a cocktail that some years latter would include glucose (xylose) Food processing through the use of biological agents is isomerase [1, 2, 4, 5]. From then on, the trend for the historically a well-established approach. The earliest appli- design and implementation of processes and production of cations go back to 6,000 BC or earlier, with the brewing of goods anchored in the use of enzymes has steadily increased. beer, bread baking, and cheese and wine making, whereas Enzymes are currently among the well established products the first purposeful microbial oxidation dates from 2,000 in biotechnology [6], from US $1.3 billion in 2002 to US $4 BC, with vinegar production [1–3]. Coming to modern days, billion in 2007; it is expected to have reached US $5.1 billion in the late XIX, century Christian Hansen reported the use in a rough 2009 year, and is anticipated to reach $7 billion of rennet (a mixture of chymosin and pepsin) for cheese by 2013 [3, 5, 7–9]. In the overall, this pattern corresponds making, and production of bacterial amylases was started to a rise in global demand slightly exceeding 6% yearly at Takamine (latter to become part of Genencor). Pectinases [7, 9]. Part of this market is ascribed to enzymes used in were used for juice clarification in the 1930s, and for a short large-scale applications, among them are those used in food period during World War II, invertase was also used for the and feed applications [10]. These include enzymes used in production of invert sugar syrup in a process that pioneered baking, beverages and brewing, dairy, dietary supplements, the use of immobilized enzymes in the sugar industry [1]. as well as fats and oils, and they have typically been Still, the large-scale application of enzymes only became dominating one, only bested by the segment assigned to really established in the 1960s, when the traditional acid technical enzymes [11, 12]. The latter includes enzymes in hydrolysis of starch was replaced by an approach based in the detergent, personal care, leather, textile and pulp, and 2 Enzyme Research paper industries [10, 13]. A recent survey on world sales (namely, enzymes from plant or animal cells, such as trans- of enzymes ascribes 31% for food enzymes, 6% for feed glutaminase or even slow-growing microorganisms). When enzymes and the remaining for technical enzymes [11]. A successfully implemented, the undertaken approaches allow: relatively large number of companies are involved in enzyme (a) continuous operations at relatively high temperatures; manufacture, but major players are located in Europe, USA (b) eased implementation of enzyme cascade, given the and Japan. Denmark is dominating, with Novozymes (45%) reduced need for processing the reaction media (pH adjust- and Danisco (17%), moreover after the latter taking over ments; metal ion removal/addition) throughout the interme- Genencor (USA), with DSM (The Netherlands) and BASF diate steps of a multistep biotransformation (namely, starch (Germany) lagging behind, with 5% and 4% [10, 11, 14]. to high fructose syrup); and (c) the use of raw substrates, The pace of development in emerging markets is suggestive preferably as high-concentrated solutions, hence cutting that companies from India and China can join this restricted back in costs related to upstream processing and increasing party in a very near future [15–17]. productivity [4, 23, 24]. Methodologies with a high level of parallelization, anchored in computer-monitored microtiter plates equipped with optic fibers and temperature control 2. Relevant Enzymes: Tapping for have also been developed. These provide high-throughput Improved Biocatalysts capability for a speedy and detailed characterization of the performance of enzymes [25]. Particular focus was given to 2.1. General Aspects and the Screening Approach. Roughly the prediction of the long-term stability of enzymes under all classes of enzymes have an application within the food moderate conditions using short-term runs (up to 3 hours). and feed area, but hydrolases are possibly the prevalent one. One of the methodologies to obtain improved bio- Representative examples of the enzymes and their role in catalyst relies on in-vitro modifications, which will be food and feed processing are given in Table 1. The widespread addressed latter in this paper; another approach relies on use of enzymes for food and feed processing is easily under- screening efforts, which has been consistently undertaken, standable, given their unsurpassed specificity, ability to oper- as summarized recently [26–31]. Some focus is given to ate under mild conditions of pH, temperature and pressure extremophiles, particularly thermophiles, since operation while displaying high activity and turnover numbers, and at high temperatures (roughly above 45–50◦C) minimizes high biodegradability. Enzymes are furthermore generally the risk of microbial contamination, a particularly deli- considered a natural product [18, 19]. The whole contributes cate matter under continuous operation. Furthermore, the for developing sustainable and environmentally friendly extension of some reactions in relevant food applications processes, since there is a low amount of by-products, is favored at relatively high temperatures (namely, iso- hence reducing the need for complex downstream process merization of glucose to fructose), although care should operations, and the energy requirements are relatively low. be taken to avoid an operational environment that may Life-cycle assessment (LCA) has confirmed, that within the lead by-product formation (namely, Maillard reactions). range of given practical case studies, including food and feed Examples of screened enzymes include the isolation of processing, the implementation of enzyme-based technology amylases, with some of them being calcium independent has a positive impact on the environment [3]. LCA is a [32–38]; amylopullulanases [39]; fructosyltransferases [40]; methodology used to compare the environmental impact glucoamylases [41]; glucose (xylose) isomerases [42, 43]; of alternative production technologies while providing the glucosidases [44, 45]; inulinases [46–49]; levansucrases [50]; same user benefits [20]. pullulanases [51, 52]; and xylanases [53, 54]. Other examples Some of the broad generalizations on the limitations of of these enzymes, with some of which able to retain stability enzymes for application as biocatalysts in commercial scale, under temperatures of 90◦C or higher, were reviewed by namely, their high cost, low productivity and stability, and Gomes and Steiner [55]. The majority of enzymes used in narrow range of substrates, have been rebutted [21, 22]. food and feed processing is of terrestrial microbial origin, Aiming at improving the performance of biocatalysts for and screening-efforts for isolation of promising enzyme- food and feed applications, particular care has been given to producing strains have accordingly been performed in such increasing thermal stability, enhancing the range of pH with background [3, 5, 56]. From some years now, marine catalytic activity and decreasing metal ions requirements, as environment has also been tapped as a source for useful well as to overcoming the susceptibility to typical inhibitory enzymes from either microbial or higher organisms origin molecules. Some examples of strategies taken to improve the [57–60]. This latter environment has allowed the isolation performance of relevant enzymes for food and feed are given of some promising biocatalysts, such as the heat-stable in Table 2. Along with these different strategies focused on invertase/inulinase from Thermotoga neapolitana DSM 4359 the enzyme molecule (namely, protein engineering, enzyme or inulinase from Cryptococcus aureus [61–63], amylolytic immobilization), the developments in recombinant DNA enzymes, glucosidases and proteases from severalgenera [32, technology that occurred in the 1980s also had a huge 44, 45, 64, 65], esterase from Vibrio fischeri [66], and glycosyl impact on the application of enzymes in food and feed. By hydrolases [67, 68]. Other examples of useful enzymes for allowing gene cloning in microorganisms compatible with food and feed, but isolated from higher organisms [59, 69], industrial requirements, this methodology enabled cost- are given in Table 3. Some of these enzymes are actually feasible production of enzymes that were naturally pro- psychrophiles, hence performing best at low temperatures duced in conditions that prevented large-scale application [30]. Enzyme Research 3

Operation at low temperatures is also welcome since it (i) The first is directed evolution of enzymes, through also reduces the risk of microbial contamination, enables random mutagenesis and recombination, where the some processes to be carried out with minimal deterioration environmental adaptation is reproduced in-vitro in a of the raw material. These include protein processing, such much hastened timescale, towards the optimization as cheese maturing and milk coagulation with proteases [59, of the intended property. In order to control the 80]; milk processing with lactase for lactose-free milk [81– pathway of the process, either a screening test for 83]; clarification of fruit juices with pectinases to produce the assessed feature is performed after each round of clear juice [84]; or production of oligosaccharides [85]. modification, or selective pressure is applied [100– Since extremophiles are often difficult to grow under 102]. This methodology, which allows for a high typical laboratory conditions if not nonculturable at all, throughput, has been extensively applied, aiming for different approaches have been developed in order to assess more efficient biocatalysts [103–106]. Some relevant the potential of enzymes from such microorganisms. One examples in the area of food and feed processing approach relies on the generation and screening of target include the following. genes from DNA libraries, which can be obtained from mixed microbial population from environmental samples. (1) The first is the enhancement of the activity of the Recombinant microorganisms can then be obtained using hyperthermostable glucose (xylose) isomerase from mesophiles as hosts where the genes of interest from Thermotoga neapolitana at relatively low temperature extremophiles have been expressed [86]. In order to screen and pH, without decay in thermostability [107]. the huge number of DNA-libraries typically generated for The enzyme from the parent strain is highly active at 97◦C, but it retains only 10% of its activity at the intended property, high-throughput methods have been ◦ implemented [87]. These methods are also widely used when 60 C, and requires neutral pH for optimal activity. protein engineering is carried out. This will be addressed in This pattern is often reported when glucose iso- the following section. merases from hyperthermophilic strains operate in α mesophilic environments. Large-scale glucose iso- Several enzymes (namely, -amylases; pullulanases) cur- ◦ rently used in food processing, namely, in starch hydrolysis, merization is carried out at 55–60 C and slightly are actually produced by recombinant microorganisms. alkaline pH [1, 31]. This set of conditions results from the optimal range of pH (typically 7.0 to 9.0) and Despite some complexity in the implementation of their ◦ use in large-scale applications, partly resulting from lack of temperature (60 to 80 C) for glucose isomerization uniformity in the US and EU legislation, quite a few enzyme displayed by most of the glucose isomerases used, preparations have been accepted for industrial use [88, 89]. combined with process boundary conditions. The latter result from by-product and color formation occurring when the reaction is carried out at alka- 3. Improving Biocatalysts: line pH and high temperatures [31, 108]. There is therefore interest in selecting an enzyme able to Beyond Screening operate efficiently at temperatures close to those Taking advantage of the knowledge gathered on molecular currently used but at a lower pH. The mutant biology, high-throughput processing, and computer-assisted glucose isomerase 1F1 obtained by Sriprapundh and coworkers displayed a roughly 5-fold higher activity design of proteins, in-vitro improvement of biocatalysts ◦ have been consistently implemented [90–93]. Some of the at 60 C and pH 5.5, when compared with the parent research efforts in this area has focused on the biochemi- T. neapolitana isomerase, and was more thermostable cal and molecular mechanisms underlying the stability of than the wild type isomerase [104, 107]. The acti- enzymes from extremophiles [31, 94–96]. Such knowledge vation energy required by the triple 1F1 mutant is also particularly useful for protein engineering of known (V185T/L282P/F186S) was roughly half of the wild- enzymes, aiming at enhancing stability without compro- type, hence allowing for high activity at relatively mising catalytic activity [97]. Enhancing the stability of low temperatures [107]. The encouraging results enzymes is of paramount importance when implementation obtained suggest the soundness of the approach to of industrial processes is foreseen, since it allows for reducing obtain a mutant glucose isomerase competitive with those currently used, while being able to operate in a the amount of enzyme used in the process. Given that ◦ thermostability is determined by a series of short- and slightly acidic environment and 60 C. long-range interactions, it can be improved by several (2) The second is the enhancement of the thermostability substitutions of amino acids in a single mutant, where the of the maltogenic amylase from Thermus sp. IM6501 combination of each individual effect is usually roughly [109], of the amylosucrase from Neisseria polysac- additive [98]. The targeted improvements have not been charea [110], of the glucoamylase from Aspergillus restricted to thermostability, but they have also addressed niger [111], of a phytase from Escherichia coli [112, other features, such as broadening the range of pH where the 113], and of a xylanase from Bacillus subtilis [114]. enzyme is active, or lessening the temperature of operation Amylases and glucoamylases are enzymes used in while retaining high activity [91, 99]. starch processing, which involves temperatures typ- Two methodologies can be used for protein engineering ically in excess of 60◦C; hence, improving thermal [97]. stability without decreasing enzyme activity is of 4 Enzyme Research

Table 1: An overview of enzymes used in food and feed processing (adapted from [10, 12, 13, 68]).

Class Enzyme Role Glucose oxidase Dough strengthening Oxidoreductases Laccases Clarification of juices, flavor enhancer (beer) Lipoxygenase Dough strengthening, bread whitening Cyclodextrin Cyclodextrin production Glycosyltransferase Transferases Fructosyltransferase Synthesis of fructose oligomers Transglutaminase Modification of viscoelastic properties, dough processing, meat processing Starch liquefaction and sachcarification Increasing shelf life and improving quality by retaining moist, elastic and soft Amylases nature Bread softness and volume, flour adjustment, ensuring uniform yeast fermentation Juice treatment, low calorie beer Viscosity reduction in lupins and grain legumes used in animal feed, enhanced Galactosidase digestibility Hydrolases Glucanase Viscosity reduction in barley and oats used in animal feed, enhanced digestibility Glucoamylase Saccharification Invertase Sucrose hydrolysis, production of invert sugar syrup Lactase Lactose hydrolysis, whey hydrolysis Cheese flavor, in-situ emulsification for dough conditioning, support for lipid Lipase digestion in young animals, synthesis of aromatic molecules Protein hydrolysis, milk clotting, low-allergenic infant-food formulation, Proteases (namely, chymosin, papain) enhanced digestibility and utilization, flavor improvement in milk and cheese, meat tenderizer, prevention of chill haze formation in brewing Pectinase Mash treatment, juice clarification Peptidase Hydrolysis of proteins (namely, soy, gluten) for savoury flavors, cheese ripening Phospholipase In-situ emulsification for dough conditioning Phytases Release of phosphate from phytate, enhanced digestibility Pullulanase Saccharification Xylanases Viscosity reduction, enhanced digestibility, dough conditioning Lyases Acetolactate decarboxylase Beer maturation Isomerases Xylose (Glucose) isomerase Glucose isomerization to fructose

relevance. Starch liquefaction is performed at 105◦C the wild-type ThMA was fully inactivated in less in the presence of α-amylase, upon which the effluent than 1 minute. However, one of the mutations reaction stream has to be cooled to 60◦C, so that glu- most accountable for enhanced thermal stability, coamylases can be used. In order to avoid, or at least M375T, close to the active site, also led to a 23% minimize, the cooling step, thermostable glucoamy- decrease in specific activity, as compared to the wild lases are aimed at. Wang and coworkers obtained a type [109]. The amylosucrase engineered by Emond multiply-mutated enzyme (N20C, A27C, S30P,T62A, and coworkers was a double mutant (R20C/A451T), S119P, G137A, T290A, H391Y), which displayed a displaying a 10-fold increase in the half-life at 50◦C 5.12 kJ mol−1 increase in the free energy of thermal compared to the wild-type enzyme. Actually, the inactivation, when compared to the wild type, thus mutant was claimed to be the only amylosucrase resulting in the enhanced thermal stability of the usable at 50◦C. At the latter temperature, the mutant mutant. Furthermore specific activities and catalytic enabled the synthesis of amylose chains twice as long efficiencies remained unaltered, when mutant and as those obtained by the wild-type enzyme at 30◦C, wild type were compared [111]. Kim and coworkers for sucrose concentrations of 600 mM. The mutant obtained also a multiply-mutated amylase (R26Q, thus allowed for a process with increased yield in S169N, I333V, M375T, A398V, Q411L, P453L) which amylose chains (31 g L−1), lower risk of contami- displayed an optimal reaction temperature 15◦C nation, enhanced substrate and product solubility higher than that of the wild-type and a half-life of and overall productivity [110]. Phytases are added to roughly 170 min at 80◦C, a temperature at which animal feeds to improve phosphorus nutrition and Enzyme Research 5

Table 2: Some examples of strategies undertaken to improve the performance of enzymes with applications in food and feed.

Targeted Enzyme Role Strategy/comments Reference improvement Protein engineering through site-directed Starch liquefaction Thermostability mutagenesis. Mutant displayed increased [70] ◦ α-amylase half-life from 15 min to about 70 min (100 C). Directed evolution. After 3 rounds the mutant enzyme from S. cerevisiae displayed a 20-fold Starch liquefaction Activity [71] increase in the specific activity when compared to the wild-type enzyme. Protein engineering through site-directed Baking pH-activity profile [72] mutagenesis l-arabinose Tagatose production pH-activity profile Protein engineering through directed evolution [73] isomerase Substrate specificity, Protein engineering through site-directed Glucoamylase Starch saccharification thermostability and [74] mutagenesis pH optimum Lactase Lactose hydrolysis Thermostability Immobilization [75] Pullulanase Starch debranching Activity Protein engineering through directed evolution [76] Protein engineering through site-directed Phytase Animal feed pH-activity profile [77] mutagenesis Protein engineering through directed evolution. The turnover number on D-glucose in some mutants was increased by 30%–40% Xylose (glucose) Isomerization/epimerization pH-activity profile [78] when compared to the wild type at pH 7.3. isomerase of hexoses, pentoses and tetroses Enhanced activities are maintained between pH 6.0 and 7.5. Protein engineering through site-directed mutagenesis. The resulting mutant displayed a Substrate specificity [79] 3-fold increase in catalytic efficiency with L-arabinose as substrate.

Table 3: Examples of enzymes isolated from various marine higher organisms with potential of application in food and feed (adapted from [68, 69]).

Class Enzyme Source Muscles of atka mackerel (Pleurogrammus azonus), botan shrimp (Pandalus nipponensis), carp Transferases Transglutaminase (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), scallop (Patinopecten yessoensis). Gilt-head (sea) bream (Sparus aurata), found in Mediterranean sea and coastal North Atlantic Ocean. Amylase Turbot (Scophthalmus maximus), found mostly in Northeast Atlantic Ocean, Baltic, Black and Mediterranean seas, and Southeast the Pacific Ocean Hydrolases Deepwater redfish (Sebastes mentella, found in North Atlantic). Chymotrypsin Atlantic cod (Gadus morhua), crayfish, white shrimp. Pepsin Arctic capelin (Mallotus villosus), Atlantic cod (Gadus morhua). Marine sponges Spheciospongia vesperia, found in Caribbean sea and South Atlantic, close to Brazil, and Geodia cydonium, found in Northeast Atlantic Ocean and Mediterranean sea. Protease Mangrove crab (Scylla serrata), found in estuaries and mangroves of Africa, Asia and Australia. Sardine Orange roughy (Hoplostethus atlanticus)

to reduce phosphorus excretion, by promoting the animals [115]. E. coli phytases, which are appealing to hydrolysis of phytate into myoinositol and inorganic industrial application, due to the acidic pH optimum, phosphate. Thermal stable enzymes are needed, since speciﬁcity phytate, and resistance to pepsin digestion, feed pelleting is carried out at high temperature were thus engineered in order to improve their (60 to 80◦C). Phytases produced by thermophiles thermal stability, without compromising the kinetic do not provide a suitable approach, since they have parameters. As a result, mutants were obtained, low activity at the physiological temperature of with roughly 20% increased thermostability at 80◦C 6 Enzyme Research

improved overall catalytic efficiency (kcat,turnover as expressed by decreases in KM of roughly 35% number/KM, Michaelis constant) within 50 to 150%, and 25%, as compared to the wild-type enzyme. as compared to the wild type. No significant changes Furthermore, the overall catalytic efficiency of the in the pH activity profile were observed, but for mutants increased 1.4- and 1.6-fold as compared to some mutants, containing a K46E substitution, that the wild type. displayed a decrease in activity at pH 5.0 [112, 113]. Xylanases catalyze the cleavage of β1,4 bonds in xylan Other examples can be found elsewhere [120, 121]. polymers. Accordingly, these enzymes can be used in dough making, in baking, in brewing and in (ii) The second methodology underlines that rational animal feed compositions. When the latter contain pinpoint modifications in one or more amino acids cereals (namely, barley, maize, rye or wheat), or cereal are made, where these changes are predicted to bring by-products, xylanases improve the break-down of along the envisaged improvement in the targeted plant cell walls, which favors the ingestion of plant enzyme function. The alterations promoted are per- nutrients by the animals and consequently enhances formed based on the growing knowledge on the feed consumption and growth rate. Furthermore, structure and functions of enzyme. Information on the use of xylanases decreases the viscosity of xylan- this matter mostly comes from bioinformatics, which containing feeds [116, 117]. As referred for phytases, provides data on amino-acid propensities and on the formulation of commercial feed often involves protein sequences. Adequate processing of the data steps at high temperatures. Xylanases added to the enable the output of generalized rules predicting ff the formulations hence have to withstand these the e ect of mutations on enzyme properties. Also conditions, while they are to display high activity used are molecular potential functions, which, once ◦ ff at about 40 C, which is the temperature in the implemented, enable the prediction of the e ect intestine of animals. However, most xylanases are of mutations in enzyme structure [97]. Compu- inactive at temperatures exceeding 60◦C, hence the tational tools used for enzyme engineering have need for enhancing thermal stability [114, 117]. been recently reviewed [122]. Enzyme engineering Miyazaki and coworkers obtained a triple-mutant through molecular simulations requires structural xylanase (Q7H, N8F, and S179C) which retained full data from the native enzyme, which can be preferably activity for 2 hours at 60◦C, whereas the wild-type obtained from crystallography or NMR. Otherwise enzyme was inactivated within 5 minutes under the a model is built based on known enzyme structures same conditions. The mutation also led to a 10◦C with homologous sequences [90]. Computational increase in the optimal temperature for reaction and methods are also welcome in directed evolution, enhanced activity at higher temperatures, albeit at the as a tool to better lead the random mutagenesis cost of decreased activity at lower temperatures, as [97]. Ultimately this approach is put into practice compared to the wild-type enzyme [114]. by producing a site-directed mutant, where selected amino acids are replaced with those suggested from (3) Third is the enhancement of the activity of the the outcome of modeling. amylosucrase from Neisseria polysaccharea [118]. Some relevant examples of this strategy in the area Amylosucrases can be used for the modification or of food and feed processing are given. These mostly synthesis of amylose-type polymers from sucrose, but aim to improve thermal stability and/or catalytic effi- their industrial application is somehow thwarted by ciency and/or to modify the range of pH/temperature the low catalytic efficiency on sucrose and by side where the enzyme is active—goals that were already reactions leading to the formation of sucrose isomers. referred to when examples of enzyme modifications Van der Veen and co-works engineered mutant using random mutagenesis were addressed. enzymes through error-prone PCR that displayed increases in activity up to 5-fold and in overall (1) The first example underlines the enhancement of the catalytic efficiency up to 2-fold, when compared to thermostability of the recombinant glucose (xylose) the wild-type enzyme. Furthermore, the mutants isomerase from Actinoplanes missouriensis [123, 124] were able to produce amylose polymers from 10 mM and of glucose (xylose) isomerase from Streptomyces sucrose on, unlike the wild-type enzyme [118]. Their diastaticus [125]; of amylases from Bacillus spp. [126, work provides an illustrative example on the use 127]; and of glucoamylase from Aspergillus awamori of random mutagenesis and recombination for the [128]. The mutant isomerase from A. missouriensis enhancement of the catalytic properties of enzymes displayed an enhanced thermal stability, alongside with application on food and feed. Another example with improved stability at different pH, as compared was provided by Tian and coworkers who engineered with the original enzyme, with no changes in catalytic a phytase from Aspergillus niger 113 through gene properties [123, 124].Thedoublemutantisomerase shuffling, to obtain mutants with enhanced catalytic (G138P, G247D) displayed a 2.5-fold increase in properties [119]. Hence, K41E and E121F substitu- half-life, and additionally a 45% increase in the tions allowed for increases in the specific activity of specific activity, when compared to the wild type. 2.5- and 3.1-fold, and of affinity for sodium phytate, Such features were ascribed to increased molecular Enzyme Research 7

rigidity due to the introduction of a proline in the random mutation of glucose isomerases was the turn of a random coil [125]. Multiply-mutated addressed. Hence, again arises the need for enzymes amylases obtained by Declerck and coworkers dis- able to isomerize l-arabinose in an acidic environ- played considered enhanced thermal stability. Based ment and at relatively low temperature, 60 to 70◦C. on the temperature at which amylase initial activity Operation within the latter temperature range also is reduced by 50% for a 10-minute incubation, rules away the use of divalent ions, which stabilize this parameter went as high as 106◦C, as compared isomerases at high temperatures [133, 134]. Rhimi to 83◦C for the wild-type strain. Furthermore, the and coworkers engineered two individual mutants, thermal stabilization was not accompanied by a harboring each N175H and Q268K mutations. These decrease in the catalytic activity [126]. The work by led to broader optimal temperature range within 50 Lin and coworkers on amylase mutants from Bacillus to 65◦C and to enhanced stability in acidic media, sp. strain TS-23 highlighted the relevance of E219 respectively, when compared to the wild type. An for the thermal stability of the enzyme [127]. The engineered double mutant, harboring both modifi- mutated glucoamylases engineered by Liu and Wang cations, displayed optimal activity within a pH range allowed to establish the role of several intermolecular of 6.0 to 7.0 and a temperature range within 50– interactions in thermal stability of these enzymes. 65◦C. Such set of operational conditions matches the Thermostable enzymes were obtained through the targeted goals and again shows that the basis for pH- introduction of disulfide bonds in highly flexible activity profile and thermostability in l-arabinose region in the polypeptide chain of the enzyme, as well isomerase are quite independent and compatible. as by the introduction of more hydrophobic residues- Cumulative enhancements in both properties in the stabilized α-helices. Data gathered also showed that same enzyme were thus possible [134]. A similar care had to be taken not to disrupt the hydrogen bond pattern was also observed in the previous example and salt linkage network in the catalytic center as a dedicated to a mutant phytase. result of mutagenesis, for this could lead to a decrease (4) The fourth example underlines the modification of in the specific activity and overall catalytic efficiency the product profile of inulosucrase from Lactobacillus [128]. reuteri [135]andfromB. subtilis [136]. Inulosucrases (2) The second example underlines the enhancement of are used to synthesize fructooligosaccharides or fruc- the pH-activity profile and of the thermostability tan polymer from sucrose. The transglycosylation of phytase from A. niger. This was achieved by catalyzed by the inulosucrase from L. reuteri leads combining several individual mutations that allowed to a wide range of fructooligosaccharides alongside for mutants that were quite active at pH 3.5. Effi- with minor amounts of an inulin polymer. In order cient operation in the stomach of simple-stomached to minimize the dispersion in the products obtained, animals where phytate hydrolysis mostly occurs at a mutants R423K and W271N were obtained, which pH around 3.5, and the wild type was ineffective, was allowed the synthesis of a significant amount of thus enabled. Furthermore, the hydrolytic activity of polymer and a lower amount of oligosaccharide, the mutants at pH 3.5 exceeded in roughly 1.5-fold without significantly affecting the catalytic activity, that of the parent one at pH 5.5, which was the when compared with the wild type. The data gathered optimum of the latter. Mutants also retained higher showed that the −1 subsite in the inulosucrase residual activity after incubation within 70 to 100◦C, from L. reuteri has a key role in the determination as compared to the wild type. The work demonstrates of the size of the products obtained [135]. Ortiz- that cumulative improvements in pH activity and Soto and coworkers also showed that the product thermostability through mutation are compatible in profile of transfructosylation reactions could be this phytase; see [129]. adequately tuned through modification of target residues of an inulosucrase from B. subtilis. These (3) The third example underlines the modification of authors established the effect of mutations on the the temperature- and pH activity profile of the l- reaction specificity (hydrolysis/transfructosylation), arabinose isomerase from Bacillus stearothermophilus molecular weight and acceptor specificity. For exam- US100 [130]. l-Arabinose isomerases catalyze the ple, engineered mutants R360S, Y429N and R433A conversion of l-arabinose to l-ribulose in-vivo, but only synthesized oligosaccharides, whereas the wild in-vitro they also isomerize d-galactose into d- type synthesized levan, since the former are more tagatose [130]. The latter keto-hexose is being used hydrolytic. On the other hand these mutations as a low-calorie bulk sweetener, since its taste and reduced the affinity for sucrose, and thermal stability, sweetness are roughly equivalent to sucrose, but the when compared to the wild type [136]. caloric value is only 30% of that of sucrose [131, 132]. Although several thermostable l-arabinose iso- (5) The fifth example underlines the enhancement of merases have been isolated and characterized, most of the product profile of cyclodextrin glycosyltrans- these display an alkaline pH optimum. For industrial ferases (CGTase) from differentgenera [137, 138]. application this presents the same drawbacks of These enzymes promote the production of cyclodex- by-product and color formation referred to when trins, α(1 → 4) linked oligosaccharides form starch, 8 Enzyme Research

through an intramolecular transglycosylation reac- be minimized. Along with this, immobilization prevents tion. In the process, a starch oligosaccharide is cleaved denaturation by autolysis or organic solvents, and can bring and cleaved and the resulting reducing-end sugar along thermal, operational and storage stabilization, pro- is transferred to the non-reducing-end sugar of the vided that immobilization is adequately designed [142, 143]. same chain [137]. The resulting cyclodextrin may Immobilization has some intrinsic drawbacks, namely, mass consist of six, seven or eight, which are accord- transfer limitations, loss of activity during immobilization ingly termed α, β,orγ-cyclodextrin, respectively. procedures, particularly due to chemical interaction or Given their ability to form inclusion complexes with steric blocking of the active site; the possibility of enzyme small hydrophobic molecules, they are of interest leakage during operation; risk of support deterioration for both industrial and research applications. Wild- under operational conditions, due to mechanical or chemical type CGTases typically produce a mixture of the stress; and a (still) relative empirical methodology, which three cyclodextrins when incubated with starch. The may hamper scale up. Economical issues are furthermore purification of a given cyclodextrin from the reaction to be taken into consideration when commercial processes mixture requires several additional steps, including are envisaged, although immobilization can prove critical for selective complexation with organic solvents, which economic viability if costly enzymes are used. Still, the cost may prove restrictive for cyclodextrin applications of the support, immobilization procedure and processing the involving human consumption [139, 140]. There biocatalyst once exhausted, up- and downstream processing is therefore a clear interest in obtaining a mutant of the bioconversion systems, and sanitation requirements CGTase capable of producing a particular type of have to be taken into consideration. In the overall, the cyclodextrin in a high rate. Van der Veen and cowork- enhanced stability allowing for consecutive reuse leads to −1 −1 ers engineered a double-mutant (Y89D/S146P) of high specific productivity (massproduct massbiocatalyst), which CGTase from Bacillus circulans which displayed a 2- influences biocatalyst-related production costs [1, 142]. fold increase in the production of α-cyclodextrin and A typical example is the output of immobilized glucose a marked decrease in β-cyclodextrin when compared isomerase, allowing for 12,000–15,000 kg of dry-product to the wild type. From the data gathered, the high-fructose corn syrup (containing 42% fructose) per authors suggested that hydrogen bonds (S146) and kilogram of biocatalyst, throughout the operational lifetime hydrophobic interactions (Y89), are likely to play of the biocatalyst [144]. Increased thermal stability, allowing a key role in to the size of cyclodextrin products forroutinereactoroperationabove60◦C minimizes the risks formed, and that changes in sugar-binding subsites of microbial growth, hence leading to lower risks of microbial −3and−7 may result in mutant CGTases with altered growth and to less demanding sanitation requirements, since product specificity [137]. Li and coworkers were also cleaning needs of the reactor are less frequent [1, 144]. able to obtain CGTase mutants from Paenibacillus A rule of thumb suggesting that the enzyme costs should macerans strain JFB05-01 with increased specificity be a few percent of the total production costs has been for α-cyclodextrin, through mutations at subsite established [142]. The half-life of the bioreactor is also a −3. In particular, double mutant D372K/Y89R dis- critical issue when evaluating the economical feasibility of a played a 1.5-fold increase in the production of bioconversion process, longer half-lives favoring process eco- α-cyclodextrin, and a significant (roughly 45%) nomics. Examples of commercial bioreactors depict half-lives decrease in the production of β-cyclodextrin when of several months to years, and the same packing can work compared to the wild-type enzyme [138]. throughout some months to years. Among this group, are immobilized enzyme reactors packed with glucose isomerase The two methods are not mutually exclusive and meth- for the production of high-fructose corn syrup; lactase for odologies for engineering of enzymes can assemble both lactose hydrolysis, for the production of whey hydrolysates strategies [141]. and for the production of tagatose; aminoacylase for the Upon identification of the most adequate enzyme, this production of amino acids; isomaltulose synthase for the can be formulated adequately for better process integration. production of isomaltulose; invertase for the production One of the most widely considered approaches for such of inverted sugar syrup; lipases for the interesterification formulation is enzyme immobilization. of edible oils, ultimately targeted at the production of trans-free fat, of cocoa butter equivalents, and of modified 4. Immobilization triacylglycerols; and β-fructofuranosidase for the production of fructooligosaccharides [144–146]. On the other hand, There are several issues that can be lined up to sustain despite the technical advantages of immobilization, the large- enzyme immobilization. It allows for high-enzyme load scale liquefaction of starch to dextrins by α-amylases is with high activity within the bioreactor, hence leading performed by free enzymes, given the low cost of the enzyme to high-volumetric productivities; it enables the control [18]. of the extension of the reaction; downstream process is Immobilization can be performed by several methods, simplified, since biocatalyst is easily recovered and reused; namely, entrapment/microencapsulation, binding to a solid the product stream is clear from biocatalyst; continuous carrier, and cross-linking of enzyme aggregates, resulting in operation (or batch operation on a drain-and-fill basis) and carrier-free macromolecules [142]. The latter presents an process automation is possible; and substrate inhibition can alternative to carrier-bound enzymes, since these introduce Enzyme Research 9 a large portion of noncatalytic material. This can account for sucrose hydrolysis [160]. The stability of an amylase to about 90% to more than 99% of the total mass of immobilized biocatalyst was further enhanced with the the biocatalysts, resulting in low space-time yields and addition of 1% silica gel to the alginate prior to gelation, as productivities, and often leads to the loss of more than reflected by the use of the biocatalyst in 20 cycles of opera- 50% native activity, which is particularly noticeable at high tion, while retaining more than 90% of the initial efficiency enzyme loadings [142]. A broad, generalized overview of the [159]. Several enzymes, namely, chymosin, cyprosin, lactase, advantages and drawbacks of the different immobilization Neutrase, trypsin, have also been immobilized in liposomes, approachesisgiveninTable4. A typical example of the [161]. In a particularly favored technique immobilization of patterns suggested by data in Table 4 was observed by Abdel- enzymes in liposomes, known as dehydration-rehydration Naby when evaluating the immobilization of α-amylase vesicles (DRVs), small (diameters usually below 50 nm) through different methods [147]. Details on the different unilamellar vesicles (SUVs) is prepared in distilled water methods, as well as some illustrative examples of their and mixed with an aqueous solution of the enzyme to applications, are given hereafter. be encapsulated. The resulting vesicle suspension is then Entrapment/(micro)encapsulation, where the enzyme is dehydrated under freeze drying or equivalent method. Upon contained within a given structure. This can be: a polymer rehydration, the resulting DRVs are multilamellar and larger network of an organic polymer or a sol-gel; a membrane (from 200 nm to a little above 1000 nm) than the original device such as a hollow fiber or a microcapsule; or a (reverse) SUVs, and can capture solute molecules [161, 162]. Recent micelle. Apart from the hollow fiber, the whole process work in this particular application has used lactase as of immobilization is performed in-situ. The polymeric enzyme model and has focused on the optimization and network is formed in the presence of the enzyme, leading characterization of the liposome-based immobilized system to supports that are often referred to as beads or capsules. [163, 164]. If liposome-based biocatalysts are used in a Still, the latter term could preferably be used when the core process under continuous operation, biocatalyst separation and the boundary layer(s) are made of different materials, has to be integrated (namely, using an ultra-filtration namely, alginate and poly-l-lysine. Although direct contact membrane). In a different concept, based in batch mode, with an adverse environment is prevented, mass transfer liposome-encapsulated lactase was incorporated in milk. limitations may be relevant, enzyme loading is relatively After ingestion, the vesicles are disrupted in the stomach low, and leakage, particularly of smaller enzymes from by the presence of bile salts, allowing in-situ degradation of hydrogels (namely, alginate, gelatin), may occur. This may lactose [165]. Cocktails of enzymes, namely, Flavourzyme, be minimized by previously cross-linking the enzyme with bacterial proteases and Palatase M (a commercial lipase multifunctional agent (namely, glutaraldehyde) [148, 149] preparation), were immobilized in liposomes and success- or by promoting cross-linkage of the matrix after the fully used to speed up cheddar cheese ripening [166]. entrapment [150]. The use of LentiKats, a polyvinyl-alcohol- Encapsulation in lipid vesicles has been proved a mild based support in lens-shaped form, has been used for several method, providing high protection against proteolysis. There applications in carbohydrate processing. Among these are is however some lack of consensus on the feasibility of its the synthesis of oligosaccharides with dextransucrase [149], application on large scale, as well as on the effectiveness maltodextrin hydrolysis with glucoamylase [151], lactose of the methodology for controlled release of enzymes [156, hydrolysis with lactase [152], and production of invert sugar 157, 161, 163, 167]. Containment within an ultra-filtration syrup with invertase [153]. In these processes the biocatalyst (UF) membrane allows the enzyme to perform in a fully could be effectively reused or operated in a continuous fluid environment; hence, with little loss (if any) of catalytic manner. Methodologies for large scale production of these activity. However, the membrane still presents a boundary supports have been implemented [154, 155]. Flavourzyme, for overall mass transfer of substrate/products and enzyme (a fungal protease/peptidase complex) entrapped in calcium molecules are prone to interact with the membrane material. alginate [156], k-carragenan, gellan, and higher melting-fat This feature is enhanced along with the hydrophobicity fraction of milk fat [157], was effectively used in cheese of the membrane, hence immobilization in membrane ripening, in order to speed up the process, while avoiding devices may have some adsorptive nature, a feature that the problems associated with the use of free enzyme. These will be addressed in (ii). Besides, regular replacement of include deficient enzyme distribution, reduced yield and the membrane may be required. Enzyme containment by a poor-quality cheese, partly ascribed to excessive proteolysis membrane has been used for the continuous production of and whey contamination. The enzyme complex is released in galactooligosaccharides from lactose. The reaction, with up a controlled manner due to pressure applied during cheese to 80% lactose conversion out of a substrate concentration curd [156]. of 250 gL−1, was carried out in a perfectly mixed reactor and Calcium alginate beads were also used to immobilize enzyme was recovered in a 10 kDa nominal molecular weight glucose isomerase [158]andα-amylase for starch hydrolysis cutoff. The resulting product presented some similarities to to whey [159]. In the latter work, the authors observed that the commercially available Vivinal prebiotic [168]. Within increasing the concentration of CaCl2 and of sodium alginate the same methodology, a hollow-fiber module was used to to 4% and 3%, respectively, enzyme leakage was minimized contain lactase, in order to carry out lactose hydrolysis in (a common drawback of hydrogels) while allowing for high continuous operation. A conversion rate close to 95% in skim activity and stability. This effect was also observed in a milk was observed for an initial substrate concentration close previous work where alginate-entrapped inulinase was used to 40 gL−1 [169]. 10 Enzyme Research

Table 4: A generalized characterization of immobilization methods.

Immobilization method Parameter Carrier binding CLEAs, CLECs Entrapment Covalent Ionic Adsorption Activity High High Low Intermediate/High High Range of application Low Intermediate Intermediate Low Intermediate/High Immobilization efficiency Low Intermediate High Intermediate Intermediate Cost Low Low High Intermediate Low Preparation Easy Easy Difficult Intermediate Intermediate/Difficult Substrate specificity Cannot be changed Cannot be changed Can be changed Cannot be changed Can be changed Regeneration Possible Possible Impossible Impossible Impossible

Binding to a solid carrier, where enzyme-support inter- onto dried oxidized bagasse [180], onto polyglutaraldehyde- action can be of covalent, ionic, or physical nature. The latter activated gelatin [181], or onto macroporous copolymer comprehends hydrophobic and van der Waals interactions. of ethylene glycol dimethacrylate and glycidyl methacrylate These are of weak nature and easily allow for enzyme leakage through the carbohydrate moiety of the enzyme [182]; glu- from the support, namely, after environmental shifts in pH, coamylase or invertase immobilized onto montmorillonite ionic strength, temperature or even as a result of flow rate K-10 activated with aminopropyltriethoxysilane and glu- or abrasion. On the other hand, desorption can be turned taraldehyde [183, 184]; and invertase immobilized on nylon- into an advantage if performed under a controlled manner, 6 microbeads, previously activated with glutaraldehyde and since it enables the expedite removal of spent enzyme and using PEI as spacer [185, 186]; on polyurethane treated its replacement with fresh enzyme [170]. A recent paper with hydrochloric acid, polyethylenimine and glutaralde- by Gopinath andSugunan illustrates the increased trend for hyde [187]; on poly(styrene-2-hydroxyethyl methacrylate) leakage when adsorption is compared with covalent binding, microbeads activated with epichlorohydrin [188]; or on using α-amylase as model enzyme [171]. Curiously, the poly(hydroxyethyl methacrylate)/glycidyl methacrylate films first reported application of enzyme immobilization was of [189]. Within this methodology for immobilization, high- invertase onto activated charcoal [172]. Recently invertase light should be given to the introduction of commer- was immobilized in different types of sawdust, aiming at its cial supports (namely, Eupergit, Sepabeads) with a high application for sucrose hydrolysis. When wood shavings were density of epoxide functional groups aimed at multipoint used as support, the immobilized invertase retained 90% attachment, typically with the ε-amino group of lysine, to of the original activity after 20 cycles of 15 minutes, each confer high rigidity to the enzyme molecule, hence enhanc- under consecutive batch operation; and it retained 65% of ing stabilization [190, 191]. This methodology has been the original activity after 10 hours of continuous operational used for lactase immobilization in magnetic poly(GMA- regime in a column reactor [173]. Anther example is the MMA), formed from monomers of glycidylmethacrylate immobilization of pectinase in egg shell for the preparation and ethylmethacrylate, and cross-linked with ethyleneglycol of low-methoxyl pectin. The immobilized biocatalyst could dimethacrylate [192]; for the immobilization of cyclodextrin be reused for 32 times at 30◦C, and it was used in a glycosyltransferases to glyoxylagarose supports for the pro- fluidized-bed reactor, operated at an optimum flow rate of duction of cyclodextrins [193]; or for the immobilization of 5mLh−1 and 35◦C[174]. Other examples are the surface dextransucrase on Eupergit C [194]. Ionic binding to a car- immobilizations of α-amylase on alumina [175] and in rier involves interaction of negatively or positively charged zirconia [176]. Covalent binding is the strongest form of groups of the carrier with charged amino-acid residues enzyme linking to a solid support. It involves chemically on the enzyme molecules [195]. Ionic interaction may be reactive sites of the protein such as amino groups, carboxyl favored if enzyme leakage is not an issue, since it allows groups, and phenol residues of tyrosine; sulfhydryl groups; for support regeneration, unlike immobilization by covalent or the imidazole group of histidine. The binding can be binding. Ion-exchanger resins are typical supports for ionic carried out by several methods; among them are amide binding; among them are derivatives of cross-linked polysac- bond formation, alkylation and arylation, or UGI reaction. charides, namely, carboxymethyl- (CM-) cellulose, CM- However, this often brings along loss of activity during the Sepharose, diethylaminoethyl- (DEAE-) cellulose, DEAE- process of immobilization, due to support binding to critical Sephadex, quaternary aminoethyl anion exchange- (QAE- residues for enzyme activity, and steric hindrance, among ) cellulose, QAE-dextran, QAE-Sephadex; derivatives of others. Examples include the immobilization of α-amylase synthetic polymers, namely, Amberlite, Diaion, Dowex, [177] and of levansucrase [178] on glutaraldehyde-treated Duolite; and resins coated with ionic polymers, namely, chitosan beads, through the glutaraldehyde reaction between polyethylenimine (PEI) [196]. Recent examples include the the free amino groups of chitosan and the enzyme molecule; immobilization of invertase in Dowex [197], in Duolite the immobilization of pectinase onto Amberlite IRA900 Cl [198], in poly(glycidyl methacrylate-co-methyl methacry- through glutaraldehyde cross-linking [179]; glucoamylase late beads grafted with PEI [199], and in epoxy(amino) Enzyme Research 11

Sepabeads [200]; lactase immobilization in PEI-grafted those processed by commercially available enzyme Sepabeads [201]; fructosyltransferase in DEAE-cellulose for preparations (either free, carrier free, or carrier- the production of fructosyl disaccharides [202]; glucose bound), while achieving the same 45% yield in isomerase in DEAE-cellulose [203] or in Indion 48-R [204]; fructose, under similar operational condition [212]. glucoamylase onto SBA-15 silica [205] and in epoxy(amino) Sepabeads [200]. Ionic binding to Sepabeads-like supports (6) Sixth, CLECs of glucose isomerase packed in a has acknowledged multipoint attachment nature. Enzyme column were also used for the concentration/puri- molecules can be modified chemically or genetically mod- fication of xylitol from dilute or impure solutions. ified to enhance immobilization efficiency, an approach fol- The approach was based on the high specificity lowed by Kweon and coworkers, who obtained a cyclodextrin of the enzyme crystals towards xylitol, allowing its glycosyltransferase fused with 10 lysine residues to improve separation from other sugars, including the nat- ionic binding to SP-Sepharose [206]. ural substrates, xylose and glucose. Recovery of the adsorbed xylitol was achieved by elution with Carrier-free macroparticles, where a bifunctional reagent 2+ (namely, glutaraldehyde), is used to cross-link enzyme aggre- CaCl2 solutions, with Ca being acknowledged to gates (CLEAs) or crystals (CLECs), leading to a biocatalyst inactivate glucose isomerase [213]. displaying highly concentrated enzyme activity, high stability and low production costs [142, 207]. The use of CLEAs Each method for enzyme immobilization has a unique is favored given the lower complexity of the process. This nature. Therefore, despite the potential of immobilization to approach is recent, as compared with entrapment and improve enzyme performance by enhancing activity, stability, or specificity, no specific approach tackles simultaneously binding to a solid carrier, and there are still relatively few ff examples of its application to enzymes used in the area of these di erent features. A careful evaluation and charac- food processing. Among those are following. terization of the methodology addressed is thus required, which can be significantly fastened by high-throughput (1) First is the immobilization of Pectinex Ultra SP- approaches [214]. Again, the feasibility of its application to L, a commercial enzyme preparation containing reactor configuration and mode of operation has also to be pectinase, xylanase, and cellulose activities [208]. The considered in the selection process of the most adequate CLEA biocatalyst displayed a slight (30%) in the immobilized biocatalyst for a given bioconversion. Vmax, maximal reaction rate/KM ratio, but a significant enhancement in thermal stability (a roughly 10- 4.1. Typical Bioreactors. The most common form of enzy- fold increase in half-life), when the pectinase activity matic reactors for continuous operation is the packed-bed of the immobilized biocatalyst was compared with setup, basically a cylindrical column holding a fixed bed the free form. of catalyst particles (Figure 1). These should not have sizes (2) Second is the immobilization of lactase for the below 0.05 mm, in order to keep the pressure drop within hydrolysis of lactose, where, under similar opera- reasonable limits. Commercially available carriers such as tional conditions as for the free enzyme, the CLEA Eupergit C have particle sizes of roughly 0.1 mm [215]. yielded 78% monosaccharides in 12 h as compared Commonly operated in down-flow mode, the range of flow to 3.9% of the free form [209]. ratesusedmustbesuchastoprovideacompromisebetween ff (3) Third, CLEAs of glucoamylase, formed by either reasonable pressure drop, minimal di usion layer and high glutaraldehyde or diimidates, namely, dimethylmal- conversion yield. Minimization of external mass-transfer onimidate, dimethylsuccinimidate, and dimethylglu- resistances with enhanced flow rates can be considered, tarimidate, led to biocatalysts with improved thermal leading to the fluidized-bed reactor. This is basically a stability as compared to the free form (over 2-fold variation of the packed-bed reactor, but operated in up- increase in half-lives) [210]. flow mode, where the biocatalyst particles are not in close contact which each other; hence, pressure drop is low, and (4) Fourth, CLEAs of wild type and two mutant levan- accordingly are pumping costs. The residence time allowed sucrases were assayed for oligosaccharides/levan and by the flow rates required for fluidization may however for fructosyl-xyloside synthesis. Although the specific result in low conversion yields. This can be overcome by activity of the three free enzymes was 1.25- to 3- operating a battery of reactor or by operation in recycle fold higher than the corresponding CLEAs, these mode [216]. Bioconversions with free enzymes are carried displayed a 40- to 200-fold higher specific activity out in stirred tanks. When on their own, they are restricted than the equivalent Eupergit-C-immobilized enzyme to batch mode, but when coupled to a membrane setup preparations. Furthermore, all CLEA preparations with suitable cutoff, they can be integrated in a continuous displayed enhanced thermal stability when compared process, since the enzymes are rejected by the membrane, with the corresponding free enzymes [211]. which acts as an immobilization device, whereas the product (5) Fifth are CLECs of glucose isomerase, aimed at the (and unconverted substrate) freely permeates. Shear stress conversion of glucose into fructose for the pro- induced by stirring creates a hazardous environment for ductionofhighfructosecornsyrup.Whenplaced immobilized biocatalysts, particularly when hydrogels are in a packed-bed, the resulting enzyme preparation considered, since they are prone to abrasion. In order to allowed for flow rates that matched or even exceeded overcome this, a basket reactor was developed, but is seldom 12 Enzyme Research

Fluid in Fluid out Fluid in Fluid out

Fluid in

Fluid out (product rich)

Biocatalyst Ultrafiltration unit recycle Free enzyme Free enzyme Fluid out Fluid in Immobilized enzyme Immobilized enzyme (a) (b) (c) Perfectly mixed reactor with recycle (d) Stirred basket reac- (e) Stirred batch reactor Packed- Fluidized- tor bed bed reactor reactor Figure 1: Examples of bioreactor configurations commonly used in bioconversion processed involving free or immobilized enzymes. Reactors (a) to (d) are depicted under continuous mode of operation, whereas reactor (e) is depicted. used, possibly due to mass transfer resistances associated the food and feed sector, since most products are of relatively [18]. low added value. Therefore, there is no universal support and method for enzyme immobilization aimed at application in food and feed (let alone the overall range of possible 5. Conclusions and Future Perspectives fields of use), and the immobilized biocatalyst fit for a given process and product may be totally unsuitable for another. Theintegrationofenzymesinfoodandfeedprocessesisa Given the diversity of enzyme nature and applications this well-established approach, but evidence clearly shows that pattern is unlikely to be reversed. Hence, it can be foreseen dedicated research efforts are consistently being made as that efforts will be towards the development of immobilized to make this application of biological agents more effective biocatalyst with suitable chemical, physical, and geometric and/or diversified. These endeavors have been anchoring characteristics, which can be produced under mild condi- in innovative approaches for the design of new/improved tion, that can be used in different reactor configurations and biocatalysts, more stable (to temperature and pH), less that comply with the economic requirements for large-scale dependent on metal ions and less susceptible to inhibitory application. All these strategies either isolated or preferably agents and to aggressive environmental conditions, while suitably integrated have been put into practice in food and maintaining the targeted activity or evolving novel activities. feed, to improve existing processes or to implement new This is of particular relevance for application in the food ones, with the latter often combined with the output of new and feed sector, for it allows enhanced performance under goods, resulting from novel enzymatic activities. Given the operational conditions that minimize the risk of microbial recent developments in this field, this trend is foreseen to be contamination. It also favors process integration, by allowing further implemented. the concerted use of enzymes that naturally have diverse requirements for effective application. Such progresses have been made through the ever-continuing developments in Acknowledgment molecular biology, the accumulated evolutionary enzyme engineering expertise, the (bio)computational tools, and the Pedro Fernandes acknowledges Fundaçao˜ para a Cienciaˆ e a implementation of high-throughput methodologies, with Tecnologia (Portugal) for financial support under program high level of parallelization, enabling the efficient and timely Cienciaˆ 2007. screening/characterization of the biocatalysts. Alongside with these strategies, the immobilization of enzymes has also been a key supporting tool for rendering these proteins fit References for industrial application, while simultaneously enabling the [1] D. Vasic-Racki, “History of industrial biotransformations— improvement of their catalytic features. Again, and despite dreams and realities,” in Industrial Biotransformations,A. the developments made in this particular field, there is still Liese, K. Seelbach, and C. Wandrey, Eds., pp. 1–35, Wiley- the lack of a set of unanimously applicable rules for the VCH, Weinheim, Germany, 2nd edition, 2006. selection of carrier and method of enzyme immobilization, [2] P.B. Poulsen and H. Klaus Buchholz, “History of enzymology which furthermore encompass both technical and economic with emphasis on food production,” in Handbook of Food requirements. The latter can be particularly restrictive in Enzymology,J.R.Whitaker,A.G.J.Voragen,andD.W.S. Enzyme Research 13

Wong, Eds., pp. 11–20, Marcel Dekker, New York, NY, USA, [18] A. Illanes, Enzyme Biocatalysis—Principles and Applications, 2003. Springer, New York, NY, USA, 2008. [3] T. Schafer,¨ O. Kirk, T. V. Borchert et al., “Enzymes for [19] A. S. Bommarius and B. R. Riebel, Biocatalysis: Fundamentals technical applications,” in Biopolymers, S. R. Fahnestock and and Applications, Wiley-VCH, Weinheim, Germany, 2004. S. R. Steinbuchel,¨ Eds., pp. 377–437, Wiley-VCH, Weinheim, [20] K. Oxenbøll and S. Ernst, “Environment as a new perspective Germany, 2002. on the use of enzymes in the food industry,” Food Science and [4] P. Fernandes, “Enzymes in sugar industries,” in Enzymes in Technology, vol. 22, no. 1, pp. 35–37, 2008. Food Processing: Fundamentals and Potential Applications,P. [21] J. D. Rozzell, “Commercial scale biocatalysis: myths and Panesar, S. S. Marwaha, and H. K. Chopra, Eds., pp. 165–197, realities,” Bioorganic and Medicinal Chemistry, vol. 7, no. 10, I.K. International Publishing House, New Delhi, India, 2010. pp. 2253–2261, 1999. [5] M. Leisola, J. Jokela, O. Pastinen, O. Turunen, and H. Schoe- [22] H. E. Schoemaker, D. Mink, and M. G. WubboLts, “Dis- maker, “Industrial use of enzymes,” in Encyclopedia of Life pelling the myths—biocatalysis in industrial synthesis,” Sci- Support Systems (EOLSS),O.O.P.Hanninen¨ and M. Atalay, ence, vol. 299, no. 5613, pp. 1694–1697, 2003. Eds., pp. 1–25, EOLSS, Oxford, UK, 2002. [23] R. H. Sajedi, H. Naderi-Manesh, K. Khajeh et al., “A Ca- [6] J. Norus, “Building sustainable competitive advantage from independent α-amylase that is active and stable at low knowledge in the region: the industrial enzymes industry,” pH from the Bacillus sp. KR-8104,” Enzyme and Microbial European Planning Studies, vol. 14, no. 5, pp. 681–696, 2006. Technology, vol. 36, no. 5-6, pp. 666–671, 2005. [7]E.P.S.BonandM.A.Ferrara,Bioethanolproduction [24] X. D. Liu and Y. Xu, “A novel raw starch digesting α-amylase via enzymatic hydrolysis of cellulosic biomass, Document from a newly isolated Bacillus sp. YX-1: puriﬁcation and prepared for “The Role of Agricultural Biotechnologies characterization,” Bioresource Technology, vol. 99, no. 10, pp. for Production of Bioenergy in Developing Countries” 4315–4320, 2008. an FAO seminar held in Rome on 12 October 2007, [25] K. Rachinskiy, H. Schultze, M. Boy, U. Bornscheuer, and J. http://www.fao.org/biotech/seminaroct2007.htm. Buchs,¨ “”Enzyme Test Bench,” a high-throughput enzyme [8]H.ElEnshasy,A.Abuoul-Enein,S.Helmy,andY.ElAzaly, characterization technique including the long-term stability,” “Optimization of the industrial production of alkaline Biotechnology and Bioengineering, vol. 103, no. 2, pp. 305– protease by Bacillus licheniformis in diﬀerent production 322, 2009. scales,” Australian Journal of Applied Science, vol. 2, pp. 583– [26] C. M. M. C. Andrade, N. Pereira Jr., and G. Antranikian, 593, 2008. “Extremely thermophilic microorganisms and their poly- [9] Freedonia Group Inc. World Enzymes—Industry Study with merhydrolytic enzymes,” Revista de Microbiologia, vol. 30, no. Forecasts for 2013 & 2018: Study #2506, August 2009, 4, pp. 287–298, 1999. http://www.freedoniagroup.com/brochure/25xx/2506smwe [27] H. Sun, P. Zhao, X. Ge et al., “Recent advances in microbial .pdf. raw starch degrading enzymes,” Applied Biochemistry and [10] P. Binod, R. R. Singhania, C. R. Soccol, and A. Pandey, Biotechnology, vol. 160, no. 4, pp. 988–1003, 2009. “Industrial enzymes,” in Advances in Fermentation Technol- [28] C. Bertoldo and G. Antranikian, “Starch-hydrolyzing ogy,A.Pandey,C.Larroche,C.R.Soccol,andC.-G.Dussap, enzymes from thermophilic archaea and bacteria,” Current Eds., pp. 291–320, Asiatech Publishers, New Delhi, India, Opinion in Chemical Biology, vol. 6, no. 2, pp. 151–160, 2002. 2008. [29] J. Synowiecki, B. Grzybowska, and A. Zdziebło, “Sources, [11] R. M. Berka and J. R. Cherry, “Enzyme biotechnology,” in properties and suitability of new thermostable enzymes Basic Biotechnology, C. Ratledge and B. Kristiansen, Eds., pp. in food processing,” Critical Reviews in Food Science and 477–498, Cambridge University Press, Cambridge, UK, 3rd Nutrition, vol. 46, no. 3, pp. 197–205, 2006. edition, 2006. [30] M .M. Kristjansson´ and A. Asgeirsson,´ “Properties of [12]O.Kirk,T.V.Borchert,andC.C.Fuglsang,“Industrial extremophilic enzymes and their importance in food science enzyme applications,” Current Opinion in Biotechnology, vol. and technology,” in Handbook of Food Enzymology,J.R. 13, no. 4, pp. 345–351, 2002. Whitaker, A. G. J. Voragen, and D. W. S. Wong, Eds., pp. 77– [13] T. Schafer,¨ T. W. Borchert, V. S. Nielsen et al., “Industrial 100, Marcel Dekker, New York, NY, USA, 2003. enzymes,” Advances in Biochemical Engineering/Biotechnolo- [31] C. Vieille and G. J. Zeikus, “Hyperthermophilic enzymes: gy, vol. 105, pp. 59–131, 2006. sources, uses, and molecular mechanisms for thermostabil- [14] J. Ogawa and S. Shimizu, “Industrial microbial enzymes: ity,” Microbiology and Molecular Biology Reviews, vol. 65, no. their discovery by screening and use in large-scale production 1, pp. 1–43, 2001. of useful chemicals in Japan,” Current Opinion in Biotechnol- [32] S. H. Brown, H. R. Costantino, and R. M. Kelly, “Character- ogy, vol. 13, no. 4, pp. 367–375, 2002. ization of amylolytic enzyme activities associated with the [15] A. K. Chandel, R. Rudravaram, L. V. Rao, P. Ravindra, and hyperthermophilic archaebacterium Pyrococcus furiosus,” M. L. Narasu, “Industrial enzymes in bioindustrial sector Applied and Environmental Microbiology,vol.56,no.7,pp. development: an Indian perspective,” Journal of Commercial 1985–1991, 1990. Biotechnology, vol. 13, no. 4, pp. 283–291, 2007. [33] N. Goyal, J. K. Gupta, and S. K. Soni, “A novel raw starch [16] D. Carrez and W. Soetaert, “Looking ahead in Europe: white digesting thermostable α-amylase from Bacillus sp. I-3 and biotech by 2025,” Industrial Biotechnology, vol. 1, pp. 95–101, its use in the direct hydrolysis of raw potato starch,” Enzyme 2005. and Microbial Technology, vol. 37, no. 7, pp. 723–734, 2005. [17] Research and markets (2010). Future of Enzymes in China to [34] B. Arikan, “Highly thermostable, thermophilic, alkaline, SDS 2020, http://www.researchandmarkets.com/reportinfo.asp? and chelator resistant amylase from a thermophilic Bacillus cat id=0&report id=1202421&q=future%20of%20enzymes sp. isolate A3-15,” Bioresource Technology,vol.99,no.8,pp. &p=1. 3071–3076, 2008. 14 Enzyme Research

[35]G.D.Haki,A.J.Anceno,andS.K.Rakshit,“AtypicalCa2+- [48] A. D. Sharma and P. K. Gill, “Purification and characteri- independent, raw-starch hydrolysing α-amylase from Bacillus zation of heat-stable exo-inulinase from Streptomyces sp,” sp. GRE1: characterization and gene isolation,” World Journal Journal of Food Engineering, vol. 79, no. 4, pp. 1172–1178, of Microbiology and Biotechnology, vol. 24, no. 11, pp. 2517– 2007. 2524, 2008. [49] D. M. Lima, R. Q. Oliveira, A. P.T. Uetanabaro, A. Goes-Neto,´ [36] M. Ballschmiter, O. Futterer,¨ and W. Liebl, “Identification C. A. Rosa, and S. A. Assis, “Thermostable inulinases secreted and characterization of a novel intracellular alkaline α- by yeast and yeast-like strains from the Brazilian semi-arid amylase from the hyperthermophilic bacterium Thermotoga region,” International Journal of Food Sciences and Nutrition, maritima MSB8,” Applied and Environmental Microbiology, vol. 60, supplement 7, pp. 63–71, 2009. vol. 72, no. 3, pp. 2206–2211, 2006. [50] Y. B. Ammar, T. Matsubara, K. Ito et al., “Characterization [37] P. Dheeran, S. Kumar, Y. K. Jaiswal, and D. K. Adhikari, of a thermostable levansucrase from Bacillus sp. TH4-2 “Characterization of hyperthermostable α-amylase from capable of producing high molecular weight levan at high Geobacillus sp. IIPTN,” Applied Microbiology and Biotechnol- temperature,” Journal of Biotechnology, vol. 99, no. 2, pp. 111– ogy, vol. 86, pp. 1857–1866, 2010. 119, 2002. [38] J. L. Uma Maheswar Rao and T. Satyanarayana, “Biophysical [51] S. H. Brown and R. M. Kelly, “Characterization of amylolytic α α and biochemical characterization of a hyperthermostable enzymes, having both -1,4 and -1,6 hydrolytic activity, and Ca2+-independent α-amylase of an extreme ther- from the thermophilic archaea Pyrococcus furiosus and mophile Geobacillus thermoleovorans,” Applied Biochemistry Thermococcus litoralis,” Applied and Environmental Micro- and Biotechnology, vol. 150, no. 2, pp. 205–219, 2008. biology, vol. 59, no. 8, pp. 2614–2621, 1993. [39] S. M. Noorwez, M. Ezhilvannan, and T. Satyanarayana, [52] A. Kunamneni and S. Singh, “Improved high thermal “Production of a high maltose-forming, hyperthermostable stability of pullulanase from a newly isolated thermophilic and Ca2+-independent amylopullulanase by an extreme Bacillus sp. AN-7,” Enzyme and Microbial Technology, vol. 39, thermophile Geobacillus thermoleovorans in submerged fer- no. 7, pp. 1399–1404, 2006. mentation,” Indian Journal of Biotechnology,vol.5,no.3,pp. [53] C. Winterhalter and W. Liebl, “Two extremely thermostable 337–345, 2006. xylanases of the hyperthermophilic bacterium Thermotoga [40] S. Hernalsteens and F. Maugeri, “Properties of thermostable maritima MSB8,” Applied and Environmental Microbiology, extracellular FOS-producing fructofuranosidase from cryp- vol. 61, no. 5, pp. 1810–1815, 1995. tococcus sp,” European Food Research and Technology, vol. [54] R. Khandeparkar and N. B. Bhosle, “Purification and char- 228, no. 2, pp. 213–221, 2008. acterization of thermoalkalophilic xylanase isolated from the Enterobacter sp. MTCC 5112,” Research in Microbiology, vol. [41] M.-S. Kim, J.-T. Park, Y.-W. Kim et al., “Properties of 157, no. 4, pp. 315–325, 2006. a novel thermostable glucoamylase from the hyperther- [55] J. Gomes and W. Steiner, “The biocatalytic potential of mophilic archaeon sulfolobus solfataricus in relation to extremophiles and extremozymes,” Food Technology and starch processing,” Applied and Environmental Microbiology, Biotechnology, vol. 42, no. 4, pp. 223–235, 2004. vol. 70, no. 7, pp. 3933–3940, 2004. [56] S. Linko, “Novel approaches in microbial enzyme produc- [42] K. Srih-Belghith and S. Bejar, “A thermostable glucose tion,” Food Biotechnology, vol. 3, pp. 31–43, 1989. isomerase having a relatively low optimum pH: study of [57] N. F. Haard, “A review of proteotlytic enzymes from marine activity and molecular cloning of the corresponding gene,” organisms and their application in the food industry,” Journal Biotechnology Letters, vol. 20, no. 6, pp. 553–556, 1998. of Aquatic Food Product Technology, vol. 1, pp. 17–35, 1992. [43] R. K. Bandlish, J. M. Hess, K. L. Epting, C. Vieille, and R. M. [58] M. Chandrasekaran, “Industrial enzymes from marine Kelly, “Glucose-to-fructose conversion at high temperatures microorganisms: the Indian scenario,” Journal of Marine with xylose (glucose) isomerases from Streptomyces murinus Biotechnology, vol. 5, no. 2-3, pp. 86–89, 1997. and two hyperthermophilic Thermotoga species,” Biotechnol- [59] F. Shahidi and Y. V. A. Janak Kamil, “Enzymes from fish ogy and Bioengineering, vol. 80, no. 2, pp. 185–194, 2002. and aquatic invertebrates and their application in the food [44] H. R. Costantino, S. H. Brown, and R. M. Kelly, “Purifi- industry,” Trends in Food Science and Technology, vol. 12, no. α cation and characterization of an -glucosidase from a 12, pp. 435–464, 2001. hyperthermophilic archaebacterium, Pyrococcus furiosus, [60] R. S. Rasmussen and M. T. Morrissey, “Marine biotechnology exhibiting a temperature optimum of 105 to 115◦C,” Journal for production of food ingredients,” Advances in Food and of Bacteriology, vol. 172, no. 7, pp. 3654–3660, 1990. Nutrition Research, vol. 52, pp. 237–292, 2007. [45]S.W.M.Kengen,E.J.Luesink,A.J.M.Stams,andA. [61] L. Dipasquale, A. Gambacorta, R. A. Siciliano, M. F. Mazzeo, J. B. Zehnder, “Purification and characterization of an and L. Lama, “Purification and biochemical characterization extremely thermostable β-glucosidase from the hyperther- of a native invertase from the hydrogen-producing Thermo- mophilic archaeon Pyrococcus furiosus,” European Journal of toga neapolitana (DSM 4359),” Extremophiles, vol. 13, no. 2, Biochemistry, vol. 213, no. 1, pp. 305–312, 1993. pp. 345–354, 2009. [46]K.Kato,T.Araki,T.Kitamura,N.Morita,M.Moori,andY. [62] W. Liebl, D. Brem, and A. Gotschlich, “Analysis of the gene Suzuki, “Purification and properties of a thermostable inuli- for β-fructosidase (invertase, inulinase) of the hyperther- nase (β-D-fructan fructohydrolase) from bacillus stearother- mophilic bacterium Thermotoga maritima, and characteri- mophilus KP1289,” Starch/Starke¨ , vol. 51, no. 7, pp. 253–258, sation of the enzyme expressed in Escherichia coli,” Applied 1999. Microbiology and Biotechnology, vol. 50, no. 1, pp. 55–64, [47] P. K. Gill, R. K. Manhas, and P. Singh, “Comparative 1998. analysis of thermostability of extracellular inulinase activity [63] J. Sheng, Z. Chi, J. Li, L. Gao, and F. Gong, “Inulinase produc- from Aspergillus fumigatus with commercially available tion by the marine yeast Cryptococcus aureus G7a and inulin (Novozyme) inulinase,” Bioresource Technology, vol. 97, no. hydrolysis by the crude inulinase,” Process Biochemistry, vol. 2, pp. 355–358, 2006. 42, no. 5, pp. 805–811, 2007. Enzyme Research 15

[64] B. R. Mohapatra, M. Bapuji, and A. Sree, “Production of [80]Q.-F.Wang,Y.-H.Hou,Z.Xu,J.-L.Miao,andG.-Y. industrial enzymes (Amylase, Carboxymethylcellulase and Li, “Purification and properties of an extracellular cold- Protease) by bacteria isolated from marine sedentary organ- active protease from the psychrophilic bacterium Pseudoal- isms,” Acta Biotechnologica, vol. 23, no. 1, pp. 75–84, 2003. teromonas sp. NJ276,” Biochemical Engineering Journal, vol. [65] E. Legin, C. Ladrat, A. Godfroy, G. Barbier, and F. Duchiron, 38, no. 3, pp. 362–368, 2008. “Thermostable amylolytic enzymes of thermophilic microor- [81] T. Nakagawa, R. Ikehata, M. Uchino, T. Miyaji, K. Takano, ganisms from deep sea hydrothermal vents,” Comptes Rendus and N. Tomizuka, “Cold-active acid β-galactosidase activity de l’Academie des Sciences - Serie III, vol. 320, no. 11, pp. 893– of isolated psychrophilic-basidiomycetous yeast Guehomyces 898, 1997. pullulans,” Microbiological Research, vol. 161, no. 1, pp. 75– [66] P. Ranjitha, E. S. Karthy, and A. Mohankumar, “Purification 79, 2006. and partial characterization of esterase from marine vibrio [82] P. Hildebrandt, M. Wanarska, and J. Kur, “A new cold- fischeri,” Modern Applied Science, vol. 3, pp. 73–82, 2009. adapted β-D-galactosidase from the Antarctic Arthrobacter [67] A. Giordano, G. Andreotti, A. Tramice, and A. Trincone, sp. 32c—gene cloning, overexpression, purification and “Marine glycosyl hydrolases in the hydrolysis and synthesis properties,” BMC Microbiology, vol. 9, article 151, 2009. of oligosaccharides,” Biotechnology journal,vol.1,no.5,pp. [83] A. Hoyoux, J.-M. François, and P. Dubois, Cold-active beta- 511–530, 2006. galactosidase, the process for its preparation and the use [68] A. Tramice, E. Pagnotta, I. Romano, A. Gambacorta, and thereof. Patent US 6727084, 2004. A. Trincone, “Transglycosylation reactions using glyco- [84] T. Nakagawa, T. Nagaoka, S. Taniguchi, T. Miyaji, and N. syl hydrolases from Thermotoga neapolitana, a marine Tomizuka, “Isolation and characterization of psychrophilic hydrogen-producing bacterium,” Journal of Molecular Catal- yeasts producing cold-adapted pectinolytic enzymes,” Letters ysis B, vol. 47, no. 1-2, pp. 21–27, 2007. in Applied Microbiology, vol. 38, no. 5, pp. 383–387, 2004. [69] A. Gudmundsdottir´ and J. B. Bjarnason, “Applications of cold [85] A. R. D. Mahmoud and A. W. Helmy, “A novel cold-active adapted proteases in the food industry,” in Novel Enzyme and alkali-stable β-glucosidase gene isolated from the marine Technology for Food Application, R. Rastall, Ed., pp. 205–221, bacterium martelella mediterranea,”ˆ Australian Journal of Woodhead Publishing Limited, Cambridge, UK, 2008. Basic and Applied Sciences, vol. 3, pp. 3808–3817, 2009. [70] M. B. Ali, B. Khemakhem, X. Robert, R. Haser, and S. [86] S. Fujiwara, “Extremophiles: developments of their special Bejar, “Thermostability enhancement and change in starch functions and potential resources,” Journal of Bioscience and hydrolysis profile of the maltohexaose-forming amylase Bioengineering, vol. 94, no. 6, pp. 518–525, 2002. of Bacillus stearothermophilus US100 strain,” Biochemical [87] T. H. Richardson, X. Tan, G. Frey et al., “A novel, high Journal, vol. 394, no. 1, pp. 51–56, 2006. performance enzyme for starch liquefaction. Discovery and [71]D.W.S.Wong,S.B.Batt,C.C.Lee,andG.H.Robertson, optimization of a low pH, thermostable α-amylase,” The “High-activity barley α-amylase by directed evolution,” Pro- Journal of Biological Chemistry, vol. 277, no. 29, pp. 26501– tein Journal, vol. 23, no. 7, pp. 453–460, 2004. 26507, 2002. [72] S. Danielsen and H. Lundqvist, Bacterial alpha-amylase [88] Z. S. Olempska-Beer, R. I. Merker, M. D. Ditto, and M. variants. WO Patent 2008/000825, 2008. J. DiNovi, “Food-processing enzymes from recombinant [73] D. K. Oh, H. J. Oh, H. J. Kim, J. Cheon, and P. Kim, microorganisms-a review,” Regulatory Toxicology and Phar- “Modification of optimal pH in l-arabinose isomerase from macology, vol. 45, no. 2, pp. 144–158, 2006. Geobacillus stearothermophilus for d-galactose isomeriza- [89] A. Spok,¨ “Safety regulations of food enzymes,” Food Technol- tion,” Journal of Molecular Catalysis B,vol.43,no.1–4,pp. ogy and Biotechnology, vol. 44, no. 2, pp. 197–209, 2006. 108–112, 2006. [90] J. Vieceli, J. Mullegger,¨ and A. Tehrani, “Computer-assisted [74] M. J. Allen, T,-Y. Fang, Y. Li et al., “Protein engineering of design of industrial enzymes: the resurgence of rational glucoamylase to increase pH optimum, substrate specificity design and in silico mutagenesis,” Industrial Biotechnology, and thermostability,” United States Patents No. 6,537,792, vol. 2, no. 4, pp. 303–308, 2006. 2003. [91] A. S. Bommarius and B. R. Riebel, Biocatalysis. Fundamentals [75] A. Dwevedi and A. M. Kayastha, “Stabilization of β- and Applications, Wiley-VCH, Weinheim, Germany, 2004. galactosidase (from peas) by immobilization onto Amberlite [92] P. Fernandes and J. M. S. Cabral, “Applied biocatalysis: an MB-150 beads and its application in lactose hydrolysis,” overview,” in Industrial Biotechnology, W. Soetaert and E. Journal of Agricultural and Food Chemistry, vol. 57, no. 2, pp. J. Vandamme, Eds., pp. 227–250, Wiley-VCH, Weinheim, 682–688, 2009. Germany, 2010. [76] G. England, M. Kolkman, B. S. Miller, and C. Vroeman, [93] S. Bershtein and D. S. Tawfik, “Advances in laboratory Pullulanase variants with increased productivity. Patent WO evolution of enzymes,” Current Opinion in Chemical Biology, 2008024372A2, 2008. vol. 12, no. 2, pp. 151–158, 2008. [77] A. Tomschy, R. Brugger, M. Lehmann et al., “Engineering [94] W. F. Li, X. X. Zhou, and P. Lu, “Structural features of of phytase for improved activity at low pH,” Applied and thermozymes,” Biotechnology Advances,vol.23,no.4,pp. Environmental Microbiology, vol. 68, no. 4, pp. 1907–1913, 271–281, 2005. 2002. [95] S. Trivedi, H. S. Gehlot, and S. R. Rao, “Protein thermosta- [78] J. Cha and C. A. Batt, “Lowering the pH optimum of D- bility in Archaea and Eubacteria,” Genetics and Molecular xylose isomerase: the effect of mutations of the negatively Research, vol. 5, no. 4, pp. 816–827, 2006. charged residues,” Molecules and Cells, vol. 8, no. 4, pp. 374– [96] S. D’Amico, J.-C. Marx, C. Gerday, and G. Feller, “Activity- 382, 1998. stability relationships in extremophilic enzymes,” The Journal [79] J. Karimaki,¨ T. Parkkinen, H. Santa et al., “Engineering the of Biological Chemistry, vol. 278, no. 10, pp. 7891–7896, 2003. substrate specificity of xylose isomerase,” Protein Engineering, [97] P. A. Dalby, “Engineering enzymes for biocatalysis,” Recent Design and Selection, vol. 17, no. 12, pp. 861–869, 2004. Patents on Biotechnology, vol. 1, no. 1, pp. 1–9, 2007. 16 Enzyme Research

[98] M. Lehmann and M. Wyss, “Engineering proteins for stability,” Trends in Biotechnology, vol. 14, no. 6, pp. 183–190, thermostability: the use of sequence alignments versus 1996. rational design and directed evolution,” Current Opinion in [116] N. Kulkarni, A. Shendye, and M. Rao, “Molecular and Biotechnology, vol. 12, no. 4, pp. 371–375, 2001. biotechnological aspects of xylanases,” FEMS Microbiology [99] D. W. S. Wong, “Recent advances in enzyme development,” Reviews, vol. 23, no. 4, pp. 411–456, 1999. in Handbook of Food Enzymology,J.R.Whitaker,A.G.J. [117] M. Bauer, M. R. Bedford, and D. A. Pulliam, Microbially Voragen, and D. W. S. Wong, Eds., pp. 379–387, Marcel expresses xylanases and their use as feed additives and other Dekker, New York, NY, USA, 2003. uses. Patent US US2008187627A1, 2008. [100] V. G. H. Eijsink, S. Gaseidnesa,˚ T. V. Borchert, and B. van den [118] B. A. van der Veen, G. Potocki-Veron´ ese,` C. Albenne, G. Burg, “Directed evolution of enzyme stability,” Biomolecular Joucla, P. Monsan, and M. Remaud-Simeon, “Combina- Engineering, vol. 22, no. 1–3, pp. 21–30, 2005. torial engineering to enhance amylosucrase performance: [101] C. A. Tracewell and F. H. Arnold, “Directed enzyme evo- construction, selection, and screening of variant libraries for lution: climbing fitness peaks one amino acid at a time,” increased activity,” FEBS Letters, vol. 560, no. 1–3, pp. 91–97, Current Opinion in Chemical Biology, vol. 13, no. 1, pp. 3–9, 2004. 2009. [119] Y.-S. Tian, R.-H. Peng, J. Xu et al., “Mutations in two amino [102] S. Sen, V. V. Dasu, and B. Mandal, “Developments in acids in phyI1s from Aspergillus niger 113 improve its phytase directed evolution for improving enzyme functions,” Applied activity,” World Journal of Microbiology and Biotechnology, Biochemistry and Biotechnology, vol. 143, no. 3, pp. 212–223, vol. 26, no. 5, pp. 903–907, 2009. 2007. [120] J. M. Short, Directed evolution of thermophilic enzymes. [103] F. Valetti and G. Gilardi, “Directed evolution of enzymes for Patent US5,830,696, 1998. product chemistry,” Natural Product Reports,vol.21,no.4, [121] R. M. Kelly, L. Dijkhuizen, and H. Leemhuis, “Starch and pp. 490–511, 2004. α-glucan acting enzymes, modulating their properties by [104] S. B. Rubin-Pitel and H. Zhao, “Recent advances in biocatal- directed evolution,” Journal of Biotechnology, vol. 140, no. 3- ysis by directed enzyme evolution,” Combinatorial Chemistry 4, pp. 184–193, 2009. and High Throughput Screening, vol. 9, no. 4, pp. 247–257, [122] J. Damborsky and J. Brezovsky, “Computational tools for 2006. designing and engineering biocatalysts,” Current Opinion in [105] M. Adamczak and S. H. Krishna, “Strategies for improving Chemical Biology, vol. 13, no. 1, pp. 26–34, 2009. enzymes for efficient biocatalysis,” Food Technology and [123] W. J. Quax, N. T. Mrabet, R. G. M. Luiten, P. W. Biotechnology, vol. 42, no. 4, pp. 251–264, 2004. Schuurhuizen, P. Stanssens, and I. Lasters, “Enhancing the [106] N. J. Turner, “Directed evolution drives the next generation thermostability of glucose isomerase by protein engineering,” of biocatalysts,” Nature Chemical Biology,vol.5,no.8,pp. Nature Biotechnology, vol. 9, no. 8, pp. 738–742, 1991. 567–573, 2009. [124] R. G. M. Luiten, W. J. Quax, P. W. Schuurhuizen, and N. [107] D. Sriprapundh, C. Vieille, and J. G. Zeikus, “Directed evo- Mrabet, Novel glucose isomerase enzymes and their use. lution of Thermotoga neapolitana xylose isomerase: high Patent EP0351029 (A1), 1990. activity on glucose at low temperature and low pH,” Protein [125] G. P.Zhu, C. Xu, M. K. Teng et al., “Increasing the thermosta- Engineering, vol. 16, no. 9, pp. 683–690, 2003. bility of D-xylose isomerase by introduction of a proline into [108] S. H. Bhosale, M. B. Rao, and V. V. Deshpande, “Molecular the turn of a random coil,” Protein Engineering,vol.12,no.8, and industrial aspects of glucose isomerase,” Microbiological pp. 635–638, 1999. Reviews, vol. 60, no. 2, pp. 280–300, 1996. [126] N. Declerck, M. Machius, P. Joyet, G. Wiegand, R. Huber, [109] Y.-W. Kim, J.-H. Choi, J.-W. Kim et al., “Directed evolution and C. Gaillardin, “Hyperthermostabilization of Bacillus of Thermus maltogenic amylase toward enhanced thermal licheniformis α-amylase and modulation of its stability over resistance,” Applied and Environmental Microbiology, vol. 69, a50◦C temperature range,” Protein Engineering, vol. 16, no. no. 8, pp. 4866–4874, 2003. 4, pp. 287–293, 2003. [110] S. Emond, I. Andre,´ K. Jaziri et al., “Combinatorial engi- [127] L.-L. Lin, J.-S. Liu, W.-C. Wang, S.-H. Chen, C.-C. Huang, neering to enhance thermostability of amylosucrase,” Protein and H.-F. Lo, “Glutamic acid 219 is critical for the ther- Science, vol. 17, no. 6, pp. 967–976, 2008. mostability of a truncated α-amylase from alkaliphilic and [111] Y. Wang, E. Fuchs, R. da Silva, A. McDaniel, J. Seibel, and thermophilic Bacillus sp. strain TS-23,” World Journal of C. Ford, “Improvement of Aspergillus niger glucoamylase Microbiology and Biotechnology, vol. 24, no. 5, pp. 619–626, thermostability by directed evolution,” Starch/Starke¨ , vol. 58, 2008. no. 10, pp. 501–508, 2006. [128] H.-L. Liu and W.-C. Wang, “Protein engineering to improve [112] M.-S. Kim and X. G. Lei, “Enhancing thermostability of the thermostability of glucoamylase from Aspergillus Escherichia coli phytase AppA2 by error-prone PCR,” Applied awamori based on molecular dynamics simulations,” Protein Microbiology and Biotechnology, vol. 79, no. 1, pp. 69–75, Engineering, vol. 16, no. 1, pp. 19–25, 2003. 2008. [129] W. Zhang and X. G. Lei, “Cumulative improvements of ther- [113] M.-S. Kim, J. D. Weaver, and X. G. Lei, “Assembly of mostability and pH-activity profile of Aspergillus niger PhyA mutations for improving thermostability of Escherichia coli phytase by site-directed mutagenesis,” Applied Microbiology AppA2 phytase,” Applied Microbiology and Biotechnology, vol. and Biotechnology, vol. 77, no. 5, pp. 1033–1040, 2008. 79, no. 5, pp. 751–758, 2008. [130] M. Rhimi, N. Aghajari, M. Juy et al., “Rational design of [114] K. Miyazaki, M. Takenouchi, H. Kondo, N. Noro, M. Suzuki, Bacillus stearothermophilus US100 l-arabinose isomerase: and S. Tsuda, “Thermal stabilization of Bacillus subtilis potential applications for d-tagatose production,” Biochimie, family-11 xylanase by directed evolution,” The Journal of vol. 91, no. 5, pp. 650–653, 2009. Biological Chemistry, vol. 281, no. 15, pp. 10236–10242, 2006. [131] D.-K. Oh, “Tagatose: properties, applications, and biotech- [115] C. Vieille and J. G. Zeikus, “Thermozymes: identifying nological processes,” Applied Microbiology and Biotechnology, molecular determinants of protein structural and functional vol. 76, no. 1, pp. 1–8, 2007. Enzyme Research 17

[132] F. Jørgensen, O. C. Hansen, and P. Stougaard, “Enzy- [146] T. Nakakuki, “Development of functional oligosaccharides in matic conversion of D-galactose to D-tagatose: heterolo- Japan,” Trends in Glycoscience and Glycotechnology, vol. 15, gous expression and characterisation of a thermostable L- no. 82, pp. 57–64, 2003. arabinose isomerase from Thermoanaerobacter mathranii,” [147] M. A. Abdel-Naby, A. M. Hashem, M. A. Esawy, and Applied Microbiology and Biotechnology,vol.64,no.6,pp. A. F. Abdel-Fattah, “Immobilization of Bacillus subtilis α- 816–822, 2004. amylase and characterization of its enzymatic properties,” [133] P. Kim, “Current studies on biological tagatose production Microbiological Research, vol. 153, no. 4, pp. 319–325, 1999. using L-arabinose isomerase: a review and future perspec- [148] D. Brady and J. Jordaan, “Advances in enzyme immobilisa- tive,” Applied Microbiology and Biotechnology, vol. 65, no. 3, tion,” Biotechnology Letters, vol. 31, no. 11, pp. 1639–1650, pp. 243–249, 2004. 2009. [134] M. Rhimi, E. B. Messaoud, M. A. Borgi, K. B. khadra, [149] A. G. de Segura, M. Alcalde, F. J. Plou, M. Remaud-Simeon, and S. Bejar, “Co-expression of l-arabinose isomerase and P. Monsan, and A. Ballesteros, “Encapsulation in LentiKats d-glucose isomerase in E. coli and development of an of dextransucrase from Leuconostoc mesenteroides NRRL B- efficient process producing simultaneously d-tagatose and d- 1299, and its effect on product selectivity,” Biocatalysis and fructose,” Enzyme and Microbial Technology,vol.40,no.6,pp. Biotransformation, vol. 21, no. 6, pp. 325–331, 2003. 1531–1537, 2007. [150] S. A. de Assis, B. S. Ferreira, P. Fernandes, D. G. Guaglianoni, [135] L. K. Ozimek, S. Kralj, T. Kaper, M. J. E. C. Van Der Maarel, J. M. S. Cabral, and O. M. M. F. Oliveira, “Gelatin- and L. Dijkhuizen, “Single amino acid residue changes in immobilized pectinmethylesterase for production of low subsite - 1 of inulosucrase from Lactobacillus reuteri 121 methoxyl pectin,” Food Chemistry, vol. 86, no. 3, pp. 333–337, strongly influence the size of products synthesized,” FEBS 2004. Journal, vol. 273, no. 17, pp. 4104–4113, 2006. [151] M. Rebros,ˇ M. Rosenberg, Z. Mlichova,´ L. Kristofˇ ´ıkova,´ and [136] M. E. Ortiz-Soto, M. Rivera, E. Rudino-Pi˜ nera,˜ C. Olvera, M. Paluch, “A simple entrapment of glucoamylase into and A. Lopez-Mungu´ ´ıa, “Selected mutations in Bacillus LentiKats as an efficient catalyst for maltodextrin hydroly- subtilis levansucrase semi-conserved regions affecting its sis,” Enzyme and Microbial Technology, vol. 39, no. 4, pp. 800– biochemical properties,” Protein Engineering, Design and 804, 2006. Selection, vol. 21, no. 10, pp. 589–595, 2008. [152] Z. Grosova,´ M. Rosenberg, M. Rebros,ˇ M. Sipocz,ˇ and B. [137] B. A. van der Veen, J. C.M. Uitdehaag, D. Penninga et al., Sedlaćkovˇ a,´ “Entrapment of β-galactosidase in polyvinylal- “Rational design of cyclodextrin glycosyltransferase from cohol hydrogel,” Biotechnology Letters, vol. 30, no. 4, pp. 763– Bacillus circulans strain 251 to increase α-cyclodextrin 767, 2008. production,” Journal of Molecular Biology, vol. 296, no. 4, pp. [153] M. Rebros,ˇ M. Rosenberg, Z. Mlichova,´ and L. Kristofˇ ´ıkova,´ 1027–1038, 2000. “Hydrolysis of sucrose by invertase entrapped in polyvinyl [138] Z. Li, J. Zhang, M. Wang et al., “Mutations at subsite -3 in alcoholhydrogelcapsules,”Food Chemistry, vol. 102, no. 3, cyclodextrin glycosyltransferase from Paenibacillus macerans pp. 784–787, 2007. enhancing α-cyclodextrin specificity,” Applied Microbiology [154] M. Schlieker and K.-D. Vorlop, “A novel immobilization and Biotechnology, vol. 83, no. 3, pp. 483–490, 2009. method for entrapment LentiKats,” in Immobilization of [139] B. A. Van der Veen, J. C. M. Uitdehaag, B. W. Dijkstra, and L. Enzymes and Cells, J. M. Guisan, Ed., pp. 333–343, Humana Dijkhuizen, “Engineering of cyclodextrin glycosyltransferase Press, Totowa, NJ, USA, 2nd edition, 2006. reaction and product specificity,” Biochimica et Biophysica [155] R. Stloukal, M. Rosenberg, and M. Rebros, Industrial Acta, vol. 1543, no. 2, pp. 336–360, 2000. production of biocatalysts in the form of enzymes or [140] Z. Li, M. Wang, F. Wang et al., “γ-Cyclodextrin: a review on microorganisms immobilized in polyvinyl alcohol gel. Patent enzymatic production and applications,” Applied Microbiol- US 2009/0061499 A1, 2009. ogy and Biotechnology, vol. 77, no. 2, pp. 245–255, 2007. [156] K. Anjani, K. Kailasapathy, and M. Phillips, “Microencapsu- [141] K. Fujii, H. Minagawa, Y. Terada et al., “Use of random lation of enzymes for potential application in acceleration of and saturation mutageneses to improve the properties of cheese ripening,” International Dairy Journal, vol. 17, no. 1, Thermus aquaticus amylomaltase for efficient production of pp. 79–86, 2007. cycloamyloses,” Applied and Environmental Microbiology, vol. [157] K. Kailasapathy and S. H. Lam, “Application of encapsulated 71, no. 10, pp. 5823–5827, 2005. enzymes to accelerate cheese ripening,” International Dairy [142] R. A. Sheldon, “Enzyme immobilization: the quest for Journal, vol. 15, no. 6-9, pp. 929–939, 2005. optimum performance,” Advanced Synthesis and Catalysis, [158] H. Tumturk, G. Demirel, H. Altinok, S. Aksoy, and N. vol. 349, no. 8-9, pp. 1289–1307, 2007. Hasirci, “Immobilization of glucose isomerase in surface- [143] C. Mateo, J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan, modified alginate gel beads,” Journal of Food Biochemistry, and R. Fernandez-Lafuente, “Improvement of enzyme activ- vol. 32, no. 2, pp. 234–246, 2008. ity, stability and selectivity via immobilization techniques,” [159] G. Rajagopalan and C. Krishnan, “Immobilization of malto- Enzyme and Microbial Technology, vol. 40, no. 6, pp. 1451– oligosaccharide forming α-amylase from Bacillus subtilis 1463, 2007. KCC103: properties and application in starch hydrolysis,” [144] H. E. Swaisgood, “Use of immobilized enzymes in the food Journal of Chemical Technology and Biotechnology, vol. 83, no. industry,” in Handbook of Food Enzymology,J.R.Whitaker,A. 11, pp. 1511–1517, 2008. G. J. Voragen, and D. M. S. Wong, Eds., pp. 359–366, Marcel [160] R. Catana, B. S. Ferreira, J. M. S. Cabral, and P. Fernandes, Dekker, New York, NY, USA, 2003. “Immobilization of inulinase for sucrose hydrolysis,” Food [145] M. K. Walsh, “Immobilized enzyme technology for food Chemistry, vol. 91, no. 3, pp. 517–520, 2005. applications,” in Novel Enzyme Technology for Food Appli- [161] P. Walde and S. Ichikawa, “Enzymes inside lipid vesicles: cation, R. Rastall, Ed., pp. 60–84, Woodhead Publishing preparation, reactivity and applications,” Biomolecular Engi- Limited, Cambridge, UK, 2007. neering, vol. 18, no. 4, pp. 143–177, 2001. 18 Enzyme Research

[162] Z. Grosova,´ M. Rosenberg, and M. Rebros,ˇ “Perspectives Amberlite MB 150 and chitosan beads: a comparative study,” and applications of immobilised β-galactosidase in food Journal of Molecular Catalysis B, vol. 49, no. 1–4, pp. 69–74, industry—a review,” Czech Journal of Food Sciences, vol. 26, 2007. no. 1, pp. 1–14, 2008. [178] M. A. Esawy, D. A. R. Mahmoud, and A. F. A. Fattah, [163] J. M. Rodriguez-Nogales and A. Delgadillo, “Stability “Immobilisation of Bacillus subtilis NRC33a levansucrase and catalytic kinetics of microencapsulated β-galactosidase and some studies on its properties,” Brazilian Journal of in liposomes prepared by the dehydration-rehydration Chemical Engineering, vol. 25, no. 2, pp. 237–246, 2008. method,” Journal of Molecular Catalysis B, vol. 33, no. 1-2, [179] Z. S. Csanadi´ and C. S. Sisak, “Immobilization of Pectinex pp. 15–21, 2005. Ultra SP-L pectinase and its application to production of [164] J. M. Rodr´ıguez-Nogales and A. D. Lopez,´ “A novel approach fructooligosaccharides,” Acta Alimentaria,vol.35,no.2,pp. to develop β-galactosidase entrapped in liposomes in order 205–212, 2006. to prevent an immediate hydrolysis of lactose in milk,” [180] S. Varavinit, N. Chaokasem, and S. Shobsngob, “Covalent International Dairy Journal, vol. 16, no. 4, pp. 354–360, 2006. immobilization of a glucoamylase to bagasse dialdehyde [165] C.-K. Kim, H.-S. Chung, M.-K. Lee, L.-N. Choi, and M.- cellulose,” World Journal of Microbiology and Biotechnology, H. Kim, “Development of dried liposomes containing β- vol. 17, no. 7, pp. 721–725, 2001. galactosidase for the digestion of lactose in milk,” Interna- [181] A. Tanriseven and Z. Olc¨ ¸er, “A novel method for the tional Journal of Pharmaceutics, vol. 183, no. 2, pp. 185–193, immobilization of glucoamylase onto polyglutaraldehyde- 1999. activated gelatin,” Biochemical Engineering Journal, vol. 39, [166] E. E. Kheadr, J. C. Vuillemard, and S. A. El-Deeb, “Impact of no. 3, pp. 430–434, 2008. liposome-encapsulated enzyme cocktails on cheddar cheese [182] N. Milosavic,´ R. Prodanovic,´ S. Jovanovic,´ and Z. Vujciˇ c,´ ripening,” Food Research International, vol. 36, no. 3, pp. 241– “Immobilization of glucoamylase via its carbohydrate moi- 252, 2003. ety on macroporous poly(GMA-co-EGDMA),” Enzyme and [167] C.-K. Kim, H.-S. Chung, M.-K. Lee, L.-N. Choi, and M.- Microbial Technology, vol. 40, no. 5, pp. 1422–1426, 2007. H. Kim, “Development of dried liposomes containing β- [183] G. Sanjay and S. Sugunan, “Fixed bed reactor performance galactosidase for the digestion of lactose in milk,” Interna- of invertase immobilized on montmorillonite,” Catalysis tional Journal of Pharmaceutics, vol. 183, no. 2, pp. 185–193, Communications, vol. 7, no. 12, pp. 1005–1011, 2006. 1999. [184] G. Sanjay and S. Sugunan, “Glucoamylase immobilized on [168] S. Chockchaisawasdee, V. I. Athanasopoulos, K. Niranjan, montmorillonite: influence of nature of binding on surface and R. A. Rastall, “Synthesis of galacto-oligosaccharide from properties of clay-support and activity of enzyme,” Journal of lactose using β-galactosidase from kluyveromyces lactis: Porous Materials, vol. 14, no. 2, pp. 127–136, 2007. studies on batch and continuous UF membrane-fitted biore- [185] L. Amaya-Delgado, M. E. Hidalgo-Lara, and M. C. Montes- actors,” Biotechnology and Bioengineering,vol.89,no.4,pp. Horcasitas, “Hydrolysis of sucrose by invertase immobilized 434–443, 2005. on nylon-6 microbeads,” Food Chemistry,vol.99,no.2,pp. 299–304, 2006. [169] S. Novalin, W. Neuhaus, and K. D. Kulbe, “A new innovative [186] V. Vallejo-Becerra, J. M. Vasquez-Bahena,´ J. A. Santiago- process to produce lactose-reduced skim milk,” Journal of Hernandez,´ and M. E. Hidalgo-Lara, “Immobilization of the Biotechnology, vol. 119, no. 2, pp. 212–218, 2005. recombinant invertase INVB from Zymomonas mobilis on [170] G. F. Bickerstaff, “Immobilization of enzymes and cells: some Nylon-6,” Journal of Industrial Microbiology and Biotechnol- practical considerations,” in Immobilization of Enzymes and ogy, vol. 35, no. 11, pp. 1289–1295, 2008. Cells,G.F.Bickerstaff, Ed., pp. 1–11, Humana Press, Totowa, [187] P. G. Cadena, R. A. S. Jeronimo, J. M. Melo, R. A. Silva, J. L. NJ, USA, 1997. Lima Filho, and M. C. B. Pimentel, “Covalent immobilization [171] S. Gopinath and S. Sugunan, “Leaching studies over immo- α of invertase on polyurethane, plast-film and ferromagnetic bilized -amylase. Importance of the nature of enzyme Dacron,” Bioresource Technology, vol. 101, pp. 1595–1602, attachment,” Reaction Kinetics and Catalysis Letters, vol. 83, 2009. no. 1, pp. 79–83, 2004. [188] H. Altinok, S. Aksoy, H. Tumt¨ urk,¨ and N. Hasirci, “Covalent ffi [172] J. M. Nelson and E. G. Gri n, “Adsorption of invertase,” immobilization of invertase on chemically activated poly Journal of the American Chemical Society,vol.38,no.5,pp. (styrene-2-hydroxyethyl methacrylate) microbeads,” Journal 1109–1115, 1916. of Food Biochemistry, vol. 32, no. 3, pp. 299–315, 2008. [173] D. A. R. Mahmoud, “Immobilization of Invertase by a New [189] G. Bayramoglu,ˇ S. Akgol,¨ A. Bulut, A. Denizli, and M. Y. Economical Method Using Wood Sawdust Waste,” Australian Arica, “Covalent immobilisation of invertase onto a reactive Journal of Applied Science, vol. 1, pp. 364–372, 2007. film composed of 2-hydroxyethyl methacrylate and glycidyl [174] A. Nighojkar, S. Srivastava, and A. Kumar, “Production methacrylate: properties and application in a continuous of low methoxyl pectin using immobilized pectinesterase flow system,” Biochemical Engineering Journal, vol. 14, no. 2, bioreactors,” Journal of Fermentation and Bioengineering, vol. pp. 117–126, 2003. 80, no. 4, pp. 346–349, 1995. [190] R. Fernandez-Lafuente, “Hyperstabilization of a ther- [175] R. Reshmi, G. Sanjay, and S. Sugunan, “Enhanced activity mophilic esterase by multipoint covalent attachment,” and stability of α-amylase immobilized on alumina,” Catal- Enzyme and Microbial Technology, vol. 17, no. 4, pp. 366–372, ysis Communications, vol. 7, no. 7, pp. 460–465, 2006. 1995. [176] R. Reshmi, G. Sanjay, and S. Sugunan, “Immobilization of [191] C. Mateo, V. Grazu,´ B. C. C. Pessela et al., “Advances in the α-amylase on zirconia: a heterogeneous biocatalyst for starch design of new epoxy supports for enzyme immobilization- hydrolysis,” Catalysis Communications, vol. 8, no. 3, pp. 393– stabilization,” Biochemical Society Transactions,vol.35,no.6, 399, 2007. pp. 1593–1601, 2007. [177] P. Tripathi, A. Kumari, P. Rath, and A. M. Kayastha, “Immo- [192] G. Bayramoglu, Y. Tunali, and M. Y. Arica, “Immobilization bilization of α-amylase from mung beans (Vigna radiata)on of β-galactosidase onto magnetic poly(GMA-MMA) beads Enzyme Research 19

forhydrolysisoflactoseinbedreactor,”Catalysis Commu- cellulase activities,” Chemistry Central Journal, vol. 1, no. 1, nications, vol. 8, no. 7, pp. 1094–1101, 2007. article 16, 2007. [193] S. A. Ferrarotti, J. M. Bolivar, C. Mateo, L. Wilson, J. M. [209] R. Gaur, H. Pant, R. Jain, and S. K. Khare, “Galacto- Guisan, and R. Fernandez-Lafuente, “Immobilization and oligosaccharide synthesis by immobilized Aspergillus oryzae stabilization of a cyclodextrin glycosyltransferase by covalent β-galactosidase,” Food Chemistry, vol. 97, no. 3, pp. 426–430, attachment on highly activated glyoxyl-agarose supports,” 2006. Biotechnology Progress, vol. 22, no. 4, pp. 1140–1145, 2006. [210] K. Tatsumoto, K. K. Oh, J. O. Baker, and M. E. Him- [194] A. G. de Segura, M. Alcalde, M. Yates et al., “Immobilization mel, “Enhanced stability of glucoamylase through chemical of dextransucrase from Leuconostoc mesenteroides NRRL B- crosslinking,” Applied Biochemistry and Biotechnology, vol. 512F on eupergit C supports,” Biotechnology Progress, vol. 20, 20-21, no. 1, pp. 293–308, 1989. no. 5, pp. 1414–1420, 2004. [211] M. E. Ortiz-Soto, E. Rudino-Pi˜ nera,M.E.Rodriguez-˜ [195] L. Cao, Carrier-Bound Immobilized Enzymes—Principles, Alegria, and A. L. Munguia, “Evaluation of cross-linked ag- Applications and Design, Wiley-VCH, Weinheim, Germany, gregates from puriﬁed Bacillus subtilis levansucrase mutants 2005. for transfructosylation reactions,” BMC Biotechnology, vol. 9, [196] C. Mateo, B. C. C. Pessela, M. Fuentes et al., “Very Strong But article 68, 2009. Reversible Immobilization of Enzymes on Supports Coated [212] K. Visuri, Preparation of cross-linked glucose isomerase With Ionic Polymers,” in Immobilization of Enzymes and crystals. Patent US 5437993, 1995. Cells, J. M. Guisan, Ed., pp. 205–216, Humana Press, Totowa, [213] O. Pastinen, K. Visuri, and M. Leisola, “Xylitol puriﬁcation NJ, USA, 2nd edition, 2006. by cross-linked glucose isomerase crystals,” Biotechnology [197] E. J. Tomotani and M. Vitolo, “Method for immobilizing Techniques, vol. 12, no. 7, pp. 557–560, 1998. invertase by adsorption on Dowex anionic exchange resin,” [214] B. Brandt, A. Hidalgo, and U. T. Bornscheuer, “Immobi- Brazilian Journal of Pharmaceutical Sciences,vol.42,no.2,pp. lization of enzymes in microtiter plate scale,” Biotechnology 245–249, 2006. Journal, vol. 1, no. 5, pp. 582–587, 2006. [198] L. D. S. Marquez, B. V. Cabral, F. F. Freitas, V. L. Cardoso, [215] L. Cao, Carrier-bound immobilized enzymes: principles, appli- and E. J. Ribeiro, “Optimization of invertase immobilization cations and design, Wiley-VCH, Weinheim, Germany, 2005. by adsorption in ionic exchange resin for sucrose hydrolysis,” [216] W. Pitcher, “Design and operation of immobilized enzyme Journal of Molecular Catalysis B, vol. 51, no. 3-4, pp. 86–92, reactors,” in Enzymes for Industrial Reactors, R. Messing, Ed., 2008. pp. 151–199, Academic Press, New York, NY, USA, 1975. [199] M. Y. Arica and G. Bayramoglu,ˇ “Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA-MMA) beads for sucrose hydrolysis,” Journal of Molecular Catalysis B, vol. 38, no. 3-6, pp. 131–138, 2006. [200] C. Mateo, R. Torres, G. Fernandez-Lorente´ et al., “Epoxy- amino groups: a new tool for improved immobilization of proteins by the epoxy method,” Biomacromolecules, vol. 4, no. 3, pp. 772–777, 2003. [201] B. C. C. Pessela, R. Fernandez-Lafuente,´ M. Fuentes et al., “Reversible immobilization of a thermophilic β-galactosidase via ionic adsorption on PEI-coated Sepabeads,” Enzyme and Microbial Technology, vol. 32, no. 3-4, pp. 369–374, 2003. [202] E. B. Rathbone, A. J. Hacking, and P. S. J. Cheetham, Process for the preparation of fructosyl disaccharides. Patent US 4617269, 1986. [203] R. L. Antrim and A. L. Auterinen, “A new regenerable immobilized glucose isomerase,” Starch/Starke¨ , vol. 38, pp. 132–137, 1986. [204] S. M. Gaikwad and V. V. Deshpande, “Immobilization of glucose isomerase on Indion 48-R,” Enzyme and Microbial Technology, vol. 14, no. 10, pp. 855–858, 1992. [205] J. M. Gomez,M.D.Romero,T.M.Fern´ andez,´ and S. Garc´ıa, “Immobilization and enzymatic activity of β-glucosidase on mesoporous SBA-15 silica,” JournalofPorousMaterials.In press. [206] D.-H. Kweon, S.-G. Kim, N. S. Han, J. H. Lee, K. M. Chung, and J.-H. Seo, “Immobilization of Bacillus macerans cyclodextrin glycosyltransferase fused with poly-lysine using cation exchanger,” Enzyme and Microbial Technology, vol. 36, no. 4, pp. 571–578, 2005. [207] J. J. Roy and T. E. Abraham, “Strategies in making cross- linked enzyme crystals,” Chemical Reviews, vol. 104, no. 9, pp. 3705–3721, 2004. [208] S. Dalal, A. Sharma, and M. N. Gupta, “A multipur- pose immobilized biocatalyst with pectinase, xylanase and SAGE-Hindawi Access to Research Enzyme Research Volume 2010, Article ID 480923, 8 pages doi:10.4061/2010/480923

Review Article Some Nutritional, Technological and Environmental Advances in the Use of Enzymes in Meat Products

Anne y Castro Marques, Mario´ Roberto Marostica´ Jr., and Glaucia´ Maria Pastore

School of Food Engineering, University of Campinas, Monteiro Lobato st., 80, 13083-862 Campinas, SP, Brazil

Correspondence should be addressed to Mario´ Roberto Marostica´ Jr., [email protected]

Received 11 June 2010; Revised 31 August 2010; Accepted 14 September 2010

Academic Editor: Cristina M. Rosell

Copyright © 2010 Anne y Castro Marques et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The growing consumer demand for healthier products has stimulated the development of nutritionally enhanced meat products. However, this can result in undesirable sensory consequences to the product, such as texture alterations in low-salt and low- phosphate meat foods. Additionally, in the meat industry, economical aspects have stimulated researchers to use all the animal parts to maximize yields of marketable products. This paper aimed to show some advances in the use of enzymes in meat processing, particularly the application of the proteolytic enzymes transglutaminase and phytases, associated with nutritional, technological, and environmental improvements.

1. Introduction Faced with new market trends, is it possible to produce meat products that meet all the market requirements Meat products consumption (including beef, pork, mutton, (healthy, with good sensory properties, low cost, and envi- goat and poultry) has increased gradually, particularly ronmental friendly)? The aim of this paper is to show some in developing countries. Studies estimate that the world advances related to this topic, focusing on the application of consumption of meat products will reach 40 kg per capita in proteolytic enzymes, transglutaminase and phytases in meat 2020 [1]. The processes involved in the conversion of muscle products. to meat are complex. The chemical and physical properties of muscle tissue and the associated connective tissue are 2. The Use of Proteolytic Enzymes in determinant on meat quality [2]. Meat Products The growing consumer demand for healthier products has stimulated the development of nutritionally enhanced Of all the attributes of meat quality, consumers rate ten- meat foods. In order to achieve these nutritionally enhanced derness as the most important. Tenderness is a charac- meat foods, changes such as the use of improved raw mate- teristic resulting from the interaction of actomyosin effect rials, reformulation of products, and technological processes of myofibrillar proteins, the bulk density effect of fat, are necessary [3]. These improvements, however, can bring and the background effect of connective tissue. There are undesirable consequences to the product, such as texture several ways to tenderize meat, chemically or physically, alterations in low-salt and low-phosphate meat foods [4, 5]. which mainly reduce the amounts of detectable connective Additionally, high costs have stimulated researchers to use tissue without causing extensive degradation of myofibrillar all animal parts, including muscles of poorer technological proteins. Treatment by proteolytic enzymes is one of the quality, to maximize the yield of marketable products. This most popular methods of meat tenderization [8, 9]. has required the development of methods to restructure Proteolytic enzymes are a multifunctional class of enz- low-valued cuts and trimmings, improving appearance and ymes, with physiological functions that range from gener- texture and increasing market value [6, 7]. alized protein digestion to more specific regulated processes 2 Enzyme Research such as the activation of zymogens, blood coagulation, com- food industry to tenderize meat and as an additive in flour plement activation, inflammation process, and liberation of and in beer manufacturing [18, 19]. However, papain has physiological peptides from the precursor proteins. They are a tendency to overtenderize the meat surface, making it frequently used in food processing [10]. “mushy”, which has limited its use as a commercial meat The first variation of meat tenderness is due to the tenderizer [10]. Herranz et al. [20] used papain (300 units/kg complex endogenous calpain-calpastatin, which acts in mus- of papain) to increase the amount of free amino acids in dry cle tissue after slaughter. Calpains are calcium-dependent fermented sausages. These precursors of volatile compounds, proteases that degrade myofibrillar proteins (tropomyosin, responsible for the ripened flavor, were tested in presence of troponin T, troponin I, C-protein, connectin, and desmin). Lactococcus lactis subsp. cremoris NCDO 763, its intracellular Calpastastin, in turn, inactivates calpains, decreases the cell free extract (ICFE), and α-ketoglutarate. The results, myofibrillar degradation, and thus reduces the tenderness. however, did not show any important activity related to Calpastatin effect is finished after calpastatin is inactivated amino acid breakdown, and the sensory analysis showed by cooking. The concentration of the enzymes varies among that neither the addition of the extract nor its use together breeds of species, determining the higher or lower meat with papain or a-ketoglutarate lead to an improvement in tenderness, due to increased or reduced proteolysis of the sensory quality of the experimental sausages. Recently, myofibrillar proteins [11, 12]. Shimizu et al. [21] evaluated the antithrombotic activity Several examples of proteases application in meat prod- of papain-hydrolyzate from defatted pork meat (crude and ucts can be found in the literature. Benito et al. [13] showed peptides purified by cation exchange chromatography) “in that the fungal protease EPg222 hydrolyzed myofibrillar vivo”. The initial peptide fraction with an average molecular proteins of whole pieces of meat with 5% NaCl, favoring weight of 2500 showed antithrombotic activity after oral tenderization and improving texture of the product. Accord- administration to mice at 210 mg/kg. The fraction with an ing to the authors, salt and curing agents at the level found average molecular weight of 2517, further purified by cation in dry cured meat products are powerful inhibitors of the exchange chromatography, showed antithrombotic activity ff former endogenous enzymes. The e ect of this protease after oral administration at 70 mg/kg. Antithrombotic activ- may be of great interest to counterbalance the increase of ity of the last peptide fraction was equivalent to that of hardness reported in these products as a consequence of aspirin at 50 mg/kg body weight. protein denaturation. Nalinanon et al. [14] used pepsin to Bromelain (or bromelin, EC 3.4.4.24) is a group of pro- obtain fish gelatin, from bigeye snapper skin, as an alternative teolytic enzymes present in large quantities in fruit, leaves, for porcine and bovine gelatin. Thiansilakul et al. [15] and stems of the Bromeliacea family, of which pineapple produced a protein hydrolyzate (derived from round scad) (Ananas comosus) is the most commonly known [22, 23]. with Flavourzyme protease addition that could be used as This enzyme, like other proteases, degrades myofibrillar an emulsifier and as a foaming agent with antioxidative proteins and collagen, often resulting in overtenderization activities in food systems. of meat [24]. Ionescu et al. [25]investigatedbromelain The quest for valuable proteases with distinct specificity use in adult beef, with the best results at 10 mg/100 g for industrial applications is always a continuous challenge. meat, with tenderization time 24 hours at 4◦C, followed by Proteolytic enzymes from plant sources have received special thermal treatment by increasing 1◦C/min until 70◦C(when attention for being active over a wide range of temperatures enzyme inactivation occurs). These conditions improved and pH [16, 17]. beef tenderness. The name ficin (EC 3.4.22.3) refers to the endoprote- olytic enzymes from trees of the genus Ficus. These enzymes The ideal meat tenderizer would be a proteolytic enzyme have different properties. The most extensively studied ficins with specificity for collagen and elastin in connective tissue, are the cysteine proteases found in the latex of Ficus glabrata at the relatively low pH of meat, which would act either and Ficus carica. Proteases from other species are less known. at the low temperature at which meat is stored or at the In 2008, a new protease from Ficus racemosa was identified. high temperature achieved during cooking [26]. Qihe et al. The protein has a molecular weight of 44.500 ± 500 Da, pH [8] investigated elastase from Bacillus sp. EL31410, applied optima between pH 4.5 and 6.5 and maximum activity at to beef tenderization, in comparison with other nonspecific 60 ± 0.5◦C. These unique properties indicate this protease proteases, such as papain, and evaluated the feasibility of to be distinct from other known ficins. Applications of this using it for this purpose. The samples were treated for 4 enzyme include its use as meat tenderizers, removal of chill hours in different enzyme solutions and then were stored at haze in beer, improvement in the processing of cereals, and 4◦C for 24, 48, and 72 hours. A marked decrease in hardness plant and milk clotting enzymes for novel dairy products was observed in the meat with papain and elastase and higher [16, 18]. sensory scores for tenderness were obtained from the meat Papain (EC 3.4.22.4) is a nonspecific thiol protease and treated with enzymes. However, the scores given for juiciness the major protein constituent of the latex in the tropical and taste were lower than those of the control. Rapid increase plant Carica papaya. The enzyme has high thermal and of fragmentation of myofibrils from the enzyme-treated meat pressure stability, requiring intense process conditions for was observed in the first 24 hours of storage, especially adequate inactivation (to achieve 95% inactivation of papain for papain-treated meat. Meantime, elastin of myofibrillar at 900 MPa and 80◦C, 22 minutes of processing is required). structure was selectively degraded by elastase when stored at Due to its proteolytic properties, it is widely used in the 4◦C for 48 hours as shown by electron microscopy. These Enzyme Research 3

Table 1: Use of proteolytic enzymes for bioactive peptides production in meat foods.

Product Conditions Results Reference Sardinelle proteins were digested by proteases and the ACE Sardine: heads and viscera. ACE of protein inhibitory activity was markedly increased. The degrees of Enzymes: alcalase, chymotrypsin, hydrolysates hydrolysis and the inhibitory activities of ACE increased with Bacillus licheniformis NH1 from sardine increasing proteolysis time. The sardinelle hydrolysis with the [32] protease, Aspergillus clavatus ES1 (Sardinella crude enzyme extract from sardine viscera resulted in the protease and sardine viscera aurita) production of the hydrolysate with the highest ACE inhibitory protease. activity. Enzyme: Bacillus sp. SM98011 ACE of protein protease (diluted to 4000 U/mL The hydrolysate of shark meat was rich with ACE inhibitory hydrolysates with distilled water). peptides, and 3 novel peptides with high ACE inhibitory activity [33] from shark meat Enzyme/substrate concentration: were identified (Cys-Phe, Glu-Tyr, and Phe-Glu). hydrolysate 1:5w/v. ACE of protein Enzymes: trypsin, chymotrypsin, The most active hydrolysate was obtained with the crude protease hydrolysates sardinelle protease, cuttlefish extract from the hepatopancreas of cuttlefish (64.47 ± 1.0% at from muscle of protease and smooth hounds 2mgofdryweight/mL)withadegreeofhydrolysisof8%.Three [34] cuttlefish (Sepia protease. Enzyme/substrate novel peptides with high ACE-inhibitory activity were formed: officinalis) concentration: 3 U/mg. Val-Tyr-Ala-Pro, Val-Ile-Ile-Phe, and Met-Ala-Trp.

findings suggest that Bacillus elastase could be a promising insoluble keratin proteins, can be applied in the conversion substitute for papain as a favorable meat tenderizer. of large amounts of chicken feather waste generated from Sullivan et al. [17] studied the tenderization extent poultry into highly digestible animal feed [30, 31]. (Warner-Bratzler shear and sensory evaluation) and mode Proteases are also being used for other purposes, such of action (myofibrillar or collagen degradation) of seven as the production of bioactive peptides against hypertension enzyme randomized treatments (papain, ficin, bromelain, [32–34] and reduction of the power of allergenic meat foods homogenized fresh ginger, Bacillus subtilis protease, and [35]. The angiotensin I-converting enzyme (ACE), a trans- two Aspergillus oryzae proteases) in Triceps brachii and membrane dipeptidyl peptidase which degrades bradykinin, Supraspinatus. Except for ginger treatment, all steaks treated shows the potential of cleaving any peptide, including with enzymes showed improvement in both sensory and vasoactive peptides such as angiotensin-I. The nutritional instrumental tenderness analysis. If this enzyme could be therapy approach and the use of nutraceuticals from meat purified further, applications in meat would be promising. is a good way of continual healthcare for patients with Among the results presented, papain was the enzyme that hypertension [36]. These studies are more detailed in Table 1. caused the greatest tenderness in meat, but juiciness and textural changes were negatively affected. The authors also 3. The Use of Transglutaminase in concluded that all enzyme treatments resulted in increased Meat Products tenderness with no difference between high- and low- connective tissue muscles. Transglutaminase (TGase; protein-glutamine γ-glutamyltra- Kiwifruit has also been studied as a source of actinidin, nsferase, EC 2.3.2.13) is an enzyme with the ability to an important proteolytic enzyme. Han et al. [10]investigated improve the functional characteristics of protein such as the ability of prerigor infusion of kiwifruit juice (10% body texture, flavor, and shelf life. TGase initially attracted interest weight) to improve the tenderness of lamb. The enhanced because of its capacity to reconstitute small pieces of meat proteolytic activity in lamb carcass was associated with into a steak. It can adhere to the bonding surfaces of food significant degradation of the myofibrillar proteins, resulting such as meat, fish, eggs, and vegetables as a thin layer, and in new peptides and activation of m-calpain during post- it exhibits strong adhesion in small amounts. The enzyme mortem ageing. Thus, kiwifruit juice is a powerful and easily catalyses acyl transfer reactions between the γ-carboxyamide prepared meat tenderizer, which could contribute efficiently group of peptide bound glutamine residues and a variety and effectively to the meat tenderization process. However, of primary amines, including the ε-amino group of lysine studies have show kiwifruit to cause allergic reactions, and residues, resulting in the formation of high molecular actinidin to be one of most important allergens, both in weight polymers. In the presence of primary amines, TGase children and adults [27–29]. Thus, caution should be taken can cross-link the amines to the glutamines of a protein when considering tenderizing meat using kiwifruit juice. (acyl-transfer reaction). In the absence of lysine residues Currently, besides the extensive use in meat processes, or other primary amines, water will react as a nucleophile, proteases are being investigated with the aim of transforming resulting in deamidation of glutamines. All three of these the byproducts of these processes. For example, keratinases, TGase reactions can modify the functional properties of serine proteases which are capable of degrading hard and food proteins [4, 37, 38]. 4 Enzyme Research

Table 2: Studies using microbial tranglutaminase (MTGase) in meat food.

Product Conditions Results Reference MTGase affected the breaking strength score in both meat types, especially for beef cooked at 80◦C. The functional Proportion of MTGase to MHC∗ = properties of MTGase make it a good protein-binding agent, 1 : 500. Heat treatment: 40◦C/30 minutes Chicken and positively helping the functionality of proteins to improve using a thermo-minder; 80◦C/30 [2] beef sausages the texture and gelation of sausages. Some variation in gel minutes, using a water bath shaker. improvement level between chicken and beef sausages were ∗MHC: myosin heavy chain. observed, in response to MTGase, as well as to the original glutamyl and lysine contents. Treatments: no treatment (control); immersion in a saline (NaCl with 200 ppm of KNO3 and 100 ppm of NaNO ) aqueous solution (3%, w/v) for 2 MTGase provided enough stable cross-links in the course of 10 minutes at 4◦C; and even distribution the salting and drying processes. The highest binding force of a mixture of salts (NaCl with 200 ppm Dry-cured ham and rate were obtained by treating the meat surface with a [42] of KNO and 100 ppm of NaNO )onthe 3 2 mixture of salts (NaCl including KNO and NaNO ) then surfaces for 1 minute and after 10 3 2 adding MTGase. minutes of setting time. Binding temperature: 0◦C, 7◦Cand24◦C. MTGase: powder and liquid (MTGase at 0.1% in solution of NaCl 3%). MTGase led to an increase in hardness and a considerable High pressure treatment (300 MPa, 25◦C, Fish (Trachurus decrease in elasticity and breaking deformation. MTGase 15 minutes), combined with a prior or a spp., horse activity was greater when setting was applied before [45] subsequent setting step (25◦C, 2 hours), mackerel) pressurization than after; moreover, there was no synergism 1.5% chitosan and/or 0.02% MTGase. derived from the addition of chitosan and MTGase together. Preparation A: MTG (1 g/100 g) and maltodextrin (99 g/100 g); Preparation B: MTG with sodium caseinate (SC) led to a slight increase in Ground beef MTG (0.5 g/100 g), SC (60 g/100 g) and peak temperature (Tmax) values of myosin. MTGase [51] maltodextrin (39.5 g/100 g). MTG: treatment caused a slight decrease in Tmax values of myosin product weight/meat weight. MTG affected consistency and overall acceptability of the Phosphate-free product. Salt levels: 2% product. MTG had no effect on firmness, juiciness, color, Restructured and 1%; MTG: 0%, 0.075% and 0.15%. odor, taste and saltiness. MTG can be used at a level of 0.15% cooked pork [37] Processing conditions: 72◦C/65 minutes with reduced salt level (1%) and processing at 72◦C/65 shoulder and 78◦C/65 minutes. minutes to produce phosphate-free restructured cooked pork shoulder with acceptable sensory attributes.

TGase is widely distributed among mammals, plants, resulted from protein aggregation in food is highly related invertebrates, amphibians, fish, birds, and microorganisms, to the enzymes reactions as well as the biological activities of but the extremely high cost of transglutaminase from animal some additives [2]. The TGase catalyses the interconnections origin has hampered its wider application and has initiated of myofibrils, improves the gel elasticity of meat protein, efforts to find an enzyme of microbial origin. The industrial and forms a protein-rotein network. Gel strength is further production of transglutaminase is done mainly from a enhanced by heat treatment subsequent to the action of variant of Streptoverticillium mobaraense (namely MTGase). TGase [46]. The pH optimum of MTGase is around 5 to 8. However, Herrero et al. [47] determined the effect of adding even at pH 4 or 9, MTGase still expresses some enzymatic differentlevelsofMTGasetomeatsystems(meatemulsion activity. The optimum temperature for enzymatic activity at 0.0%, 0.05%, and 0.10%). This addition produced a is 50◦C, and MTGase shows activity even during chilling significant increase in hardness, springiness, and cohesive- temperatures (under 4◦C); this property is used to bind raw ness. Data revealed secondary structural changes in meat pieces of meat under refrigeration to produce restructured proteins due to MTGase action; significant correlations were meat products [4, 5, 38–40]. found between these secondary structural changes in meat TGase has been widely applied in meat products such as proteins and the textural properties of meat systems. Fort chicken and beef sausages [2], ham [41, 42], doner¨ kebab et al. [48] studied the heat-induced gelling properties, at [43], frankfurters [44], fish [45], and so forth. Some studies acid pH, of porcine plasma previously treated with MTGase with TGase in meat food are detailed in Table 2. under high pressure (HP), when kept under refrigeration An important functional property of transglutaminase is conditions for different times. The results indicated that the ability to induce gelation in meat foods. The gelation although the cross-linking activity of MTGase was enhanced Enzyme Research 5 under pressure, consequently improving the thermal gel which could act as a glue to bind restructured meat pieces texture, the most significant effects, particularly on gel together. hardness, were obtained by keeping the treated plasma Colmenero et al. [44] observed that the combination of solutions under refrigeration for at least 2 hours before TGase with caseinate, KCl, or fibre (caseinate > KCl > fibre) gelation. Literature also shows a species-specific variation in led to harder, springier, and chewier frankfurters with better the ability of MTGase to catalyze the cross-linking of muscle water- and fat-binding properties (emulsion stability and proteins. Proteins in chicken, beef, and pork respond differ- cooking loss) than those made with TGase only. According ently to MTGase, generating different products (polymers) to the authors, caseinate has proven to be a good substrate and, consequently, differ in terms of both rheological and for TGase, facilitating cross-linking and promoting the physiochemical properties [39, 49]. formation of a much more stable gel matrix during heating. The fact that MTGase reacts differently to myofibrils Some previous studies reported several problems related to of different species may be because of the variation in moisture loss of meat products induced by TGase. Hong et muscle physiology and morphogenesis, the identity of free al. [52], however, suggested that the combination of TGase amino acids, especially those with the ability to react with with sodium alginate can improve water-binding ability and MTGase, the amount and distance between transferable produce cold-set myofibrillar protein gelation at an even amino acids, and the amount of MTGase inhibitors. It is lower salt level than TGase alone. In the meat processing necessary to understand the protein reactions induced by industry, cold-set meat binding is a useful technique for MTGase binding in meat proteins because of the important making raw meat products [53]. economic benefits of using it to improve the textural quality Because of the many promising applications of MTGase of meat products [46]. catalyzed modification of food proteins, attention should be TGase application in low-salt and phosphate-free meat focused on the nutritional value of resultant cross-linked products has been extensively investigated. Dry-cured meat proteins. It is obvious that modified MTGase and native and restructured meat products are traditionally prepared proteins differ only with respect to ε(γ-glutamyl) lysine using high salt and phosphate contents, which, with the aid bonds, and the rest is totally the same. The cross-linked of mechanical action, promote the extraction of myofibrillar proteins can be readily absorbed in the body [4]. A study proteins; upon cooking, these form a stable protein matrix conducted by our research group showed that MTGase did with a beneficial effect on product characteristics, such as not interfere on the protein quality of soy protein isolate in cohesion and cook yield. The exclusion of salt and phosphate growing Wistar rats (unpublished data). led to products with poor physicochemical properties. Addition of transglutaminase has been proposed as a means of inducing gelation, reducing or eliminating the need to add 4. Phytase: Environment Approach and Use as NaCl and phosphate products. Furthermore, combinations Feed Additive in Meat Animal Production of TGase with suitable nonmeat ingredients are also needed to overcome the problems in NaCl-free meat products [4– Phytase is not an ingredient largely used in meat products 6, 41, 44, 50]. Askin et al. [43] indicated that the MTGase formulation; however, some environmental approaches and with sodium caseinate (SC) or nonfat dry milk could be used its use as additive in meat animal production should be to produce salt-free low-fat turkey doner¨ kebab (a Middle mentioned. East product); the results were more significant when the Nowadays, producers and consumers require more than enzyme was used with SC. Trespalacios et al. [3] showed that a good sensory and nutritional product: they are also the simultaneous application of MTGase and high pressure concerned about the impact of the food chain on the (700 and 900 MPa) on chicken batters with the addition of environment. So, not only should the appearance or shelf egg proteins, low salt and no phosphates resulted in increased life of meat foods be taken into consideration, but also cutting force, hardness, and chewiness of gels. the resources used for the production and the conse- TGase has been tested with other ingredients in meat quent damages to the environment. Phytase (myo-inositol products. Aktas¸ et al. [51], for example, showed that the com- hexaphosphate phosphohydrolase) has been used in order to bination of MTGase with SC could form more cross-linking reduce costs of meat food production, as well as to reduce bonds between meat proteins in ground beef than when used environmental contamination by excrement generated dur- separately; therefore, usage of MTGase with SC may be more ing the production of animals, including swines, poultry, and suitable in restructuring meat products. Carballo et al. [50] fish. Phytase is the enzyme used to hydrolyse the phytate analyzed the effect of microbial transglutaminase/sodium molecule and release phosphorus [54–56]. Microbial phytase caseinate (MTGase/SC-1.5 g/100 g) systems on meat batter has the ability of hydrolyzing dietary phytate, the salt of characteristics (water-binding and textural properties of raw phytic acid (myo-inositol hexaphosphate; IP6), to liberate six and cooked products) in the presence of NaCl (1.5 g/100 g) phosphorus and inositol in the gastrointestinal tract [57]. and sodium tripolyphosphate (0.5 g/100 g) for pork, chicken, Typically, swine and poultry diets contain around and lamb. Products combining salts and MTGase/SC had 10 g kg−1 phytate-bound phosphorus (phytate-P), but it is higher hardness and chewiness, and the efficiency of the only partially used by the animals because they do not MTGase/SC system as a texture conditioner of cooked generate sufficient endogenous phytase activity. The phytase products varied with the meat source. They concluded supplementation can enhance P absorption and reduce P that transglutaminase with caseinate form a viscous sol excretion, which are both nutritionally and ecologically 6 Enzyme Research beneficial [54]. Brenes et al. [58]conductedanexperiment technology are important to optimize existing processes, as to study the effect of microbial phytase supplementation well as to develop new methods of application. (0, 200, 400, and 600 U/kg) in chicks fed different levels of available phosphorus. The bone status is very critical in Acknowledgment poultry production, because phosphorus deficiency results in breakage or bone defect during processing. The treatment The authors thank CNPq (Conselho Nacional de Desenvolvi- of poultry feed with phytase increased weight gain; feed mento Cient´ıfico e Tecnologico)´ for financial support. consumption; Ca, P, and Zn retention; tibia ash, tibia Ca, P, and Zn contents; tibia weight; plasma Ca, P,Mg, Zn, and total References protein content; and serum aspartate aminotransferase, ala- nine aminotransferase, and lactate dehydrogenase activities. [1]C.L.Delgado,C.B.Courbois,andM.W.Rosegrant,“Global Phytase supplementation reduced linearly serum alkaline food demand and the contribution of livestock as we enter the phosphatase activity. In conclusion, the results indicate that new millennium. Markets and Structural Studies Division,” the addition of phytase to maize and soybean low-available International Food Policy Research Institute, p. 36, 1998. phosphorus meals improves the performance and increases [2] A. M. Ahhmed, S. Kawahara, K. Ohta, K. Nakade, T. Soeda, Ca, P, and Zn utilization in chicks. and M. Muguruma, “Differentiation in improvements of gel Fish meal is becoming an increasingly expensive resource strength in chicken and beef sausages induced by transglutam- as the world demand is rising. Much of the current research inase,” Meat Science, vol. 76, no. 3, pp. 455–462, 2007. [3] P. Trespalacios and R. Pla, “Synergistic action of transglu- in commercial fish feed formulation is therefore focusing on taminase and high pressure on chicken meat and egg gels in how to replace fish meal by cheaper and more readily avail- absence of phosphates,” Food Chemistry, vol. 104, no. 4, pp. able protein sources of plant origin, and good availability of 1718–1727, 2007. phosphorus in feed for aquatic animals is also important. [4] M. Motoki and K. Seguro, “Transglutaminase and its use for − The effect of a supplemental fungal phytase (0 or 1400 U kg 1 food processing,” Trends in Food Science and Technology, vol. − feed 1) on performance and phosphorus availability on 9, no. 5, pp. 204–210, 1998. juvenile rainbow trout fed diets with a high inclusion of [5] Y. Zhu and J. Tramper, “Novel applications for microbial plant based protein and on the magnitude and composition transglutaminase beyond food processing,” Trends in Biotech- of the waste phosphorus production was tested. Growth nology, vol. 26, no. 10, pp. 559–565, 2008. and feed conversion ratios were not significantly affected [6]G.S.Nielsen,B.R.Petersen,andA.J.Møller,“Impactofsalt, ff by the increased dietary phosphorus level or supplemental phosphate and temperature on the e ect of a transglutaminase fungal phytase, but this last one improved the availability of (F XIIIa) on the texture of restructured meat,” Meat Science, vol. 41, no. 3, pp. 293–299, 1995. phytate-phosphorus from an average of 6 to 64%. The fish [7] M. Dondero, V. Figueroa, X. Morales, and E. Curotto, retained 53%–79% of the ingested phosphorus, while 24%– “Transglutaminase effects on gelation capacity of thermally 44% was recovered in the feces. This study demonstrated that induced beef protein gels,” Food Chemistry,vol.99,no.3,pp. phytase supplementation will be advantageous to the fish and 546–554, 2006. the environment if supplemented to low-phosphorus diets [8] C. Qihe, H. Guoqing, J. Yingchun, and N. Hui, “Effects of containing a large share of plant-derived protein [59]. elastase from a Bacillus strain on the tenderization of beef Phytate, which cannot be digested by shrimp due to lack meat,” Food Chemistry, vol. 98, no. 4, pp. 624–629, 2006. of phytase, becomes a pollutant in the aquatic environment. [9] J. W. Savell and H. R. Cross, “The role of fat in the palatability A study with tiger shrimp (P. monodon juveniles) fed with of beef, pork, and lamb,” in Designing Foods: Animal Product soybean meal showed that although phytase supplementa- Options in the Market Place, N. R. Council, Ed., pp. 345–355, tion has no effect on shrimp growth, there is significantly National Academy Press, Washington, DC, USA, 1988. [10] J. Han, J. D. Morton, A. E. D. Bekhit, and J. R. Sedcole, “Pre- lower total phosphorus excretion, and this result is useful rigor infusion with kiwifruit juice improves lamb tenderness,” for low-pollution shrimp feed [60]. This has been driven Meat Science, vol. 82, no. 3, pp. 324–330, 2009. by recognition of the ecological need to reduce P levels in [11] B. Gerelt, H. Rusman, T. Nishiumi, and A. Suzuki, “Changes ffl e uents and an increasing scientific and practical apprecia- in calpain and calpastatin activities of osmotically dehydrated tion of the roles of phytate and phytase in animal nutrition. bovine muscle during storage after treatment with calcium,” Moreover, the recent proliferation of phytases in the market- Meat Science, vol. 70, no. 1, pp. 55–61, 2005. place has generated price reduction and facilitated their [12] R. Cheret,C.Delbarre-Ladrat,M.D.Lamballerie-Anton,and´ inclusion in pig and poultry diets [57]. V. Verrez-Bagnis, “Calpain and cathepsin activities in post mortem fish and meat muscles,” Food Chemistry, vol. 101, no. 4, pp. 1474–1479, 2007. [13] M. J. Benito, M. Rodr´ıguez, R. Acosta, and J. J. Cordoba,´ 5. Conclusion “Effect of the fungal extracellular protease EPg222 on texture of whole pieces of pork loin,” Meat Science, vol. 65, no. 2, pp. The new demands of meat products by consumers make the 877–884, 2003. food industry search continuously for higher quality, better [14] S. Nalinanon, S. Benjakul, W. Visessanguan, and H. prices, and less environmental damage. Enzyme application Kishimura, “Improvement of gelatin extraction from bigeye in the manufacturing of meat products and some possibilities snapper skin using pepsin-aided process in combination with to treat their waste have become important alternatives to protease inhibitor,” Food Hydrocolloids, vol. 22, no. 4, pp. 615– meet these needs. Further studies to apply enzymes in meat 622, 2008. Enzyme Research 7

[15] Y. Thiansilakul, S. Benjakul, and F. Shahidi, “Compositions, [31] E. Tiwary and R. Gupta, “Medium optimization for a novel functional properties and antioxidative activity of protein 58 kDa dimeric keratinase from Bacillus licheniformis ER- hydrolysates prepared from round scad (Decapterus maru- 15: biochemical characterization and application in feather adsi),” Food Chemistry, vol. 103, no. 4, pp. 1385–1394, 2007. degradation and dehairing of hides,” Bioresource Technology, [16] K. B. Devaraj, L. R. Gowda, and V. Prakash, “An unusual ther- vol. 101, no. 15, pp. 6103–6110, 2010. mostable aspartic protease from the latex of Ficus racemosa [32] A. Bougatef, N. Nedjar-Arroume, R. Ravallec-Ple´ et al., (L.),” Phytochemistry, vol. 69, no. 3, pp. 647–655, 2008. “Angiotensin I-converting enzyme (ACE) inhibitory activ- [17] G. A. Sullivan and C. R. Calkins, “Application of exogenous ities of sardinelle (Sardinella aurita) by-products protein enzymes to beef muscle of high and low-connective tissue,” hydrolysates obtained by treatment with microbial and vis- Meat Science, vol. 85, no. 4, pp. 730–734, 2010. ceral fish serine proteases,” Food Chemistry, vol. 111, no. 2, pp. [18] G. I. Katsaros, P. Katapodis, and P. S. Taoukis, “High hydro- 350–356, 2008. static pressure inactivation kinetics of the plant proteases ficin [33] H. Wu, H.-L. He, X.-L. Chen, C.-Y. Sun, Y.-Z. Zhang, and B.- and papain,” Journal of Food Engineering,vol.91,no.1,pp. C. Zhou, “Purification and identification of novel angiotensin- 42–48, 2009. I-converting enzyme inhibitory peptides from shark meat [19] H. Schmidt, “Effect of papain on different phases of prenatal hydrolysate,” Process Biochemistry, vol. 43, no. 4, pp. 457–461, ontogenesis in rats,” Reproductive Toxicology, vol. 9, no. 1, pp. 2008. 49–55, 1995. [34] R. Balti, N. Nedjar-Arroume, A. Bougatef, D. Guillochon, [20] B. Herranz, M. Fernandez,E.Hierro,J.M.Bruna,J.A.´ and M. Nasri, “Three novel angiotensin I-converting enzyme Ordońez,˜ and L. De la Hoz, “Use of Lactococcus lactis subsp. (ACE) inhibitory peptides from cuttlefish (Sepia officinalis) α cremoris NCDO 763 and -ketoglutarate to improve the using digestive proteases,” Food Research International, vol. 43, sensory quality of dry fermented sausages,” Meat Science, vol. no. 4, pp. 1136–1143, 2010. 66, no. 1, pp. 151–163, 2004. [35] M. Besler, H. Steinhart, and A. Paschke, “Stability of food [21] M. Shimizu, N. Sawashita, F. Morimatsu et al., “Antithrom- allergens and allergenicity of processed foods,” Journal of botic papain-hydrolyzed peptides isolated from pork meat,” Chromatography B, vol. 756, no. 1-2, pp. 207–228, 2001. Thrombosis Research, vol. 123, no. 5, pp. 753–757, 2009. [36] A. M. Ahhmed and M. Muguruma, “A review of meat protein [22] G. M. F. Ab´ılio, H. J. Holschuh, P. S. Bora, and E. F. hydrolysates and hypertension,” Meat Science, vol. 86, no. 1, de Oliveira, “Extraçao,˜ atividade da bromelina e analise´ de pp. 110–118, 2010. alguns parametrosˆ qu´ımicos em cultivares de abacaxi,” Revista [37]M.A.Dimitrakopoulou,J.A.Ambrosiadis,F.K.Zetou,andJ. Brasileira de Fruticultura, vol. 31, no. 4, pp. 1117–1121, 2009. G. Bloukas, “Effect of salt and transglutaminase (TG) level and [23] A. M. F. Fileti, G. A. Fischer, and E. B. Tambourgi, “Neural processing conditions on quality characteristics of phosphate- modeling of bromelain extraction by reversed micelles,” free, cooked, restructured pork shoulder,” Meat Science, vol. Brazilian Archives of Biology and Technology,vol.53,no.2,pp. 70, no. 4, pp. 743–749, 2005. 455–463, 2010. [38] L. Cui, G. Du, D. Zhang, H. Liu, and J. Chen, “Purification and [24] J. A. Melendo, J. A. Beltran,´ I. Jaime, R. Sancho, and P. characterization of transglutaminase from a newly isolated Roncales,´ “Limited proteolysis of myofibrillar proteins by Streptomyces hygroscopicus,” Food Chemistry, vol. 105, no. 2, bromelain decreases toughness of coarse dry sausage,” Food pp. 612–618, 2007. Chemistry, vol. 57, no. 3, pp. 429–433, 1996. ff [39] A. M. Ahhmed, T. Nasu, D. Q. Huy, Y. Tomisaka, S. Kawahara, [25] A. Ionescu, I. Aprodu, and G. Pascaru, “E ect of papain and ff bromelin on muscle and collagen proteins in beef meat,” The and M. Muguruma, “E ect of microbial transglutaminase on Annals of the University Dunarea de Jos of Galati. Fascicle VI— the natural actomyosin cross-linking in chicken and beef,” Food Technology, New Series, pp. 9–16, 2008. Meat Science, vol. 82, no. 2, pp. 170–178, 2009. [26] B. Gerelt, Y. Ikeuchi, and A. Suzuki, “Meat tenderization by [40] M. Castro-Briones, G. N. Calderon,´ G. Velazquez, M. S. Rubio, proteolytic enzymes after osmotic dehydration,” Meat Science, M. Vazquez,´ and J. A. Ram´ırez, “Mechanical and functional vol. 56, no. 3, pp. 311–318, 2000. properties of beef products obtained using microbial trans- [27] L. Chen, J. S. Lucas, J. O. Hourihane, J. Lindemann, S. L. glutaminase with treatments of pre-heating followed by cold Taylor, and R. E. Goodman, “Evaluation of IgE binding to binding,” Meat Science, vol. 83, no. 2, pp. 229–238, 2009. ff proteins of hardy (Actinidia arguta), gold (Actinidia chinensis) [41] E. Fulladosa, X. Serra, P. Gou, and J. Arnau, “E ects of and green (Actinidia deliciosa) kiwifruits and processed hardy potassium lactate and high pressure on transglutaminase kiwifruit concentrate, using sera of individuals with food restructured dry-cured hams with reduced salt content,” Meat allergies to green kiwifruit,” Food and Chemical Toxicology, vol. Science, vol. 82, no. 2, pp. 213–218, 2009. 44, no. 7, pp. 1100–1107, 2006. [42] M. D. Romero de Avila,´ J. A. Ordońez,˜ L. de la Hoz, A. M. [28] M. Bublin, C. Radauer, A. Knulst et al., “Effects of gastroin- Herrero, and M. I. Cambero, “Microbial transglutaminase testinal digestion and heating on the allergenicity of the kiwi for cold-set binding of unsalted/salted pork models and allergens Act d 1, actinidin, and Act d 2, a thaumatin-like restructured dry ham,” Meat Science, vol. 84, no. 4, pp. 747– protein,” Molecular Nutrition and Food Research, vol. 52, no. 754, 2010. 10, pp. 1130–1139, 2008. [43] O. O. Askin and B. Kilic, “Effect of microbial transglutaminase, [29] M. Bublin, M. Pfister, C. Radauer et al., “Component- sodium caseinate and non-fat dry milk on quality of salt- resolved diagnosis of kiwifruit allergy with purified natural free, low fat turkey doner¨ kebab,” LWT - Food Science and and recombinant kiwifruit allergens,” Journal of Allergy and Technology, vol. 42, no. 10, pp. 1590–1596, 2009. Clinical Immunology, vol. 125, no. 3, pp. 687–694, 2010. [44] F. J. Colmenero, M. J. Ayo, and J. Carballo, “Physicochemical [30] A. A. Khardenavis, A. Kapley, and H. J. Purohit, “Processing of properties of low sodium frankfurter with added walnut: effect poultry feathers by alkaline keratin hydrolyzing enzyme from of transglutaminase combined with caseinate, KCl and dietary Serratia sp. HPC 1383,” Waste Management,vol.29,no.4,pp. fibre as salt replacers,” Meat Science, vol. 69, no. 4, pp. 781–788, 1409–1415, 2009. 2005. 8 Enzyme Research

[45] M. C. Gomez-Guill´ en,´ P. Montero, M. Teresa Solas, and and phosphorus availability by phosphorus-depleted juvenile M. Perez-Mateos,´ “Effect of chitosan and microbial trans- rainbow trout (Oncorhynchus mykiss), and on the magnitude glutaminase on the gel forming ability of horse mackerel and composition of phosphorus waste output,” Aquaculture, (Trachurus spp.) muscle under high pressure,” Food Research vol. 286, no. 1-2, pp. 105–112, 2009. International, vol. 38, no. 1, pp. 103–110, 2005. [60] P. Biswas, A. K. Pal, N. P. Sahu, A. K. Reddy, A. K. Prusty, and [46] A. M. Ahhmed, R. Kuroda, S. Kawahara et al., “Dependence S. Misra, “Lysine and/or phytase supplementation in the diet of microbial transglutaminase on meat type in myofibrillar of Penaeus monodon (Fabricius) juveniles: effect on growth, proteins cross-linking,” Food Chemistry, vol. 112, no. 2, pp. body composition and lipid profile,” Aquaculture, vol. 265, no. 354–361, 2009. 1–4, pp. 253–260, 2007. [47] A. M. Herrero, M. I. Cambero, J. A. Ordońez,˜ L. de la Hoz, and P. Carmona, “Raman spectroscopy study of the structural effect of microbial transglutaminase on meat systems and its relationship with textural characteristics,” Food Chemistry, vol. 109, no. 1, pp. 25–32, 2008. [48]N.Fort,J.P.Kerry,C.Carretero,A.L.Kelly,andE.Saguer, “Cold storage of porcine plasma treated with microbial transglutaminase under high pressure. Effects on its heat- induced gel properties,” Food Chemistry, vol. 115, no. 2, pp. 602–608, 2009. [49]N.Fort,T.C.Lanier,P.M.Amato,C.Carretero,andE.Saguer, “Simultaneous application of microbial transglutaminase and high hydrostatic pressure to improve heat induced gelation of pork plasma,” Meat Science, vol. 80, no. 3, pp. 939–943, 2008. [50] J. Carballo, J. Ayo, and F. J. Colmenero, “Microbial transglutaminase and caseinate as cold set binders: influence of meat species and chilling storage,” LWT - Food Science and Technology, vol. 39, no. 6, pp. 692–699, 2006. [51] N. Aktas¸ and B. Kiliç, “Effect of microbial transglutaminase on thermal and electrophoretic properties of ground beef,” LWT—Food Science and Technology, vol. 38, no. 8, pp. 815– 819, 2005. [52] G. P. Hong and K. B. Chin, “Effects of microbial transglutaminase and sodium alginate on cold-set gelation of porcine myofibrillar protein with various salt levels,” Food Hydrocolloids, vol. 24, pp. 444–451, 2009. [53] K. Suklim, G. J. Flick Jr., J. E. Marcy, W. N. Eigel, C. G. Haugh, and L. A. Granata, “Effect of cold-set binders: alginates and microbial transglutaminase on the physical properties of restructured scallops,” Journal of Texture Studies,vol.35,no.6, pp. 634–642, 2004. [54] P. H. Selle, A. J. Cowieson, and V. Ravindran, “Consequences of calcium interactions with phytate and phytase for poultry and pigs,” Livestock Science, vol. 124, no. 1–3, pp. 126–141, 2009. [55] S.-J. Lim and K.-J. Lee, “Partial replacement of fish meal by cottonseed meal and soybean meal with iron and phytase supplementation for parrot fish Oplegnathus fasciatus,” Aqua- culture, vol. 290, no. 3-4, pp. 283–289, 2009. [56] G. Q. Lan, N. Abdullah, S. Jalaludin, and Y. W. Ho, “In vitro and in vivo enzymatic dephosphorylation of phytate in maize-soya bean meal diets for broiler chickens by phytase of Mitsuokella jalaludinii,” Animal Feed Science and Technology, vol. 158, no. 3-4, pp. 155–164, 2010. [57] P. H. Selle and V. Ravindran, “Phytate-degrading enzymes in pig nutrition,” Livestock Science, vol. 113, no. 2-3, pp. 99–122, 2008. [58]A.Brenes,A.Viveros,I.Arija,C.Centeno,M.Pizarro,and C. Bravo, “The effect of citric acid and microbial phytase on mineral utilization in broiler chicks,” Animal Feed Science and Technology, vol. 110, no. 1–4, pp. 201–219, 2003. [59] J. Dalsgaard, K. S. Ekmann, P. B. Pedersen, and V. Verlhac, “Effect of supplemented fungal phytase on performance SAGE-Hindawi Access to Research Enzyme Research Volume 2010, Article ID 174354, 9 pages doi:10.4061/2010/174354

Review Article Enzymatic Strategies to Detoxify Gluten: Implications for Celiac Disease

Ivana Caputo,1, 2 Marilena Lepretti,1 Stefania Martucciello,1 and Carla Esposito1, 2

1 Department of Chemistry, University of Salerno, 84084 Salerno, Italy 2 European Laboratory for the Investigation of Food-Induced Diseases, University Federico II, 80131 Naples, Italy

Correspondence should be addressed to Carla Esposito, [email protected]

Received 4 July 2010; Accepted 14 September 2010

Academic Editor: Raﬀaele Porta

Copyright © 2010 Ivana Caputo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Celiac disease is a permanent intolerance to the gliadin fraction of wheat gluten and to similar barley and rye proteins that occurs in genetically susceptible subjects. After ingestion, degraded gluten proteins reach the small intestine and trigger an inappropriate T cell-mediated immune response, which can result in intestinal mucosal inflammation and extraintestinal manifestations. To date, no pharmacological treatment is available to gluten-intolerant patients, and a strict, life-long gluten-free diet is the only safe and efficient treatment available. Inevitably, this may produce considerable psychological, emotional, and economic stress. Therefore, the scientific community is very interested in establishing alternative or adjunctive treatments. Attractive and novel forms of therapy include strategies to eliminate detrimental gluten peptides from the celiac diet so that the immunogenic effect of the gluten epitopes can be neutralized, as well as strategies to block the gluten-induced inflammatory response. In the present paper, we review recent developments in the use of enzymes as additives or as processing aids in the food biotechnology industry to detoxify gluten.

1. Celiac Disease whereas breastfeeding exerts a protective effect and the risk of CD is reduced if children are still being breast-fed when Celiac Disease (CD) is a condition affecting 1:70–1:200 dietary gluten is introduced [9]. individuals worldwide that may be diagnosed at any age The clinical features of CD vary considerably [2]. Intesti- [1, 2]. In a population-based study, increasing prevalence nal symptoms are frequent in children diagnosed within and high incidence of CD (1:47) in elderly people (older than the first two years of life. However, asymptomatic patients 52yearsofage)havebeenremarked[3]. CD is a permanent can be found: failure to thrive, chronic diarrhoea, vomiting, food intolerance to the ingested gliadin fraction of wheat abdominal distension, muscle wasting, anorexia, and general gluten and similar alcohol-soluble proteins of barley and irritability are present in most cases. The wider use of rye in genetically susceptible subjects [4, 5]. Most patients serological screening tests is making it easier to recognize tolerate oats without any signs of intestinal inflammation extra-intestinal manifestations such as short stature, anaemia probably because oat avenins are phylogenetically more unresponsive to iron therapy, osteoporosis, ataxia, periph- distant from the analogous proteins in wheat, rye, and eral neuropathies, hypertransaminasemia, and unexplained barley [6]. Nonetheless, a few individuals with clinical oat infertility [10]. It is also noteworthy that CD is associated intolerance have avenin-reactive mucosal T cells that can with a high prevalence of concomitant autoimmune diseases cause mucosal inflammation [7]. In children prone to CD, (approximately 5–10 times greater than in the general pop- exposure to wheat, barley, and rye in the first three months ulation), such as endocrine autoimmune diseases, thyroid of life significantly increases the risk of developing CD diseases,andselectiveIgAdeficiency,aswellasofgenetic compared with exposure between 4 and 6 months [8] disorders, such as Down and Turner’s syndromes [11]. 2 Enzyme Research

Because symptoms may improve with a gluten-free diet, spermine, spermidine, and histamine. However, the absence it is thought that gluten plays a key role in the pathogenesis of suitable nucleophilic amines and a low pH favours tTG of this disease. deamidation of protein-bound glutamine residues [17]. The presence of tTG-specific autoantibodies only in patients who have gluten in their diet suggests that the 2. Gluten generation of such antibodies in CD requires gluten as The grain protein content of wheat varies between 8 and 17 an exogenous trigger. The proposed mechanism by which percent, depending on genetic make-up and external factors autoimmunity develops in CD is that the enzyme tTG gen- associated with the crop. A unique property of wheat flour erates additional antigenic epitopes by cross-linking gliadin is that, when in contact with water, the insoluble protein peptides to itself and/or to other protein substrates, and this fraction forms a viscoelastic protein mass known as gluten. stimulates mucosal T cells to produce autoantibodies against Gluten, which comprises roughly 78 to 85 percent of the total tTG and gliadin [18](Figure 1). Since the existence of tTG- wheat endosperm protein, is a very large complex mainly specific T cells in the intestinal mucosa of untreated patients composed of polymeric (multiple polypeptide chains linked is not proven, it is hypothesized that the production of anti- by disulphide (SS) bonds) and monomeric (single chain tTG antibodies is driven completely by intestinal gliadin- polypeptides) proteins known as glutenins and gliadins, specific T cells. The observation that anti-tTG antibody respectively. Gliadin consists of proteins containing α/β-, γ-, titers fall and can become undetectable during a gluten- and ω-gliadins. In contrast to α/β-andγ-gliadins, which free diet suggests that B cell activity depends on persistent form three and four intramolecular (SS) bonds, respectively, antigen presentation. In a pioneering study in 1990, Porta ω-gliadins lack cysteine residues. Glutenin is a heterogeneous et al. demonstrated that wheat glutelins and gliadins, as mixture of SS-linked polymers with a largely unknown well as purified A-gliadin, act as acyl donor substrates for polymer structure. A glutenin polymer consists of glutenin tTG [19]. In particular, by performing incubations in vitro subunits of high or low molecular weight that are connected both in the presence of radiolabeled polyamines and in their by intermolecular SS bonds. Glutenins confer elasticity, while absence, Porta et al. showed that these proteins were able to produce not only γ(glutamyl)polyamine adducts but also gliadins mainly confer viscous flow and extensibility to the ε γ gluten complex. Thus, gluten is responsible for most of the polymeric complexes, probably through intermolecular ( - viscoelastic properties of wheat flour doughs, and it is the glutamyl)lysine crosslinks. In the case of A-gliadin, the single main factor dictating the use of a wheat variety in bread and lysil residue occurring in the amino acid sequence (K-186) pasta making. Gluten viscoelasticity, for end-use purposes, is is assumed to act as an acyl acceptor site. It is worth noting commonly known as flour or dough strength [12, 13]. that the increase of both tTG activity in situ and tTG protein Gliadins and glutenins have a unique amino acid com- has been detected at critical sites of celiac mucosae, such as position with a high content of proline (15%), hydrophobic the intestinal brush border and subepithelial compartments amino acids (19%), and glutamine (35%), hence they are [20]. named prolamins. Moreover, they contain domains with The involvement of tTG in the pathogenesis of CD numerous repetitive sequences rich in those amino acids. could be also due to another distinct but interdependent Because of this glutamine- and proline-rich structure, gluten pathway via a gliadin-derived peptide deamidation reaction proteins are resistant to complete digestion by pancreatic and (Figure 1). Gluten peptides are specifically recognized by brush border proteases [14, 15]. human leukocyte antigen (HLA)-DQ2/DQ8, a class II major histocompatibility complex [21].Indeed,CDisstrongly associated with the genes encoding HLA-DQ2, and gluten- 3. Posttranslational Modification of Gluten by specific CD4+ intestinal T cells can be isolated from intestinal Tissue Transglutaminase biopsies of CD patients but not from healthy controls [21]. By contrast, there is no evidence of T cell-mediated reactivity CD is triggered by an inappropriate T cell-mediated immune against dietary gliadin in the nonceliac mucosa. Moreover, response to dietary gluten proteins. Consequently, CD gliadin-specific T lymphocytes from CD intestinal mucosa patients display various degrees of intestinal inflammation, are mainly of the Th1/Th0 phenotype, which after gliadin ranging from mild intraepithelial lymphocytosis to severe recognition, release prevalently proinflammatory cytokines subepithelial mononuclear cell infiltration that results in dominated by interferon (IFN)-γ [22] and interleukin (IL)- total villous atrophy coupled with crypt hyperplasia. The 10 [23]. It has been hypothesized that tTG might be responsi- most evident expression of autoimmunity in CD is the ble for the deamidation of specific glutamine residues within presence of serum antibodies to tissue transglutaminase naturally digested gluten peptides, especially at low pH. (tTG), the main autoantigen of endomysial antibodies [16]. Such tTG-catalyzed posttranslational modification generates tTG is a member of a Ca2+-dependent enzyme family negatively charged amino acid residues that bind with an involved in post-translational modifications of proteins. increased affinity to the HLA-DQ2 or HLA-DQ8 molecules, tTG prevalently catalyzes the formation of stable isopeptide thus potentiating T cell activation [24, 25]. Recognition of bonds between the γ-carboxamide group of the protein- the T cell epitope has been particularly difficult since gliadin’s bound glutamine residue and an appropriate amino group, peculiar amino acid composition and its high glutamine either the ε-amino group of a protein-bound lysine residue content make it an excellent tTG substrate. A 33 mer peptide, or a small biogenic amine molecule such as putrescine, containing three of the most immunogenic epitopes, was Enzyme Research 3

Gliadin peptides

Transamidation

Plasma cell K Q Deamidation (CH ) Q XP E XP 2 4 + (CH2)2 H O 2 B NH2 (CH2)2 (CH2)2 APC C +NH3 tTG O NH2 C C Ca2+

O NH2 O OH Q

+NH3 (CH2)2

T Macrophage Th1 T C K

O N (CH2)4 H tTG DQ2/DQ8 Gliadin Tcellreceptor Deamidated gliadin CD4 Transamidated gliadin Anti-tTG antibody Cytokines Gliadin-tTG complex

Figure 1: Tissue transglutaminase (tTG)-mediated post-translational modifications in celiac disease. Gliadin peptides reach the subepithelial region of the intestinal mucosa. Here, tTG deamidation of specific glutamines of gliadin peptides generates potent immunostimulatory epitopes that are presented via HLA-DQ2/DQ8 on antigen-presenting cells (APC) to CD4+ T cells. Activated gliadin-specific CD4+ Tcells produce high levels of pro-inflammatory cytokines, thus inducing a Th1 response that results in mucosal remodelling and villous atrophy. In addition, tTG transamidation activity generates tTG-gliadin complexes that bind to tTG-specific B cells, are endocytosed and processed. Gliadin-DQ2/DQ8 complexes are then presented by the tTG-specific B cells to gliadin-specific T cells, a process that leads to the production of anti-tTG antibodies.

identified as one of the main stimulators of the inflammatory filaments [31], induce maturation of bone-marrow-derived response to gluten, resistant to intestinal proteases [26, 27]. dendritic cells [32], and, by affecting epithelial growth factor- However, Camarca et al. recently demonstrated that the receptor decay, induce epithelial cell proliferation [33]. repertoire of gluten peptides recognized by adult celiac The peptide also stimulates the synthesis and release of patients is larger than had been previously thought, and it the proinflammatory cytokine IL-15 that can promote an differs from one individual to another. Indeed, they found adaptive immune response [34] involving CD4+ T cells that several active gluten peptides with a large heterogeneity of recognize various deamidated gliadin peptides [25]. Most of responses [28]. the events related to innate immune activation were inhibited Although the role of gluten in activating gluten-specific by antibodies neutralizing IL-15, thus confirming that this T lymphocytes in the lamina propria is well established, it cytokine mediates intestinal mucosal damage induced by has been demonstrated that gluten contains peptides that can ingestion of gliadin. In particular, Barone et al. investigated stimulate cells of the innate immune system. The prototype the molecular mechanisms of the gliadin-induced IL15 of innate peptides is peptide 31–43/49, which has been increase and discovered that gliadin peptide 31–43 increases shown to be toxic for CD patients both in vitro and in vivo the levels of IL-15 on the cell surface of CaCo-2 cells probably [29, 30]. Peptide 31–43/49 can reorganize intracellular actin by interfering with its intracellular trafficking [35]. 4 Enzyme Research

4. Treatment of Celiac Disease as a treatment strategy, particularly in cases of refractory sprue because of intraepithelial lymphocyte activation in To date, no pharmacological treatment is available to gluten- this condition [41], as well as antibodies to IFN-γ [42]. intolerant patients. A strict, life-long, gluten-free diet is Other promising treatment strategies are aimed at preventing the only safe and efficient treatment available, although gliadin presentation to T cells by blocking HLA-binding sites it results in a social burden. Adhering to a gluten-free and using IL-10 as a tool for promoting tolerance [23]. To diet can have a significant negative impact on perceived reverse the toxic effects induced by gliadin in human intesti- quality of life and may produce considerable psychological, nal cells and gliadin-sensitive HCD4-DQ8 mice, Pinier et al. emotional, and economic stress. Moreover, this requirement proposed a completely different strategy based on the use of for dietary compliance is made more difficult by the synthetic sequestering polymeric binders that can complex exclusion of wheat, rye, and barley from the diet, which and neutralize gliadin in situ. Coadministration of synthetic are important sources of iron, dietary fibre, and vitamin B, polymeric binders and gliadin to HLA-HCD4/DQ8 mice especially for adolescents and adults who need continuous attenuated gliadin-induced changes in the intestinal barrier monitoring by dieticians [36]. A lifelong gluten-free diet and reduced intraepithelial lymphocyte and macrophage cell can be extremely difficult since gluten may be present counts [43]. Recent new therapeutic approaches include innonstarchyfoodssuchassoysauceandbeer,aswell correction of the intestinal barrier defect against gluten entry. as in nonfood items including some medications, postage An intestinal permeability blocker (AT1001), which is an stamp glue, and cosmetics (e.g., lipstick). CD patients can, inhibitor of the zonulin pathway that acts to prevent gliadin therefore, be exposed inadvertently to gluten. Moreover, even from inducing increased intestinal permeability, is currently after many years of gluten avoidance, CD patients never in a phase IIb clinical study [44]. Finally, a vaccine that could acquire tolerance to gliadin, and re-exposure to the antigen desensitize or induce tolerance in individuals with CD has reactivates the disease. Finally, it is worth noting that a small been proposed [45, 46]. group of patients with CD (2%–5%) fail to improve clinically Besides therapeutic treatments, transgenic technology and histologically upon elimination of dietary gluten. This and breeding ancient varieties have been tried with the goal complication is referred to as refractory CD, and it imposes of developing grains that have a low or zero content of a serious risk for developing lethal enteropathy-associated T- immunotoxic sequences, but with reasonable baking quality. cell lymphoma. However, these approaches are difficult due to the number In 2000, the Codex Alimentarius Commission of the and the repetition of sequence homologies in the cereal World Health Organization and the FAO described gluten- protein family, and because cereals like wheat are hexaploid free foods consisting of, or made only from, ingredients [47, 48]. which do not contain any prolamines from wheat or any Triticum species, such as spelt, kamut or durum wheat, rye, barley, oats, or their crossbred varieties with a gluten level not 5. Enzyme Therapy exceeding 20 ppm [37, 38]. At present, gluten-free products Enzyme supplement therapies are focused on inactivating are not widely available; they are usually expensive, and they immunogenic gluten epitopes (Table 1). have poor sensory and shelf life properties. Research and development are currently focused on improving mouth- feel, flavour, and rheology of gluten-free products. Gluten is 5.1. Oral Administration of Bacterial Endopeptidases. After responsible for most of the viscoelastic properties of wheat ingestion, degraded gluten proteins reach the small intestine. flour doughs, and its absence can result in a baked bread However, because of their unusually high proline and with a crumbling texture, poor colour and other postbaking glutamine content, especially in immunodominant gliadin quality defects [37]. For all these reasons the search for peptides like the 33 mer, gluten is poorly degraded by the safe and effective therapeutic alternatives to a gluten-free enzymes present in the gastrointestinal tract. Hence, oral diet, which are compatible with a normal social lifestyle, enzyme therapy has been suggested as an alternative to the is of great importance. Advances in our understanding gluten-free diet. Promising enzymes (expressed in various of the complex mechanisms involved in CD pathogenesis microorganisms) tested are the prolyl oligopeptidases from have opened several promising avenues for therapeutic Flavobacterium meningosepticum, Sphingomonas capsulate, intervention aimed at targeting each factor involved in the and Myxococcus xanthus. These enzymes are capable of disease onset, some of which are being tested in early clinical degrading proline-containing peptides that are otherwise trials [39]. The identification of T-cell stimulatory gliadin resistant to degradation by proteases in the gastrointestinal sequences (33 mer peptide) is important so that peptide tract in vitro [49–51]. However, most of these enzymes are analogues of gliadin epitope(s) can be engineered to generate irreversibly inactivated in the stomach by pepsin and acidic peptides that exert antagonistic effects. Of course, the pH, thus failing to degrade gluten before it reaches the small chances of success of using peptide analogues to modulate intestine [49]. Encapsulation of these prolyl oligopeptidases specific immune responses could be hampered by the wide was proposed in order to protect them from gastric juices heterogeneity of the gliadin T-cell epitopes. Elucidation of [51]. However, in a recent ex vivo study, using biopsy- the hierarchy of pathogenic gliadin epitopes and their core derived intestinal tissue mounted in Ussing chambers, it was region is required before a peptide-based therapy can be observed that only high doses of prolyl oligopeptidase were designed [40]. Antibodies to IL-15 have also been proposed capable of eliminating the accumulation of immunogenic Enzyme Research 5 peptides in the serosal compartment [50]. This indicates that, method that used selected lactobacilli to hydrolyze various even if the enzyme were encapsulated, it is too inefficient Pro-rich peptides, including the 33 mer peptide, for the to degrade gluten before it reaches the proximal part of production of sourdoughs made from a mixture of wheat the duodenum, the site where gluten triggers inflammatory and nontoxic oat, buckwheat, and millet flours [56]. After T-cell responses [50]. Mitea et al. recently investigated a 24 hours of fermentation, wheat gliadins and low-molecular- new prolyl endoprotease from Aspergillus niger. This enzyme mass, alcohol-soluble polypeptides were almost completely was found to degrade gluten peptides and intact gluten hydrolyzed. Proteins extracted from sourdough were used proteins efficiently in the stomach, to such an extent that for in vitro agglutination tests on K 562(S) subclone cells of hardly any traces of gluten reached the duodenal com- human origin and to produce two types of bread, containing partment [52]. Moreover, the optimum pH of this enzyme ca. 2 g of gluten. The latter were used in an in vivo double- is compatible with that found in the stomach and the blind acute challenge of CD patients. Agglutination testing enzyme is resistant to degradation by pepsin. Finally, prolyl of K 562(S) cells and the acute in vivo challenge showed endoprotease from Aspergillus niger is derived from the food improved tolerance of breads containing 30% wheat flour grade microorganism and is available on an industrial scale. [56]. The reported data suggest that long-time fermentation These results indicate that this enzyme might be suitable in the presence of a mixture of selected lactic acid bacteria for oral supplementation to degrade gluten proteins in food seemed to be indispensable to reduce toxicity. In actual fact, before they reach the small intestine [52]. Recently, Gass different probiotic bacterial strains have their characteristic et al. evaluated a new combination therapy, consisting of set of peptidases, which may diverge considerably from two gastrically active enzymes that detoxify gluten before its each other and have variable substrate specificities. In release in the small intestine. They used a glutamine-specific line with this concept, different probiotic bacterial strains endoprotease (EP-B2; a cysteine endoprotease from ger- have been tested. Among probiotic preparations, VSL#3, minating barley seeds) and a prolyl-specific endopeptidase a highly concentrated mixture of lactic acid and bifido- from Sphingomonas capsulata, for its ability to digest gluten bacteria, was able to hydrolyze completely the α2-gliadin- under gastric conditions. Endoprotease EP-B2 extensively derived epitopes 62–75 and 33 mer (750 ppm) [57]. It is hydrolyzes the gluten network in bread into relatively interesting to underline that probiotics, defined as the short (but still inflammatory) oligopeptides, whereas prolyl- viable microorganisms that exhibit a beneficial effect on specific endopeptidase from Sphingomonas capsulata rapidly the health of the host by improving its intestinal microbial detoxifies oligopeptides after primary proteolysis at internal balance, could directly modulate the function of epithelial proline residue level to yield nontoxic metabolites [53]. cells. It has been reported that different probiotic strains, A practical advantage of this combination product is that including the VSL3# preparation, increase epithelial barrier both enzymes are active and stable in the stomach and can function by stabilizing tight junctions and inducing mucin therefore be administered as lyophilized powders or simple secretion in epithelial cells [57, 58]. Furthermore, several capsules or tablets. probiotic bacterial strains are able to protect the epithelium, presumably by the aforementioned mechanisms, from various insults, including pathogenic bacteria [59, 60]and 5.2. Pretreatment of Whole Gluten with Bacterial-Derived inflammatory cytokines [61, 62]. More recently, Rizzello et Peptidase. An alternative approach to detoxify gluten is al. showed that fermentation with a complex formula of represented by the digestion of wheat gluten peptides sourdough lactobacilli decreased the concentration of gluten with bacterial-derived peptidase during food processing and to below 10 ppm [63]. Specifically, they used a mix of ten before administration to patients. Traditional methods to sourdough lactobacilli that were selected for their peptidase prepare cereal foods, including long fermentation times by systems capability to hydrolyze Pro-rich peptides, including selected sourdough lactic acid bacteria, have mostly been the 33 mer peptide, together with fungal proteases, that are substituted by the indiscriminate use of chemical and/or routinely used as improvers in the baking industry. In this baker’s yeast leavening agents. Under these circumstances, way, wheat and rye breads or pasta, if supplemented with cereal components (e.g., proteins) are subjected to very gluten-free flour-based structuring agents, may be tolerated mild or absent degradation during manufacture, resulting, by CD patients. Agglutination testing of K 562(S) cells and probably, in reduced digestibility compared to traditional an acute in vivo challenge showed improved tolerance of and ancient sourdough baked goods [54]. Di Cagno et al. breads containing 30% wheat flour. Moreover, prolonged selected four sourdough lactobacilli (L. alimentarius 15M, in vivo challenge of CD patients confirms reduced toxicity L. brevis 14G, L. sanfranciscensis 7A, and L. hilgardii 51B) of gluten fermented with selected lactobacilli and fungal that showed considerable hydrolysis of albumin, globulin, proteases. In fact, CD patients (age >12 years old) on and gliadin fractions during wheat sourdough fermentation. a gluten-free diet for at least five years were challenged These lactobacilli had the capacity to hydrolyze the 31– with a daily intake of 10 g of hydrolysed gluten (<20 ppm 43 fragment of A-gliadin in vitro and, after hydrolysis, of gluten) for 2 months. Intestinal functional tests, as greatly reduced the agglutination of K 562(S) subclone well as CD serum antibodies (anti-tTG, anti-endomysium), cells of human myelogenous leukemia origin by a toxic and duodenal histology and immunohistochemistry at peptic-tryptic digest of gliadins [55]. On the basis of these the beginning and after 60 days of challenge showed results, and with the goal of decreasing gluten intolerance that all parameters were normal, i.e., no villous atrophy) in humans, the authors investigated a novel bread making [64]. 6 Enzyme Research

Table 1: Potential enzyme therapies for celiac disease.

Target for Status of Detoxifying agent Mechanism of action detoxification research Prolyl endopeptidases from: S. capsulate [49] Hydrolysis of Preclinical Ingested gliadin F. meningosepticum [50] proline-rich peptides of peptides M. xanthus [51] gliadin Prolyl endopeptidases from: A. niger [52] Clinical trial Prolyl endopeptidases from: S. capsulate in combination with a glutamine-specific Clinical trial endoprotease (EP)-B2 from germinating barley [53] Sourdough lactobacilli-derived peptidases [56] Hydrolysis of Clinical trial Flour proline-rich peptides of Sourdough lactobacilli-derived peptidases in gliadin Clinical trial combination with fungal proteases [64] Transamidation of Flour Transglutaminase enzymes [67] gliadin peptides with Preclinical lysine methyl ester Irreversible thiol-reactive reagents, competitive unspecific or specific Mucosal tTG Preclinical peptidic, and nonpeptidic substrates [69] tTG inhibition

The use of proteases from germinating wheat seeds has especially for flours with low gluten content and poor baking also been proposed to create safe cereal products for CD performance. patients [65, 66]. 5.4. Transglutaminase Inhibitors. tTG plays an important 5.3. Transamidation of Gliadin. tTG-catalyzed deamidation role in CD pathology as it catalyzes deamidation and of specific glutamine residues within naturally digested cross-linking of specific gluten peptides and converts them gluten peptides generates negatively charged amino acid into potent epitopes recognized by intestinal T-cells. In residues that bind with an increased affinity to the HLA- order to restrain the T cell-mediated immune response to DQ2/DQ8 molecules, thus potentiating T cell activation. dietary gluten, a different approach could be to consider Based on this assumption, Gianfrani et al. proposed an tTG as a potential therapeutic target [69]. The inhibition enzyme strategy to inactivate immunogenic peptide epitopes of the tTG-catalyzed deamidation of specific glutamine and, at the same time, to preserve the integrity of the residues within naturally digested gluten peptides might protein structure via the transamidation of wheat flour not generate negatively charged amino acid residues and with a food-grade enzyme and an appropriate amine donor therefore might not increase the binding to the HLA- [67]. Interestingly, a recent study showed that the formation DQ2/DQ8 molecules (thus potentiating T cell activation). of the DQ2-α-II epitope was blocked by 5-biotinamido Several inhibitors acting with different mechanisms that pentylamine and by monodansylcadaverine, reagents known target the TG cross-linking activity have been developed and to cross-link glutamine residues [68]. To this end, the authors tested, mainly in vitro [69, 70]. Among the tTG inhibitors treated wheat flour with tTG and lysine methyl ester; the tested we can find several nonspecific irreversible thiol- lysine-modified gliadin peptides lost almost completely their reactive reagents, also named suicide TG inhibitors, such affinity to bind to HLA-DQ2. Moreover, lysine-modified as cystamine, able to inhibit tTG via the formation of gliadin peptides caused a drastic reduction in gliadin-specific an enzyme-inhibitor complex. Furthermore, we can find IFN-γ production in intestinal T-cell lines derived from CD competitive nonpeptidic tTG amino donor substrates, such patients where the mucosal lesion was mainly induced by the as 1,4-diaminobutane (Fibrostat), which is used topically production of IFN-γ from these gluten-specific T cells. This in a clinical trial to treat abnormal wound healing [71], result suggests that transamidation neutralized the immune and competitive peptidic tTG amino donor. Finally, we can reactivity of a large repertoire of epitopes. Similar results find amine acceptor pseudosubstrates able to inhibit tTG were obtained by using microbial TG, which is different from activity by diverting it from the natural protein substrate. tTG since it is calcium independent and is a low-molecular- Recently, Hoffmann et al. used a blocking peptide approach weight protein that exhibits advantages in food industrial to reduce the processing of gliadin by tTG. The authors applications. This enzyme is commercially available as a showed that these peptides have a potential for gluten dough improver that adds stability and elasticity to the detoxification and could be evaluated as an alternative for dough. Additionally, bread volume and crumb texture are designing new food products for gluten-intolerant patients positively influenced by the addition of microbial TG, [72]. Several factors must be taken into consideration when Enzyme Research 7 designing a tTG inhibitor: inhibitory potency, optimal size [5] B. Jabri and L. M. Sollid, “Tissue-mediated control of of the compound, resistance towards intestinal proteolytic immunopathology in coeliac disease,” Nature Reviews activities (to this end, amino acid residues will be replaced Immunology, vol. 9, no. 12, pp. 858–870, 2009. by peptidomimetics), and selectivity towards tTG. In fact, [6] N. Y. Haboubi, S. Taylor, and S. Jones, “Coeliac disease and the lack of specificity limits therapeutic utility. Furthermore, oats: a systematic review,” Postgraduate Medical Journal, vol. to reduce the risk of systemic side effects, the activity of an 82, no. 972, pp. 672–678, 2006. [7] K. E. A. Lundin, E. M. Nilsen, H. G. Scott et al., “Oats induced optimal tTG inhibitor should be specifically limited to the villous atrophy in coeliac disease,” Gut, vol. 52, no. 11, pp. compartment where gliadin encounters the immune system, 1649–1652, 2003. that is, in the gut. Therefore, blocking the transamidating [8] J. M. Norris, K. Barriga, E. J. Hoffenberg et al., “Risk of celiac activity of tTG represents an attractive tool to prevent disease autoimmunity and timing of gluten introduction in immune activation. Similar approaches have already been the diet of infants at increased risk of disease,” Journal of the investigated in other diseases where tTG is involved, such American Medical Association, vol. 293, no. 19, pp. 2343–2351, as in the neurodegenerative disorders such as Parkinson, 2005. Huntington, and Alzheimer diseases, as well as in cancer and [9] A. Ivarsson, O. Hernell, H. Stenlund, and L. A. Persson, in fibrotic/scarring conditions such as diabetic nephropathy. “Breast-feeding protects against celiac disease,” American Consequently, tTG inhibitory drugs can be predicted to have Journal of Clinical Nutrition, vol. 75, no. 5, pp. 914–921, 2002. ff a wide medical application. [10] M. F. Kagno , “Overview and pathogenesis of celiac disease,” Gastroenterology, vol. 128, no. 4, pp. S10–S18, 2005. [11] A. Di Sabatino and G. R. Corazza, “Coeliac disease,” The 6. Concluding Remarks Lancet, vol. 373, no. 9673, pp. 1480–1493, 2009. [12] H. Wieser, “Chemistry of gluten proteins,” Food Microbiology, The high incidence of CD in the worldwide population is a vol. 24, no. 2, pp. 115–119, 2007. challenging task, given the negative impact of a strict gluten- [13] B. Lagrain, K. Brijs, and J. A. Delcour, “Reaction kinetics of gliadin-glutenin cross-linking in model systems and in bread free diet on the perceived quality of life of celiac patients making,” Journal of Agricultural and Food Chemistry, vol. 56, for several reasons. A life-long gluten-free diet is not easy no. 22, pp. 10660–10666, 2008. to maintain since gluten is the most common ingredient [14] L. Shan, ∅. Molberg, I. Parrot et al., “Structural basis for in the human diet. Multidisciplinary research efforts are gluten intolerance in Celiac Sprue,” Science, vol. 297, no. 5590, currently being carried out in several directions to find pp. 2275–2279, 2002. new treatment strategies in order to reduce gluten toxicity. [15] S.-W. Qiao, E. Bergseng, ∅. Molberg et al., “Antigen presenta- The use of oral proteases capable of detoxifying ingested tion to celiac lesion-derived T cells of a 33-mer gliadin peptide gluten and new food grade fermentation technologies using naturally formed by gastrointestinal digestion,” Journal of bacterial-derived endopeptidases currently represent the Immunology, vol. 173, no. 3, pp. 1757–1762, 2004. most advanced and promising strategies. [16] W. Dieterich, T. Ehnis, M. Bauer et al., “Identification of tissue transglutaminase as the autoantigen of celiac disease,” Nature Medicine, vol. 3, no. 7, pp. 797–801, 1997. Abbreviations [17] L. Lorand and R. M. Graham, “Transglutaminases: crosslinking enzymes with pleiotropic functions,” Nature Reviews CD: Celiac disease Molecular Cell Biology, vol. 4, no. 2, pp. 140–156, 2003. SS: Disulphide [18] ∅. Molberg, S. N. McAdam, and L. M. Sollid, “Role of tTG: Tissue transglutaminase tissue transglutaminase in celiac disease,” Journal of Pediatric HLA: Human leukocyte antigen Gastroenterology and Nutrition, vol. 30, no. 3, pp. 232–240, IFN: Interferon 2000. [19] R. Porta, V. Gentile, C. Esposito, L. Mariniello, and S. IL: Interleukin. Auricchio, “Cereal dietary proteins with sites for cross-linking by transglutaminase,” Phytochemistry, vol. 29, no. 9, pp. 2801– 2804, 1990. References [20] C. Esposito, F. Paparo, I. Caputo et al., “Expression and [1] L. M. Sollid and K. E. A. Lundin, “Diagnosis and treatment enzymatic activity of small intestinal tissue transglutaminase of celiac disease,” Mucosal Immunology, vol. 2, no. 1, pp. 3–7, in celiac disease,” American Journal of Gastroenterology, vol. 98, 2009. no. 8, pp. 1813–1820, 2003. [21] L. M. Sollid, “Molecular basis of celiac disease,” Annual Review [2] G. J. Tack, W. H. M. Verbeek, M. W. J. Schreurs, and C. J. J. of Immunology, vol. 18, pp. 53–81, 2000. Mulder, “The spectrum of celiac disease: epidemiology, clin- [22] E. M. Nilsen, F. L. Jahnsen, K. E. A. Lundin et al., “Gluten ical aspects and treatment,” Nature Reviews Gastroenterology induces an intestinal cytokine response strongly dominated by and Hepatology, vol. 7, no. 4, pp. 204–213, 2010. interferon gamma in patients with celiac disease,” Gastroen- [3] A. Vilppula, K. Kaukinen, L. Luostarinen et al., “Increasing terology, vol. 115, no. 3, pp. 551–563, 1998. prevalence and high incidence of celiac disease in elderly [23] V. M. Salvati, G. Mazzarella, C. Gianfrani et al., “Recombinant people: a population-based study,” BMC Gastroenterology, vol. human interleukin 10 suppresses gliadin dependent T cell 9, article 49, 2009. activation in ex vivo cultured coeliac intestinal mucosa,” Gut, [4] L. M. Sollid, “Coeliac disease: dissecting a complex inflamma- vol. 54, no. 1, pp. 46–53, 2005. tory disorder,” Nature Reviews Immunology, vol. 2, no. 9, pp. [24] ∅. Molberg, S. N. Mcadam, R. Korner¨ et al., “Tissue 647–655, 2002. transglutaminase selectively modifies gliadin peptides that are 8 Enzyme Research

recognized by gut-derived T cells in celiac disease,” Nature [41] A. Di Sabatino, R. Ciccocioppo, F. Cupelli et al., “Epithelium Medicine, vol. 4, no. 6, pp. 713–717, 1998. derived interleukin 15 regulates intraepithelial lymphocyte [25] H. Arentz-Hansen, R. Korner,¨ ∅. Molberg et al., “The Th1 cytokine production, cytotoxicity, and survival in coeliac intestinal T cell response to α-gliadin in adult celiac disease disease,” Gut, vol. 55, no. 4, pp. 469–477, 2006. is focused on a single deamidated glutamine targeted by tissue [42] L. M. Sollid and C. Khosla, “Future therapeutic options for transglutaminase,” Journal of Experimental Medicine, vol. 191, celiac disease,” Nature Clinical Practice Gastroenterology and no. 4, pp. 603–612, 2000. Hepatology, vol. 2, no. 3, pp. 140–147, 2005. [26] H. Harentz-Hansen, S. N. Mcadam, O. Molberg et al., “Celiac [43] M. Pinier, E. F. Verdu, M. Nasser-Eddine et al., “Polymeric lesion T cells recognize epitopes that cluster in regions of binders suppress gliadin-induced toxicity in the intestinal gliadins rich in proline residues,” Gastroenterology, vol. 123, epithelium,” Gastroenterology, vol. 136, no. 1, pp. 288–298, no. 3, pp. 803–809, 2002. 2009. [27] L. Shan, S.-W. Qiao, H. Arentz-Hansen et al., “Identification [44]B.M.Paterson,K.M.Lammers,M.C.Arrieta,A.Fasano, and analysis of multivalent proteolytically resistant peptides and J. B. Meddings, “The safety, tolerance, pharmacokinetic from gluten: implications for Celiac Sprue,” Journal of Pro- and pharmacodynamic effects of single doses of AT-1001 in teome Research, vol. 4, no. 5, pp. 1732–1741, 2005. coeliac disease subjects: a proof of concept study,” Alimentary [28] A. Camarca, R. P. Anderson, G. Mamone et al., “Intestinal T Pharmacology and Therapeutics, vol. 26, no. 5, pp. 757–766, cell responses to gluten peptides are largely heterogeneous: 2007. implications for a peptide-based therapy in celiac disease,” [45] P. H. R. Green and B. Jabri, “Celiac disease,” Annual Review of Journal of Immunology, vol. 182, no. 7, pp. 4158–4166, 2009. Medicine, vol. 57, pp. 207–221, 2006. [29] L. Maiuri, R. Troncone, M. Mayer et al., “In vitro activities of [46] C. L. Keech, J. Dromey, Z. Chen, R. P. Anderson, and A-gliadin-related synthetic peptides: damaging effect on the J. P. McCluskey, “Immune tolerance induced by peptide atrophic coeliac mucosa and activation of mucosal immune immunotherapy in an HLA Dq2-dependent mouse model of response in the treated coeliac mucosa,” Scandinavian Journal gluten immunity,” Gastroenterology, vol. 136, supplement 1, p. of Gastroenterology, vol. 31, no. 3, pp. 247–253, 1996. A-57, 2009. [30] P.J. Ciclitira and H. J. Ellis, “In vivo gluten ingestion in Coeliac [47] L. Spaenij-Dekking, Y. Kooy-Winkelaar, P. Van Veelen et disease,” Digestive Diseases, vol. 16, no. 6, pp. 337–340, 1998. al., “Natural variation in toxicity of wheat: potential for [31] M. G. Clemente, S. De Virgiliis, J. S. Kang et al., “Early effects selection of nontoxic varieties for celiac disease patients,” of gliadin on enterocyte intracellular signalling involved in Gastroenterology, vol. 129, no. 3, pp. 797–806, 2005. intestinal barrier function,” Gut, vol. 52, no. 2, pp. 218–223, [48] R. J. Hamer, “Coeliac disease: background and biochemical 2003. aspects,” Biotechnology Advances, vol. 23, no. 6, pp. 401–408, [32] M. Nikulina, C. Habich, S. B. Flohe,´ F. W. Scott, and H. 2005. Kolb, “Wheat gluten causes dendritic cell maturation and [49] L. Shan, T. Marti, L. M. Sollid, G. M. Gray, and C. Khosla, chemokine secretion,” Journal of Immunology, vol. 173, no. 3, “Comparative biochemical analysis of three bacterial prolyl pp. 1925–1933, 2004. endopeptidases: implications for coeliac sprue,” Biochemical [33] M. V. Barone, A. Gimigliano, G. Castoria et al., “Growth Journal, vol. 383, no. 2, pp. 311–318, 2004. factor-like activity of gliadin, an alimentary protein: implica- [50] T. Marti, ∅. Molberg, Q. Li, G. M. Gray, C. Khosla, and L. tions for coeliac disease,” Gut, vol. 56, no. 4, pp. 480–488, 2007. M. Sollid, “Prolyl endopeptidase-mediated destruction of T [34] L. Maiuri, C. Ciacci, I. Ricciardelli et al., “Association between cell epitopes in whole gluten: chemical and immunological innate response to gliadin and activation of pathogenic T cells characterization,” Journal of Pharmacology and Experimental in coeliac disease,” The Lancet, vol. 362, no. 9377, pp. 30–37, Therapeutics, vol. 312, no. 1, pp. 19–26, 2005. 2003. [51] J. Gass, J. Ehren, G. Strohmeier, I. Isaacs, and C. Khosla, [35]M.V.Barone,D.Zanzi,M.Nanayakkaraetal.,“Cooperation “Fermentation, purification, formulation, and pharmacolog- between IL15 R alfa and EGFR is responsible for gliadin pep- ical evaluation of a prolyl endopeptidase from Myxococcus tide induced and proliferation in Caco-2 cells,” in Proceedings xanthus: implications for Celiac Sprue therapy,” Biotechnology of the 42nd Espghan Annual Meeting G2-02, 2009. and Bioengineering, vol. 92, no. 6, pp. 674–684, 2005. [36] S. Errichiello, O. Esposito, R. Di Mase et al., “Celiac disease: [52] C. Mitea, R. Havenaar, J. W. Drijfhout et al., “Efficient degra- predictors of compliance with a gluten-free diet in adolescents dation of gluten by a prolyl endoprotease in a gastrointestinal and young adults,” Journal of Pediatric Gastroenterology and model: implications for coeliac disease,” Gut,vol.57,no.1,pp. Nutrition, vol. 50, no. 1, pp. 54–60, 2010. 25–32, 2008. [37] E. Gallagher, T. R. Gormley, and E. K. Arendt, “Recent [53] J. Gass, M. T. Bethune, M. Siegel, A. Spencer, and C. Khosla, advances in the formulation of gluten-free cereal-based prod- “Combination enzyme therapy for gastric digestion of dietary ucts,” Trends in Food Science and Technology, vol. 15, no. 3-4, gluten in patients with Celiac Sprue,” Gastroenterology, vol. pp. 143–152, 2004. 133, no. 2, pp. 472–480, 2007. [38] C. Hischenhuber, R. Crevel, B. Jarry et al., “Review article: safe [54] M. Gobbetti, “The sourdough microflora: interactions of amounts of gluten for patients with wheat allergy or coeliac lactic acid bacteria and yeasts,” Trends in Food Science and disease,” Alimentary Pharmacology and Therapeutics, vol. 23, Technology, vol. 9, no. 7, pp. 267–274, 1998. no. 5, pp. 559–575, 2006. [55] R. Di Cagno, M. De Angelis, P. Lavermicocca et al., “Pro- [39] D. Schuppan, Y. Junker, and D. Barisani, “Celiac disease: from teolysis by sourdough lactic acid bacteria: effects on wheat pathogenesis to novel therapies,” Gastroenterology, vol. 137, flour protein fractions and gliadin peptides involved in human no. 6, pp. 1912–1933, 2009. cereal intolerance,” Applied and Environmental Microbiology, [40]R.P.Anderson,D.A.VanHeel,J.A.Tye-Din,D.P.Jewell, vol. 68, no. 2, pp. 623–633, 2002. and A. V. S. Hill, “Antagonists and non-toxic variants of the [56] R. Di Cagno, M. De Angelis, S. Auricchio et al., “Sourdough dominant wheat gliadin T cell epitope in coeliac disease,” Gut, bread made from wheat and nontoxic flours and started vol. 55, no. 4, pp. 485–491, 2006. with selected lactobacilli is tolerated in Celiac Sprue patients,” Enzyme Research 9

Applied and Environmental Microbiology,vol.70,no.2,pp. [71] K. N. Dolyuchuk, “Topical putrescine (fibrostat) in treatment 1088–1096, 2004. of hypertrophic scars: phase II study,” Plastic and Reconstruc- [57] M. D. Angelis, C. G. Rizzello, A. Fasano et al., “VSL#3 tive Surgery, vol. 98, no. 4, p. 752, 1996. probiotic preparation has the capacity to hydrolyze gliadin [72] K. Hoffmann, M. Alminger, T. Andlid, T. Chen, O. Olsson, and polypeptides responsible for Celiac Sprue,” Biochimica et A.-S. Sandberg, “Blocking peptides decrease tissue transglu- Biophysica Acta, vol. 1762, no. 1, pp. 80–93, 2006. taminase processing of gliadin in vitro,” Journal of Agricultural [58] K. Lindfors, T. Blomqvist, K. Juuti-Uusitalo et al., “Live and Food Chemistry, vol. 57, no. 21, pp. 10150–10155, 2009. probiotic Bifidobacterium lactis bacteria inhibit the toxic effects induced by wheat gliadin in epithelial cell culture,” Clinical and Experimental Immunology, vol. 152, no. 3, pp. 552–558, 2008. [59] S. Resta-Lenert and K. E. Barrett, “Live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive Escherichia coli (EIEC),” Gut,vol.52,no.7,pp. 988–997, 2003. [60] J.-M. Otte and D. K. Podolsky, “Functional modulation of enterocytes by gram-positive and gram-negative microorganisms,” American Journal of Physiology, vol. 286, no. 4, pp. G613–G626, 2004. [61] B. Cinque, L. Di Marzio, D. N. Della Riccia et al., “Effect of Bifidobacterium infantis on interferon-γ-induced keratinocyte apoptosis: a potential therapeutic approach to skin immune abnormalities,” International Journal of Immunopathology and Pharmacology, vol. 19, no. 4, pp. 775– 786, 2006. [62] F. Yan, H. Cao, T. L. Cover, R. Whitehead, M. K. Washington, and D. B. Polk, “Soluble proteins produced by probiotic bacteria regulate intestinal epithelial cell survival and growth,” Gastroenterology, vol. 132, no. 2, pp. 562–575, 2007. [63] C. G. Rizzello, M. De Angelis, R. Di Cagno et al., “Highly efficient gluten degradation by lactobacilli and fungal proteases during food processing: new perspectives for celiac disease,” Applied and Environmental Microbiology, vol. 73, no. 14, pp. 4499–4507, 2007. [64] R. Auricchio, R. Di Mase, A. Pianese et al., “Prolonged in vivo challenge of coeliac patients confirms reduced toxicity of gluten fermented with selected lactobacilli and fungal proteases,” in Proceedings of the 13th International Celiac Disease Symposium P286, 2009. [65] T. Kiyosaki, I. Matsumoto, T. Asakura et al., “Gliadain, a gibberellin-inducible cysteine proteinase occurring in germinating seeds of wheat, Triticum aestivum L., specifically digests gliadin and is regulated by intrinsic cystatins,” The FEBS Journal, vol. 274, no. 8, pp. 1908–1917, 2007. [66] S. M. Stenman, J. I. Venal¨ ainen,¨ K. Lindfors et al., “Enzymatic detoxification of gluten by germinating wheat proteases: implications for new treatment of celiac disease,” Annals of Medicine, vol. 41, no. 5, pp. 390–400, 2009. [67] C. Gianfrani, R. A. Siciliano, A. M. Facchiano et al., “Transamidation of wheat flour inhibits the response to gliadin of intestinal T cells in celiac disease,” Gastroenterology, vol. 133, no. 3, pp. 780–789, 2007. [68] ∅. Molberg, S. McAdam, K. E. A. Lundin et al., “T cells from celiac disease lesions recognize gliadin epitopes deamidated in situ by endogenous tissue transglutaminase,” European Journal of Immunology, vol. 31, no. 5, pp. 1317–1323, 2001. [69] C. Esposito, I. Caputo, and R. Troncone, “New therapeutic strategies for coeliac disease: tissue transglutaminase as a target,” Current Medicinal Chemistry, vol. 14, no. 24, pp. 2572– 2580, 2007. [70] J. M. Wodzinska, “Transglutaminases as targets for pharmacological inhibition,” Mini-Reviews in Medicinal Chemistry, vol. 5, no. 3, pp. 279–292, 2005. SAGE-Hindawi Access to Research Enzyme Research Volume 2010, Article ID 918761, 8 pages doi:10.4061/2010/918761

Review Article Uses of Laccases in the Food Industry

Johann F. Osma,1 Jose´ L. Toca-Herrera,2 and Susana Rodrıguez-Couto´ 3, 4

1 Department of Electrical and Electronics Engineering, University of the Andes, Carrera 1 No. 18A-12, Bogota, Colombia 2 Department of Nanobiotechnology, University of Natural Resources and Applied Life Sciences (BOKU), Muthgasse 11, 1190 Vienna, Austria 3 Unit of Environmental Engineering, CEIT, Paseo Manuel de Lardizabal´ 15, 20018 San Sebastian,´ Spain 4 IKERBASQUE, Basque Foundation for Science, Alameda Urquijo 36, 48011 Bilbao, Spain

Correspondence should be addressed to Susana Rodr´ıguez-Couto, [email protected]

Received 15 June 2010; Accepted 22 August 2010

Academic Editor: Raﬀaele Porta

Copyright © 2010 Johann F. Osma et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Laccases are an interesting group of multi copper enzymes, which have received much attention of researchers in the last decades due to their ability to oxidise both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants. This makes these biocatalysts very useful for their application in several biotechnological processes, including the food industry. Thus, laccases hold great potential as food additives in food and beverage processing. Being energy-saving and biodegradable, laccase-based biocatalysts ﬁt well with the development of highly eﬃcient, sustainable, and eco-friendly industries.

1. Introduction with the addition of so-called “redox mediators”, which are low-weight molecular compounds that act as intermediate Laccases (p-diphenol:dioxygen oxidoreductases; EC 1.10.3.2) substrates for laccases, whose oxidised radical forms are able are particularly abundant in white-rot fungi, which are the to interact with the bulky or high redox potential substrate only organisms able to degrade the whole wood components targets (Figure 2(b)). [1]. Fungal laccases are secreted, glycosylated proteins with In nature, the role of laccases is to degrade lignin in order two disulphide bonds and four copper atoms distributed in to gain access to the other carbohydrates in wood (cellulose one mononuclear termed T1 (where the reducing substrate and hemicellulose). Their low substrate speciﬁcity allows place is) and one trinuclear cluster T2/T3 (where oxygen laccases to degrade compounds with a structure similar to binds and is reduced to water) [2]. Thus, electrons are lignin, such as polyaromatic hydrocarbons (PAHs), textile transferred from substrate molecules through the T1 copper dyes, and other xenobiotic compounds [2]. This together to the trinuclear T2/T3 centre. After the transfer of four with the simple requirements of laccase catalysis (presence of electrons, the dioxygen in the trinuclear centre is reduced to substrate and O2) makes laccases both suitable and attractive twomoleculesofwater[3, 4] (Figure 1). for industrial applications. From a mechanistic point of view, the reactions catalysed Typical fungal laccases are extracellular proteins of by laccases can be represented by one of the schemes approximately 60–70 kDa with acidic isoelectric point shown in Figure 2. The simplest case (Figure 2(a))is around pH 4.0 [5]. They are generally glycosylated, with an the one in which the substrate molecules are oxidised to extent of glycosylation ranging between 10 and 25% and the corresponding radicals by direct interaction with the only in a few cases higher than 30% [6, 7]. This feature may copper cluster. Frequently, however, the substrates of interest contribute to the high stability of the enzyme [8]. cannot be oxidised directly by laccases, either because they A few laccases are at present in the market for textile, are too large to penetrate into the enzyme active site or food and other industries (Table 1), and more candidates because they have a particularly high redox potential. By are being actively developed for future commercialisation mimicking nature, it is possible to overcome this limitation [9]. A vast amount of industrial applications for laccases 2 Enzyme Research

OH O• O reversible inhibition with sulphite [14]. Additionally, a O 2H O laccase has been commercialised for preparing cork stoppers 2 2 for wine bottles [15]. The enzyme oxidatively reduces 4 4 4 the characteristic cork taint and/or astringency, which is frequently imparted to aged bottled wine.

OH OH O 2.1.2. Beer Stabilisation. The storage life of beer depends on Figure 1: Reactions on phenolic compounds catalysed by laccases different factors such us haze formation, oxygen content, (extracted from [10]). and temperature. The former is produced by small quantities of naturally-occurring proanthocyanidins, polyphenols that generate protein precipitation and, therefore, the formation H2O Laccase (ox) Substrate(red) of haze [16]. This type of complex is commonly found as chill-haze and appears during cooling processes but may re- O Laccase 2 (red) Substrate(ox) dissolve at room temperature or above [12]. Even products (a) that are haze-free at the time of packing can develop this type of complex during long-term storage. Thus, the formation of H2O Laccase(ox) Mediator(red) Substrate(ox) haze has been a persistent problem in the brewing industry [17]. The use of laccases for the oxidation of polyphenols as O2 Laccase(red) Mediator(ox) Substrate(red) an alternative to the traditional treatment has been tested by (b) different authors [16, 18, 19]. However, laccases have also been used for the removal of oxygen at the end of the beer Figure 2: Schematic representation of laccase-catalysed redox cycles production process. According to Mathiasen [16], laccase for substrates oxidation in the absence (a) or in the presence (b) could be added at the end of the process in order to remove of redox mediators (extracted from [11], with kind permission of the unwanted oxygen in the finished beer, and thereby the Elsevier Ltd.) storage life of beer is enhanced. Also, a commercialised laccase preparation named “Flavourstar”, manufactured by Novozymes A/S, is marketed for using in brewing beer have been proposed which include pulp and paper, textile, to prevent the formation of off-flavour compounds (e.g., organic synthesis, environmental, food, pharmaceutical, and trans-2-nonenal) by scavenging the oxygen, which otherwise nano-biotechnology. Being energy-saving and biodegrad- would react with fatty acids, amino acids, proteins and able, laccase-based biocatalysts fit well with the development alcohol to form off-flavour precursors [20](Table1). of highly efficient, sustainable, and eco-friendly industries. This paper reviews the potential application of laccases in the food industry. The utilisation of whole laccase-producing 2.1.3. Fruit Juice Processing. Enzymatic preparations have microorganisms is not considered in the present paper. been studied since the decade of the 1930s for juice clarification [21]. The interaction between proteins and polyphenols results in the formation of haze or sediment in clear fruit 2. Application of Laccases in the Food Industry juices. Therefore, clear fruit juices are typically stabilised to delay the onset of protein-polyphenol haze formation [22]. Many laccase substrates, such as carbohydrates, unsaturated Several authors have proposed the use of laccase for the sta- fatty acids, phenols, and thiol-containing proteins, are bilisation of fruit juices [23–30]; however, results are contra- important components of various foods and beverages. dictory. On one hand, Sammartino et al. [24] compared the Their modification by laccase may lead to new functionality, treatment of apple juice with a conventional method (SO quality improvement, or cost reduction [12, 13]. 2 added as metabisulfite, polyvinylpolypyrrolidone (PVPP), bentonite) with the use of free and immobilised laccase. They 2.1. As Additives in Food and Beverage Processing. Laccases showed that the enzymatically treated juice was less stable can be applied to certain processes that enhance or modify than the one conventionally treated. Also, Giovanelli and the colour appearance of food or beverage. Ravasini [25]andGokmen¨ et al. [31] showed by stability tests of ultrafiltrated samples that laccase treatment increased 2.1.1. Wine Stabilisation. Wine stabilisation is one of the the susceptibility of browning during storage. On the other main applications of laccase in the food industry as alterna- hand, Cantarelli [30] used a mutant laccase from Polyporus tive to physical-chemical adsorbents [12]. Musts and wines versicolor to treat black grape juice. He showed a removal of are complex mixtures of different chemical compounds 50% of total polyphenols and higher stabilisation than the such as ethanol, organic acids (aroma), salts, and phenolic physical-chemical treatment. compounds (colour and taste). Polyphenol removal must The use of laccase in conjunction with a filtration be selective to avoid an undesirable alteration in the wine’s process has shown better results. Thus, Ritter et al. [27] organoleptic characteristics. Laccase presents some impor- and Maier et al. [29] obtained a stable and clear apple juice tant requirements when used for the treatment of polyphenol by applying laccase in conjunction with cross-flow-filtration removal in wines such as stability in acid medium and (ultrafiltration) in a continuous process without the addition Enzyme Research 3

Table 1: Commercial preparations based on laccases for industrial processes.

Main application Brand name Manufacturer Brewing Flavourstar Advanced Enzyme Technologies Ltd. (India) Food industry Colour enhancement in tea, etc. LACCASE Y120 Amano Enzyme USA Co. Ltd. Cork modification Suberase Novozymes (Denmark) Pulp bleaching Lignozym-process Lignozym GmbH (Germany) Paper industry Paper pulp delignification Novozym 51003 Novozymes (Denmark) Denim bleaching Bleach Cut 3-S Season Chemicals (China) Denim finishing Cololacc BB Colotex Biotechnology Co. Ltd. (Hong Kong) Denim bleaching DeniLite Novozymes (Denmark) Denim finishing Ecostone LC10 AB Enzymes GmbH (Germany) Textile Industry Denim finishing IndiStar Genencor Inc. (Rochester, USA) Denim finishing Novoprime Base 268 Novozymes (Denmark) Denim bleaching and shading Primagreen Ecofade LT100 Genencor Inc. (Rochester, USA) Denim bleaching ZyLite Zytex Pvt. Ltd. (India)

of finishing agents. Cantarelli and Giovanelli [28]reported hardening effect. Interestingly, laccase-treated flour dough that the use of laccase followed by “active” filtration or softened as a result of prolonged incubation: the extent ultrafiltration, by the addition of ascorbic acid and sulphites, of softening increasing as a function of laccase dosage. It improved colour and flavour stability in comparison to is proposed that softening phenomenon is due to radical conventional treatments. Also, Stutz [26] used laccase and catalysed breakdown of the cross-linked AX network. ultrafiltration to produce clear and stable juice concentrates Renzetti et al. [36] showed that a commercial laccase with a light colour. preparation significantly improved the bread-making perfor- ff Artik et al. [32] studied the e ect of laccase application mances of oat flour and the textural quality of oat bread on clarity stability of sour cherry juice. They found that high by increasing specific volume and lowering crumb hardness clarity was obtained by adding laccase in case of heating to ◦ and chewiness. The improved bread-making performances 50 C for 6 h and filtering through 20 kDa membrane after 1 h could be related to the increased softness, deformability and of oxidation. Also, the phenolic content decreased by around elasticity of oat batters with laccase supplementation. 70%. More recently, Neifar et al. [23] used a combined laccase- 2.1.5. Improving of Food Sensory Parameters. The physico- ultrafiltration process for controlling the haze formation and chemical deterioration of food products is a major problem browning of the pomegranate juice. The optimised treatment related to the evolution of storing and distribution systems with laccase (laccase concentration 5 U/mL; incubation ◦ and influences the consumer’s perception of the product time 300 min; incubation temperature 20 C) followed by quality. Thus, different uses of laccase have promoted ultrafiltration led to a clear and stable pomegranate juice. odour control, taste enhancement, or reduction of undesired products in several food products. 2.1.4. Baking. Laccases are currently of interest in baking due to their ability to cross-link biopolymers. The use of laccase Takemori et al. [37] used crude laccase from Coriolus in baking is reported to result in an increased strength, versicolor to improve the flavour and taste of cacao nib stability, and reduced stickiness and thereby improved and its products. Bitterness and other unpleasant tastes machinability of the dough; in addition, an increased volume were removed by the laccase treatment, and the chocolate and an improved crumb structure and softness of the baked manufactured from the cacao mass tasted better than the product were observed [33, 34]. control. Selinheimo et al. [35] showed that a laccase from the Another type of food products that may use laccase to white-rot fungus Trametes hirsuta increased the maximum improve sensory parameters is oil. Oil products may be resistance of dough and decreased the dough extensibility in deoxygenated by adding an effective amount of laccase [38]. both flour and gluten doughs. It was concluded that the effect Oils, especially vegetable oils (e.g., soybean oil), are present in of laccase was mainly due to the cross-linking of the esterified many food items such us dressings, salads, mayonnaise, and ferulic acid (FA) on the arabinoxylan (AX) fraction of dough other sauces. Soybean oil contains a large amount of linoleic resulting in a strong AX network. Gluten dough treated with and linolenic acids that can react with dissolved oxygen laccase also showed some hardening suggesting that laccase in the product producing undesirable volatile compounds. can also act to some extent on the gluten protein matrix. The Therefore, the flavour quality of some oils may be improved hardening effect of laccase was, however, clearly weaker in by eliminating the oxygen present in the oils. Other food gluten dough. Thus, the AX fraction in flour dough is the products (e.g., juices, soups, concentrates, puree, pastes, and predominant substrate for laccase, and its activity caused the sauces) can also be deoxygenated by the mean of laccase [39]. 4 Enzyme Research

Bouwensetal.[40, 41] reported that the colour of tea- inhibited the enzymatic activity, and, thus, the measurements based products could be enhanced when treated with laccase on musts and wines recently bottled were seriously affected. from a Pleurotus species. In the same way, chopped olives Di Fusco et al. [48] reported the development of an in an olive-water mixture were treated with laccase from amperometric biosensor based on laccases from T. versicolor Trametes villosa. In this case, the bitterness was considerably and T. hirsuta for the determination of polyphenol index reduced while the colour turned darker compared to the in wines. Enzymes were immobilised on carbon nanotubes controls (Novo Nordisk A/S, 1995). screen-printed electrodes using polyazetidine prepolymer Tsuchiya et al. [42] used a recombinant laccase from (PAP). They showed that biosensor performance depended Myceliophthora thermophilum and chlorogenic acid to con- on the laccase source. Thus, values obtained by using T. trol the malodour of cysteine. They showed that enzymat- hirsuta laccase were close to those determined by Folin- ically treated cysteine presented a very weak odour while Ciocalteu method whereas polyphenol index measured with the nontreated cysteine presented a strong characteristic H2S T. versicolor laccase was discordant to that found with the odour. HPLC analysis showed the reduction of more than reference assay. 50% of cysteine. Prasetyo et al. [49] studied the use of tetramethoxy azobismethylene quinone (TMAMQ) for measuring the 2.1.6. Sugar Beet Pectin Gelation. The sugar beet pectin is a antioxidant activity of a wide range of structurally diverse functional food ingredient that can form thermo-irreversible molecules present in food and humans. TMAMQ was gels. These types of gels are very interesting for the food generated by the oxidation of syringaldazine with laccases ff industry as can be heated while maintaining the gel structure. and used to detect the antioxidant activity present in di erent ff food products. Norsker et al. [43] analysed the gelling e ect of two Ibarra-Escutia et al. [50] developed and optimised an laccases and a peroxidase in food products. They found that ffi amperometric biosensor based on laccase from T. versicolor laccases were more e cient as gelling agents in luncheon for monitoring the phenolic compounds content in tea infu- meat and milk than peroxidase. In addition, in many sions. The biosensor developed showed an excellent stability countries it is prohibited to add hydrogen peroxide to food and exhibited good performance in terms of response time, products making it impossible to use peroxidases as gelling sensitivity, operational stability, and manufacturing process agents. Hence, it is more realistic to add laccase to food simplicity and can be used for accurate determination of the products. phenolic content without any pretreatment of the sample. Kuuva et al. [44] reported that by using laccases as cross- linking agents together with calcium, the ratio of covalent 2.3. Bioremediation of Food Industry Wastewater. The pres- and electrostatic cross-links of sugar beet pectin gels can be ence of phenols in agroindustrial effluents has attracted varied and it can be possible to tailor different types of gel interest for the application of laccase-based processes in structures. wastewater treatment and bioremediation. The presence of Littoz and McClements [45] showed that laccase could be phenolic compounds in drinking and irrigation water or in used to covalently cross-link beet pectin molecules adsorbed cultivated land represents a significant health and/or envi- to the surfaces of protein-coated lipid droplets at pH 4.5, ronmental hazard. With government policies on pollution thus suggesting that emulsions with improved functional control becoming more and more stringent, industries have performance could be prepared using a biomimetic approach been forced to look for more effective treatment technologies that utilised enzymes (laccases) to cross-link adsorbed for their wastewater. biopolymers. Some fraction of beer factory wastewater represents an important environmental concern due to its high content in polyphenols and dark brown colour. 2.2. Determination of Certain Compounds in Beverages. The Distillery wastewater is generated during ethanol pro- use of laccases for improving the sensing parameters of food duction from fermentation of sugarcane molasses (vinasses). products is not limited to treatment processes but also to It produces a serious ecological impact due to its high ff diagnosis systems. In this regard, di erent amperometric content in soluble organic matter and its intense dark brown biosensors based on laccases have been developed to measure colour. In fact, vinasses represent a major environmental ff polyphenols in di erent food products (e.g., wine, beer, and problem for the ethanol production industry and they are tea). Thus, Ghindilis et al. [46] showed the practical validity considered as the most aggressive by-product generated by of a biosensor based on immobilised laccase in analysing sugar-cane factories. Most of the organic matter present in ff tannin in tea of di erent brands. the vinasses can be diminished by conventional anaerobic- Montereali et al. [47] reported the detection of polyphe- aerobic digestion, but the colour is hardly removed by nols present in musts and wines from Imola (Italy) through these treatments [51] making this effluent a potential water an amperometric biosensor based on the utilisation of tyrosi- pollutant blocking out light from rivers and streams thereby nase and laccase from Trametes versicolor.Bothenzymeswere preventing oxygenation by photosynthesis and provoking immobilised on graphite screen-printed electrodes modi- their eutrophication. fied with ferrocene. Biosensors exhibited a good sampling Strong and Burgess [52] studied the fungal (Trametes behaviour compared to that obtained from spectropho- pubescens) and enzymatic (laccase from T. pubescens)reme- tometric analysis; however, the presence of SO2 clearly diation of different distillery wastewater and found that the Enzyme Research 5

Table 2: Some prices of commercially available laccases (extracted from [12], with kind permission of Elsevier Ltd).

Quantity (Units)a Price 10.000 305.00 (US$) From Agaricus bisporus 100.000 1.560.00 (US$) 10.000 250.00 (US$) From Coriolus versicolor 100.000 1.290.00 (US$) 10.000 (concentrate) 150.00 (US$) 10.000 (purified) 400.00 (US$) From Pleurotus ostreatus 100.000 (concentrate) 650.00 (US$) 100.000 (purified) 1,600.00 (US$) USBiological (www.usbio.net/) From heterologus expression of Trametes versicolor laccase in Saccharomyces cerevisiae 100 (purified) 169 (US$) Sigma-Aldrich From Rhus vernicfiera 10,000 72.30 (US$) From Agaricus bisporus (≥1.5 U/mg) 1 g 30.50 (US$) From Coriolus versicolor (≥1 U/mg) 5 g 120.90 (US$) 1 g 44.00 (US$) 10 g 358.20 (US$) Jena BioScience 100 U 15.00 (EUR) From Trametes versicolor, Coprinus cinereus and Pycnoporus cinnabarinus 1000 U 75.00 (EUR) aThe methodology and expression of laccase activity (Units) are different among the companies. fungal culture displayed much better properties than laccase usually present in OMW with oxidation percentages ranging alone in removing both the total phenolic compounds and from 60 to 100% after 24 h of laccase incubation. colour. D’Annibale et al. [63] used a laccase from the white-rot Olive mill wastewater (OMW) is a characteristic by- fungus Lentinula edodes immobilised on chitosan to treat product of olive oil production and a major environmental OMV from an olive oil mill located in Viterbo (Italy). They problem in the Mediterranean area. Thus, 30 million m3 of found that the treatment of the OMW with immobilised OMW is produced in the Mediterranean area [53]which laccase led to a partial decolouration as well as to significant generate 2.5 litres of waste per litre of oil produced [54]. abatements in its content in polyphenols, and orthodiphe- OMW contains large concentrations of phenol compounds nols combined with a decreased toxicity of the effluent. They (up to 10 g/L) [54, 55], which are highly toxic [52, 56]. Also, also showed that an oxirane-immobilised laccase from L. it has high chemical and biochemical oxygen demands (COD edodes efficiently removed the OMW phenolics [64]. and BOD, resp.) [57]. Casa et al. [65] investigated the potential of a laccase OMW is characterised by a colour variable from dark from L. edodes in removing OMW phytotoxicity. For this, red to black depending on the age and type of olive they performed germinability experiments on durum wheat processed [58], low pH value (∼5), high salt content and (Triticum durum) in the presence of different dilutions of high organic load with elevated concentrations of aromatic raw or laccase-treated OMW. The treatment with laccase compounds [59], fatty acids, pectins, sugar, tannins and resulted in a 65% and an 86% reduction in total phenols phenolic compounds, in particular polyphenols [58]. The and orthodiphenols, respectively, due to their polymerisation presence of a large number of compounds, many with as revealed by size-exclusion chromatography. In addition, polluting, phytotoxic, and antimicrobial properties [60], germinability of durum wheat seeds was increased by 57% at renders OMW a waste with high harmful effects towards a 1 : 8 dilution and by 94% at a 1 : 2 dilution, as compared to humans and environment and makes its disposal one of the the same dilutions using untreated OMW. main environmental concerns in all producing countries. Attanasio et al. [66] studied the application of a non- Martirani et al. [61] reported that the treatment of an isothermal bioreactor with laccases from T. versicolor immo- OMW effluent collected at an olive oil factory in Abruzzo bilised on a nylon membrane to detoxify OMW and showed (Italy) with a purified laccase from Pleurotus ostreatus that the technology of non-isothermal bioreactors was very significantly decreased its phenolic content (up to 90%) but useful in the treatment of OMW. no reduction of its toxicity was observed when tested on Jaouani et al. [67] studied the role of a purified laccase Bacillus cereus. from Pycnoporus coccineus in the degradation of aromatic Gianfreda et al. [62] showed that laccase from Cerrena compounds in OMW. They found that the treatment of unicolor was able to oxidise different phenolic substances OMW with laccase showed similar results to those reported 6 Enzyme Research with the fungus indicating that laccase plays an important different basidiomycetes,” Biochimie, vol. 86, no. 9-10, pp. role in the degradative process. Berrio et al. [68] studied 693–703, 2004. the treatment of OMW with a laccase from P. coccineus [7]C.G.M.DeSouzaandR.M.Peralta,“Purificationand immobilised on Eupergit C 250L. Gel filtration profiles of the characterization of the main laccase produced by the white- OMW treated with the immobilised enzyme (for 8 h at room rot fungus Pleurotus pulmonarius on wheat bran solid state temperature) showed both degradation and polymerisation medium,” Journal of Basic Microbiology, vol. 43, no. 4, pp. 278– 286, 2003. of the phenolic compounds. [8] N. Duran,´ M. A. Rosa, A. D’Annibale, and L. Gianfreda, Quaratino et al. [69] reported that phenols were the main “Applications of laccases and tyrosinases (phenoloxidases) determinants for OMW phytotoxicity and showed that the immobilized on different supports: a review,” Enzyme and use of a commercial laccase preparation (DeniLite, Novo Microbial Technology, vol. 31, no. 7, pp. 907–931, 2002. Nordisk, Denmark) might be very promising for a safer [9] A. Kunamneni, F. J. Plou, A. Ballesteros, and M. Alcalde, agronomic use of the wastewater. “Laccases and their applications: a patent review,” Recent Iamarino et al. [70] studied the capability of a laccase Patents on Biotechnology, vol. 2, no. 1, pp. 10–24, 2008. from Rhus vernicifera to degrade and detoxify two OMW [10] E. Selinheimo, Tyrosinase and laccase as novel crosslinking samples of different complexity and composition. tools for food biopolymers, Ph.D. thesis, Helsinki University of Pant and Adholeya [71] used a concentrated enzymatic Technology, Helsinki, Finland, 2008. extract from solid-state fermentation (SSF) cultures of [11] S. Riva, “Laccases: blue enzymes for green chemistry,” Trends different fungi on wheat straw to decolourise a distillery in Biotechnology, vol. 24, no. 5, pp. 219–226, 2006. [12] R. C. Minussi, G. M. Pastore, and N. Duran,´ “Potential effluent. They reported a maximum decolouration of 37% in ffl applications of laccase in the food industry,” Trends in Food the undiluted distillery e uent using the extract of Pleurotus Science and Technology, vol. 13, no. 6-7, pp. 205–216, 2002. florida EM1303 which was attributed to its high laccase [13] O. Kirk, T. V.Borchert, and C. C. Fuglsang, “Industrial enzyme production. applications,” Current Opinion in Biotechnology, vol. 13, no. 4, pp. 345–351, 2002. [14] D. Tanrioven¨ and A. Eks¸i, “Phenolic compounds in pear juice 3. Future Trends and Perspectives from different cultivars,” Food Chemistry, vol. 93, no. 1, pp. 89–93, 2005. This paper shows that laccase has a great potential applica- [15] L. S. Conrad, W. R. Sponholz, and O. Berker, “Treatment of tion in several areas of food industry. However, one of the cork with a phenol oxidizing enzyme,” US patent 6152966, limitations to the large-scale application of laccases is the lack 2000. of capacity to produce large volumes of highly active enzyme [16] T. E. Mathiasen, “Laccase and beer storage,” PCT international at an affordable cost (Table 2). The use of inexpensive sources application, WO 9521240 A2, 1995. for laccase production is being explored in recent times. In [17] I. McMurrough, D. Madigan, R. Kelly, and T. O’Rourke, this regard, an emerging field in management of industrial “Haze formation shelf-life prediction for lager beer,” Food wastewater is exploiting its nutritive potential for production Technology, vol. 53, no. 1, pp. 58–63, 1999. of laccase enzymes. Besides solid wastes, wastewater from the [18] G. Giovanelli, “Enzymic treatment of malt polyphenols for food processing industry is particularly promising for that. beer stabilization,” Industrie delle Bevande, vol. 18, pp. 497– 502, 1989. [19] M. Rossi, G. Giovanelli, C. Cantarelli, and O. Brenna, “Effects Acknowledgment of laccase and other enzymes on barley wort phenolics as a possible treatment to prevent haze in beer,” Bulletin de Liaison- This paper was financed by the Spanish Ministry of Science Groupe Polyphenols, vol. 14, pp. 85–88, 1988. and Innovation (Project CTM2008-02453/TECNO). [20] FAO, “Laccase from Myceliophthora thermophila expressed in Aspergillus oryzae,” Chemical and Technical Assessment (Cta), 2004. References [21] R. C. Minussi, M. Rossi, L. Bologna, D. Rotilio, G. M. Pastore, and N. Duran,´ “Phenols removal in musts: strategy for wine [1] T. K. Kirk and P. Fenn, Formation and Action of the Ligni- stabilization by laccase,” Journal of Molecular Catalysis B, vol. nolytic System in Basidiomycetes: Their Biology and Ecology, 45, no. 3-4, pp. 102–107, 2007. Cambridge University Press, Cambridge, UK, 1982. [22] K. J. Siebert, “Protein-polyphenol haze in beverages,” Food [2] A. M. Mayer and R. C. Staples, “Laccase: new functions for an Technology, vol. 53, no. 1, pp. 54–57, 1999. old enzyme,” Phytochemistry, vol. 60, no. 6, pp. 551–565, 2002. [23] M. Neifar, R. Elleouze-Ghorbel, A. Kamoun, et al., “Effective [3] T. Bertrand, C. Jolivalt, P. Briozzo et al., “Crystal structure of clarification of pomegranate juice using laccase treatment a four-copper laccase complexed with an arylamine: insights optimized by response surface methodology followed by into substrate recognition and correlation with kinetics,” ultrafiltration,” Journal of Food Process Engineering. In press. Biochemistry, vol. 41, no. 23, pp. 7325–7333, 2002. [24] M. Sammartino, P. Piacquadio, G. De Stefano, and V. [4]I.Bento,M.Armenia´ Carrondo, and P. F. Lindley, “Reduction Sciancalepore, “Apple juice stabilization by conventional and of dioxygen by enzymes containing copper,” Journal of Biolog- innovative methods,” Industrie delle Bevande, vol. 27, pp. 367– ical Inorganic Chemistry, vol. 11, no. 5, pp. 539–547, 2006. 369, 1998. [5] P. Baldrian, “Fungal laccases-occurrence and properties,” [25] G. Giovanelli and G. Ravasini, “Apple juice stabiliza- FEMS Microbiology Reviews, vol. 30, no. 2, pp. 215–242, 2006. tion by combined enzyme-membrane filtration process,” [6] S. V.Shleev, O. V.Morozova, O. V.Nikitina et al., “Comparison Lebensmittel-Wissenschaft und Technologie,vol.26,no.1,pp. of physico-chemical characteristics of four laccases from 1–7, 1993. Enzyme Research 7

[26] C. Stutz, “The use of enzymes in ultrafiltration,” Fruit pectin layers using laccase,” Food Hydrocolloids, vol. 22, no. 7, Processing, vol. 3, pp. 248–252, 1993. pp. 1203–1211, 2008. [27] G. Ritter, G. Maier, E. Schoepplein, and H. Dietrich, “The [46] A. L. Ghindilis, V. P. Gavrilova, and A. I. Yaropolov, “Laccase- application of polyphenoloxidase in the processing of apple based biosensor for determination of polyphenols: determi- juice,” Bulletin de Liaison-Groupe Polyphenols, vol. 16, pp. 209– nation of catechols in tea,” Biosensors and Bioelectronics, vol. 7, 212, 1992. no. 2, pp. 127–131, 1992. [28] C. Cantarelli and G. Giovanelli, “Stabilization of pome [47] M. R. Montereali, L. Della Seta, W. Vastarella, and R. and grape juice against phenolic deterioration by enzymic Pilloton, “A disposable Laccase-Tyrosinase based biosensor for treatments,” Internationale Fruchtsaft-Union, Wissenschaftlich- amperometric detection of phenolic compounds in must and Technische Commission, vol. 21, pp. 35–57, 1990. wine,” Journal of Molecular Catalysis B,vol.64,no.3-4,pp. [29] G. Maier, H. Dietrich, and K. Wucherpfenning, “Winemak- 189–194, 2010. ing without SO2-with the aid of enzymes?” Weineirtschaft- [48] M. Di Fusco, C. Tortolini, D. Deriu, and F. Mazzei, “Laccase- Technik, vol. 126, pp. 18–22, 1990. based biosensor for the determination of polyphenol index in [30] C. Cantarelli, “Trattamenti enzimatici sui costituenti fenolici wine,” Talanta, vol. 81, no. 1-2, pp. 235–240, 2010. dei mosti come prevenzione della maderizzazione,” Vini [49] E. N. Prasetyo, T. Kudanga, W. Steiner, M. Murkovic, G. S. d’Italia, vol. 3, pp. 87–98, 1986. Nyanhongo, and G. M. Guebitz, “Laccase-generated tetram- [31] V. Gokmen,¨ Z. Borneman, and H. H. Nijhuis, “Improved ethoxy azobismethylene quinone (TMAMQ) as a tool for ultrafiltration for color reduction and stabilization of apple antioxidant activity measurement,” Food Chemistry, vol. 118, juice,” Journal of Food Science, vol. 63, no. 3, pp. 504–507, 1998. no. 2, pp. 437–444, 2010. [32] N. Artik, M. Karhan, and G. Aydar, “Effects of polyphenoloxi- [50]P.Ibarra-Escutia,J.JuarezGomez,´ C. Calas-Blanchard, J. L. dase (LACCASE) application on clarity stability of sour cherry Marty,andM.T.Ram´ırez-Silva, “Amperometric biosensor juice,” Journal of Food Technology, vol. 2, no. 4, pp. 237–243, based on a high resolution photopolymer deposited onto a 2004. screen-printed electrode for phenolic compounds monitoring [33] E. Labat, M. H. Morel, and X. Rouau, “Effects of laccase and in tea infusions,” Talanta, vol. 81, no. 4-5, pp. 1636–1642, ferulic acid on wheat flour doughs,” Cereal Chemistry, vol. 77, 2010. no. 6, pp. 823–828, 2000. [51] E. Valdez and M. C. Obaya, “Estudio del tratamiento anaer- [34] J. Q. Si, “Use of laccase in baking industry,” International obico de los residuales de la industria alcoholera,” Revista patent application PCT/DK94/00232, 1994. ICIDCA, vol. 19, no. 1, pp. 7–10, 1985. [52] P. J. Strong and J. E. Burgess, “Treatment methods for wine- [35] E. Selinheimo, K. Kruus, J. Buchert, A. Hopia, and K. Autio, related and distillery wastewaters: a review,” Bioremediation “Effects of laccase, xylanase and their combination on the Journal, vol. 12, no. 2, pp. 70–87, 2008. rheological properties of wheat doughs,” Journal of Cereal [53] J. A. Fiestas Ros de Ursinos, “Differentes utilisations des Science, vol. 43, no. 2, pp. 152–159, 2006. margines,” in Proceedings of the Seminaire International sur la [36] S. Renzetti, C. M. Courtin, J. A. Delcour, and E. K. Arendt, Valorisation des Sous Produits de l’Olivier, pp. 93–110, Organi- “Oxidative and proteolytic enzyme preparations as promising sation des Nations Unies pour l’Alimentation et l’Agriculture, improvers for oat bread formulations: rheological, biochem- Monastir, Tunisia, 1981. ical and microstructural background,” Food Chemistry, vol. [54] R. Borja, A. Martin, R. Maestro, J. Alba, and J. A. Fies- 119, no. 4, pp. 1465–1473, 2010. tas, “Enhancement of the anaerobic digestion of olive mill [37] T. Takemori, Y. Ito, M. Ito, and M. Yoshama, “Flavor and taste wastewater by the removal of phenolic inhibitors,” Process improvement of cacao nib by enzymatic treatment,” Japan Biochemistry, vol. 27, no. 4, pp. 231–237, 1992. Kokai Tokkyo Koho JP 04126037 A2, 1992. [55] A. M. Klibanov, T. M. Tu, and K. P. Scott, “Peroxidase- [38] B. R. Petersen, T. E. Mathiasen, B. Peelen, and H. Andersen, catalyzed removal of phenols from coal-conversion waste “Use of laccase for deoxygenation of oil-containing product waters,” Science, vol. 221, no. 4607, pp. 259–261, 1983. such as salad dressing,” PCT international application, WO [56] P. M. Fedorak and S. E. Hrudey, “The effects of phenol and 9635768 A1, 1996. some alkyl phenolics on batch anaerobic methanogenesis,” [39] B. R. Petersen and T. E. Mathiasen, “Deoxygenation of a Water Research, vol. 18, no. 3, pp. 361–367, 1984. food item using a laccase,” PCT international application, WO [57] M. Hamdi, “Future prospects and constraints of olive mill 9631133 A1, 1996. wastewaters use and treatment: a review,” Bioprocess Engineer- [40] E. M. Bouwens, K. Trivedi, C. Van Vliet, and C. Winkel, ing, vol. 8, no. 5-6, pp. 209–214, 1993. ff “Method of enhancing color in a tea based foodstu ,” US [58] A. Jaouani, S. Sayadi, M. Vanthournhout, and M. J. Penninckx, 5879730 A, 1999. “Potent fungi for decolourisation of olive oil mill wastewaters,” [41] E. M. Bouwens, K. Trivedi, C. Van Vliet, and C. Winkel, Enzyme and Microbial Technology, vol. 33, no. 6, pp. 802–809, “Method of enhancing color in a tea based foodstuff,” 2003. European patent application; EP 760213 A1, 1997. [59] A. D’Annibale, R. Casa, F. Pieruccetti, M. Ricci, and R. [42] R. Tsuchiya, B. R. Petersen, and S. Christensen, “Oxidoreduc- Marabottini, “Lentinula edodes removes phenols from olive- tases for reduction of malodor,” US 6074631 A, 2000. mill wastewater: impact on durum wheat (Triticum durum [43] M. Norsker, M. Jensen, and J. Adler-Nissen, “Enzymatic gela- Desf.) germinability,” Chemosphere, vol. 54, no. 7, pp. 887– tion of sugar beet pectin in food products,” Food Hydrocolloids, 894, 2004. vol. 14, no. 3, pp. 237–243, 2000. [60]C.Paredes,J.Cegarra,A.Roig,M.A.Sanchez-Monedero,´ [44] T. Kuuva, R. Lantto, T. Reinikainen, J. Buchert, and K. Autio, and M. P. Bernal, “Characterization of olive mill wastewater “Rheological properties of laccase-induced sugar beet pectin (alpechin) and its sludge for agricultural purposes,” Biore- gels,” Food Hydrocolloids, vol. 17, no. 5, pp. 679–684, 2003. source Technology, vol. 67, no. 2, pp. 111–115, 1999. [45] F. Littoz and D. J. McClements, “Bio-mimetic approach to [61] L. Martirani, P. Giardina, L. Marzullo, and G. Sannia, improving emulsion stability: cross-linking adsorbed beet “Reduction of phenol content and toxicity in olive oil mill 8 Enzyme Research

waste waters with the ligninolytic fungus Pleurotus ostreatus,” Water Research, vol. 30, no. 8, pp. 1914–1918, 1996. [62] L. Gianfreda, F. Sannino, M. T. Filazzola, and A. Leonowicz, “Catalytic behavior and detoxifying ability of a laccase from the fungal strain Cerrena unicolor,” Journal of Molecular Catalysis B, vol. 4, no. 1-2, pp. 13–23, 1998. [63] A. D’Annibale, S. R. Stazi, V. Vinciguerra, E. Di Mattia, and G. Giovannozzi Sermanni, “Characterization of immobilized laccase from Lentinula edodes and its use in olive-mill wastewater treatment,” Process Biochemistry, vol. 34, no. 6-7, pp. 697–706, 1999. [64] A. D’Annibale, S. R. Stazi, V. Vinciguerra, and G. Giovannozzi Sermanni, “Oxirane-immobilized Lentinula edodes laccase: stability and phenolics removal efficiency in olive mill wastewater,” Journal of Biotechnology, vol. 77, no. 2-3, pp. 265–273, 2000. [65] R. Casa, A. D’Annibale, F. Pieruccetti, S. R. Stazi, G. G. Sermanni, and B. Lo Cascio, “Reduction of the phenolic components in olive-mill wastewater by an enzymatic treatment and its impact on durum wheat (Triticum durum Desf.) germinability,” Chemosphere, vol. 50, no. 8, pp. 959–966, 2003. [66] A. Attanasio, N. Diano, V. Grano et al., “Nonisothermal bioreactors in the treatment of vegetation waters from olive oil: laccase versus syringic acid as bioremediation model,” Biotechnology Progress, vol. 21, no. 3, pp. 806–815, 2005. [67] A. Jaouani, F. Guillen,´ M. J. Penninckx, A. T. Mart´ınez, andM.J.Mart´ınez, “Role of Pycnoporus coccineus laccase in the degradation of aromatic compounds in olive oil mill wastewater,” Enzyme and Microbial Technology, vol. 36, no. 4, pp. 478–486, 2005. [68] J. Berrio, F. J. Plou, A. Ballesteros, A.´ T. Mart´ınez, and M. J. Mart´ınez, “Immobilization of Pycnoporus coccineus laccase on Eupergit C: stabilization and treatment of olive oil mill wastewaters,” Biocatalysis and Biotransformation, vol. 25, no. 2–4, pp. 130–134, 2007. [69] D. Quaratino, A. D’Annibale, F. Federici, C. F. Cereti, F. Rossini, and M. Fenice, “Enzyme and fungal treatments and a combination thereof reduce olive mill wastewater phytotoxicity on Zea mays L. seeds,” Chemosphere, vol. 66, no. 9, pp. 1627–1633, 2007. [70] G. Iamarino, M. A. Rao, and L. Gianfreda, “Dephenolization and detoxification of olive-mill wastewater (OMW) by purified biotic and abiotic oxidative catalysts,” Chemosphere, vol. 74, no. 2, pp. 216–223, 2009. [71] D. Pant and A. Adholeya, “Concentration of fungal ligninolytic enzymes by ultrafiltration and their use in distillery effluent decolorization,” World Journal of Microbiology and Biotechnology, vol. 25, no. 10, pp. 1793–1800, 2009. SAGE-Hindawi Access to Research Enzyme Research Volume 2010, Article ID 149748, 10 pages doi:10.4061/2010/149748

Review Article Fungal Laccases: Production, Function, and Applications in Food Processing

Khushal Brijwani, Anne Rigdon, and Praveen V. Vadlani

Bioprocessing Laboratory, Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506, USA

Correspondence should be addressed to Praveen V. Vadlani, [email protected]

Received 1 July 2010; Accepted 22 August 2010

Academic Editor: Raﬀaele Porta

Copyright © 2010 Khushal Brijwani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Laccases are increasingly being used in food industry for production of cost-effective and healthy foods. To sustain this trend widespread availability of laccase and efficient production systems have to be developed. The present paper delineate the recent developments that have taken place in understanding the role of laccase action, efforts in overexpression of laccase in heterologous systems, and various cultivation techniques that have been developed to efficiently produce laccase at the industrial scale. The role of laccase in different food industries, particularly the recent developments in laccase application for food processing, is discussed.

1. Introduction 450 mV (Myceliophthora thermophila), 750 mV (Pycnoporus cinnabarinus), and 780 mV (Botrytis cinerea)[7]. Laccases, Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) is therefore, possess excellent potential to be used as processing apartofbroadgroupofenzymescalledpolyphenoloxidases aids for the food industry. containing copper atoms in the catalytic center and are The successful application of laccases in food processing usually called multicopper oxidases. Laccases contain three would require production of high amounts at reduced types of copper atoms, one of which is responsible for their costs. Several production strategies can be adopted along characteristic blue color. The enzymes lacking a blue copper with media and process optimization to achieve better atom are called yellow or white laccases. Typically laccase- process economics. Concomitantly, overexpression of laccase mediated catalysis occurs with reduction of oxygen to water in suitable host organisms would provide means to achieve accompanied by the oxidation of substrate. Laccases are thus high titers. Use of inducers could also enhance production oxidases that oxidize polyphenols, methoxy-substituted phe- capabilities [8]. nols, aromatic diamines, and a range of other compounds The main objective of this paper is to summarize the [1]. wealth of information available in the literature with regard Laccases are widely distributed in higher plants and fungi to laccase mechanism, production, and overexpression and [2] and have also been found in insects and bacteria [3]. eventually its role in food processing. Laccase has several Laccases are distributed in Ascomycetes, Deuteromycetes, food-based applications including bioremediation, beverage and Basidiomycetes, being particularly abundant in many (fruit juice, wine and beer) stabilization, uses in baking white rot fungi that are involved in lignin metabolism industry, and role in improvement of overall food quality. [4, 5]. Owing to the higher redox potential (+800 mV) The versatility of laccase in its action and its wide occurrence of fungal laccases compared to plants or bacterial laccases in several species of fungi contribute to the easy applicability they are implicated in several biotechnological applications in biotechnological processes. The present review, therefore, especially in the degradation of lignin [6]. For instance, should help to shed light on the general characteristics of redox potentials of laccases from common laccase pro- laccaseinaneﬀort to create a database that could aid usage ducing fungi are reported as 790 mV (Trametes villosa), of laccase in the food processing industry. 2 Enzyme Research

2. Mechanism of Laccase Action laccase was not demonstrated in ascomycetous yeasts, but the plasma membrane bound multicopper oxidase Fet3p from The catalysis of laccase occurs with reduction of one Saccharomyces cerevisiae shows both sequence and structural molecule of oxygen to water accompanied with one electron homology with fungal laccase [32, 33]. oxidation of a wide range of aromatic compounds which Wood rotting basidiomycetes causing white rot and a includes polyphenols [9], methoxy-substituted monophe- related group of litter decomposing saprotrophic fungi are nols, and aromatic amines [4]. This oxidation results in the most widely known species that produce appreciable generation of oxygen-centered free radical that can be quantity of laccase. Almost all species of white rot fungi converted to quinone in a second enzyme catalyzed reaction. were reported to produce laccase to varying degree [34]. Laccase catalysis occurs in three steps: (1) type I Cu reduction In case of Pycnoporus cinnabarinus laccase was described as by substrate; (2) electron transfer from type I Cu to the type the only ligninolytic enzyme produced by this species that II Cu and type III Cu trinuclear cluster; (3) reduction of was capable of lignin degradation [35]. Brown-rot fungi on oxygen to water at the trinuclear cluster [10]. the other hand are not known, in general, to carry laccase The laccase mediated catalysis can be extended to non- production capabilities. A DNA sequence with relatively high phenolic substrates by the inclusion of mediators. Mediators similarity to that of laccase was detected in Gloeophyllum are a group of low molecular weight organic compounds that trabeum that was capable of oxidizing ABTS [36]. Though can be oxidized by laccase ﬁrst forming highly active cation no laccase protein has been puriﬁed from brown-rot species, radicals capable of oxidizing nonphenolic compounds that the oxidation of syringaldazine has recently been detected laccase alone cannot oxidize (Figure 1). The most common in the brown-rot fungus Coniophora puteana [37] and the synthetic mediators are 1-hydro-xybenzotriazole (HOBT), oxidation of ABTS was reported in Laetiporus sulphureus N-hydro-xyphthalimide (NHPI), and 2,2 -azinobis-3-ethyl- [38]. thiazoline-6-sulfonat (ABTS) [11].

3. Occurrence of Laccase in Fungal Systems 4. Overexpression of Laccase Laccase activity has been demonstrated in several fungal Due to the ability of fungal laccases to oxidize phenolic species leading to the notion that most of all fungi produce and nonphenolic aromatic compounds, increased interest laccase. This, however, should not be generalized as there in the application of these enzymes for various industrial are several physiological groups of fungi that apparently applications, including food, pulping, textile, wastewater do not produce laccase. Laccase production has never treatment, and bioremediation, is growing greatly [8]. To been demonstrated in lower fungi, that is, Zygomycetes successfully utilize laccases in these applications, production and Chytridiomycetes [12]. Several reports can be referred, of large quantities at a low cost is essential. in the literature on production of laccase in ascomycetes To make laccases available for industrial applications, such as Gaeumannomyces graminis [13], Magnaporthe grisea methods to reduce costs include fermentation media opti- [14], and Ophiostoma novo-ulmi [15], Mauginella [16], mization, novel fermentation methods, and genetic mod- Melanocarpus albomyces [17], Monocillium indicum [18], ification for large scale production via eukaryotic recom- Neurospora crassa [19], and Podospora anserina [20]. In binant strains. Much research has been done to identify addition to plant pathogenic species, laccase production effective methods for mass production of laccase using was also reported for some soil ascomycete species from the the above mentioned methods. Determination of optimum genera Aspergillus, Curvularia and Penicillium [21–23], and fermentation media can easily be achieved but cofactors and in some freshwater ascomycetes [24, 25]. inducer compounds can cause an undesirable increase in Wood degrading ascomycetes like Trichoderma and cost during growth at industrial scale. Novel fermentation Botryosphaeria have been shown to have some laccase methods can also cause undesirable increases to cost due to activity. While Botryosphaeria produces constitutively a modifications to preexisting facilities. Genetic modification dimethoxyphenol oxidizing enzyme that is probably true presents a promising method of overexpression of laccase laccase [26] there are only some strains of Trichoderma for large applications. However, fungal laccases require that exhibit low level production of a syringaldazine oxi- posttranslational modifications (glycosylation), which only dizing enzyme [27]. In case of wood rotting xylariaceous eukaryotic microorganisms are capable of carrying out cre- ascomycetes, two strains of Xylaria sp. and one of Xylaria ating limitations for genetic manipulation for overexpression hypoxylon exhibited syringaldazine oxidation [28]. In com- of laccase. Laccase genes have been successfully cloned and plex liquid media, the fungi X. hypoxylon and Xylaria heterologously expressed in the filamentous fungi Aspergillus polymorpha produced appreciable titers of an ABTS oxidiz- niger, Aspergillus oryzae,andTrichoderma reesei [8]. Only a ing enzyme [29]. Furthermore, ascomycete species closely few bacterial laccases have been thoroughly studied to reveal related to wood-degrading fungi which participate in the industrial advantages over fungal laccases. Bacterial laccases decay of dead plant biomass in salt marshes have been shown have been found to be highly active and have higher stability to contain laccase genes and to oxidize syringaldazine [30]. at higher temperatures and pH values compared to fungal Basidiomycete yeast like Cryptococcus neoformans produces a laccases [39]. Laccase-like enzymes isolated from bacterial true laccase capable of oxidation of phenols and aminophe- cultures have been found to be very similar to fungal laccases; nols and is unable to oxidize tyrosine [31]. The production of however, they vary in activity [39]. Enzyme Research 3

Laccase Phenolic substrate Oxidized phenolic substrate

O2 H2O

Non-phenolic substrate Oxidized Non-phenolic substrate

Oxidized mediator Mediator

Laccase

H2O

Mediator Figure 1: Mechanism of laccase action for both phenolic and nonphenolic substrates.

Research by [40] focused on optimizing media con- molecular weight of the recombinant enzyme matched the ditions using multiple micronutrients for maximum pro- native laccase produce by T. hirsuta [41]. Further research is duction of laccase by a previously identified fungal strain still needed to ensure high laccase production using large- belonging to the genus Gandoderma and referred to as scale fermentation methods. WR-1.StrainWR-1, a white-rot fungus, was isolated from Additionally, research by [53] focused on transforming tree bark using tissue culture techniques and was found to laccase genes from Trametes versicolor into the methyltrophic produce high amounts of laccase during fermentation. WR- yeast Pichia pastoris for heterologous expression. The P. 1 was naturally found to show laccase activity of 124 U/ml, pastoris expression system is commonly used to achieve high compared to typical strains which show activity in a range expression levels of heterologous proteins. This yeast has of 4−100 U/ml. The experimental design for determining been found to achieve high cell densities during growth in the optimum media conditions included the use of the a minimal media in a short period of time. Furthermore, orthogonal matrix method. This allowed for the statistical P. pastoris has been found to be an efficient secretion evaluation of the relative importance of various nutrients system and capable of posttranslational modifications (e.g., for the highest production of laccase using submerged fer- glycosylation). After successful transformation of the P. mentation methods. It was concluded that WR-1 produced pastoris expression system, it was found that utilizing a solid- increasing amounts of laccase when grown in a starch-based state fermentation (SSF) method produced similar laccase medium with the addition of copper sulphate and 2, 5- production results compared to submerged fermentation xylidine, as a laccase production inducer. WR-1 was able to (SmF) methods [53]. increase laccase production to 692 U/ml during fermentation Recently, a few bacterial laccases have been isolated from in the optimized media, a significant increase compared to Escherichia coli, Bacillus halodurans, Thermus thermophilus, other strains under similar fermentation conditions [40]. and several species of Streptomycetes. Little is known about To help increase laccase production, research has focused the function of laccases in bacterial physiology but they are on using recombinant fungal strains for maximum produc- believed to play a role in melanin production, spore coat tion. Research by [41] successfully transferred laccase genes resistance, morphogenesis, and detoxification of copper [54]. from the basidiomycete Tramete hirsuta into the ascomycete The bacterial laccase CotA isolated from Bacillus subtilis was Penicillium canescens for heterologous expression. The fungal found to be an endospore coat protein with high thermosta- strain from the genus Penicillium was chosen due to its ability bility [55]. Utilizing bacterial laccases for industrial pro- to secrete large amounts of enzyme into culture media and it duction would allow for new biotechnological applications has been demonstrated that synthesized enzymes are safe for due to the ease of genetic improvements to expression level, human consumption. After successful transformation, it was activity, and selectivity [56]. The combination of random found that 98% of the target enzyme activity was detectable and site-directed mutagenesis was used to produce a double in the liquid culture medium. It was also found that the mutant with improved functional expression in E. coli and 4 Enzyme Research improved specific activity for different dyes [56]. Wu et 8. Influence of pH and Temperature on al. [39]isolatedanewstrainofAeromonas hydrophilia Laccase Production designated WL-11 from activated sludge in an effluent treatment plant of a textile and dyeing industry. The gene The information on effect of pH and temperature effects encoding laccase was cloned from the newly isolated strain on laccase production is scarce, but most reports indicate and successfully expressed in E. coli BL21(DE3). The recom- initial pH between 4.5 and 6.0 that is suitable for enzyme binant strain produced a high level of laccase compared to production [6]. The optimum temperature for laccase ◦ ◦ the wild type. The recombinant laccase was characterized production is between 25 Cand30 C[69]. When fungi were ◦ and could be used as a biocatalyst in biotechnological cultivated at temperatures higher than 30 C the activity of applications requiring large quantities of laccase [39]. enzyme was reduced [70].

5. Production Systems for Laccase 9. Type of Cultivation Laccases are extracellular enzymes secreted into the medium Laccases have been produced vividly in both submerged by filamentous fungi [57]. Laccases are generally produced and solid state modes of fermentation. Table 1 lists the during the secondary metabolism of different fungi. Several different cultivation techniques that have been adopted for factors including type of cultivation (submerged or solid large-scale production of laccase using wild-type filamentous state), carbon limitation, nitrogen source, and concentration fungi. In forthcoming sections, important features of laccase of microelements can influence laccase production [58]. production into two modes, submerged and solid, state will Subsequent sections delineate the role of different process be discussed. parameters in laccase production. 10. Submerged Fermentation 6. Influence of Carbon and Nitrogen Source on Laccase Production Submerged fermentation involves the cultivation of microorganisms in liquid medium containing appropriate nutrients The excessive concentrations of glucose are inhibitory to with high oxygen concentrations when operated in aerobic laccase production in various fungal strains [37]. An excess conditions. One of the major challenges in fungal submerged of sucrose also reduced the production of laccase by blocking fermentations is viscosity of broth. Mycelium formation its induction and only allowed constitutive production of during growth of fungal cells can also impede impeller action enzyme. Use of polymeric substrates like cellulose was able causing blockades resulting in oxygen and mass transfer to alleviate this problem [37]. Fungal laccases are often limitations. Different strategies have been employed to deal triggered by nitrogen depletion [59], but it was also found with oxygen and mass transfer limitations. A pulsed system ff that in some strains nitrogen had no e ect on enzyme activity developed by [71] to contain overcontrolled growth has [60]. High laccase activity was reported in some studies using been employed in decoloration of synthetic dye by the white low carbon to nitrogen ratio [61], but other studies showed rot fungus Trametes versicolor [72–75] allowing bioreactor that higher laccase production was achieved at high carbon to to operate in continuous mode for prolonged times with nitrogen ratio [62]. Laccase was also produced earlier when high efficiency. Cell immobilization is another technique the fungus was cultivated in nitrogen rich media rather than to alleviate problems associated with broth viscosity, and nitrogen-limited media [63]. oxygen and mass transfer. Schliephake et al. [42]produced laccase by Pycnoporus cinnabarinus immobilized on cubes 7. Induction of Laccase of nylon sponge in a 10-L packed bed bioreactor operated in a batch mode. Luke and Burton [44] reported that Production of laccase can be considerably enhanced by the immobilization of the fungus Neurospora crassa on addition of various supplements to the media [64]. The addi- membrane supports allowed the continuous production of tion of xenobiotic compounds such as xylidine, lignin, and laccase for the period of four months without enzyme veratryl alcohol increased and induced laccase activity [65]. deactivation. Sedarati et al. [76] compared the free cell In one study by Lu et al. [66] it was observed that addition cultures of T. versicolor with immobilized cultures using of cellobiose can induce appreciable laccase activity in some nylon mesh for the bioremediation of pentachlorophenol species of Trametes. Low concentration of copper was also (PCP) and 2,4-dichlorophenol (2,4 DCP). Authors observed shown to exhibit inducible effect on laccase activity [67]. that immobilized cultures led to efficient removal. Couto Various basidiomycetes, ascomycetes, and deuteromycetes et al. [77, 78]investigateddifferent synthetic materials as grown in sugar rich liquid medium were induced for laccase carriers for the immobilization of the white rot fungus production by the addition of 2,5-xylidine. It was explicitly Trametes hirsuta in fixed bed bioreactors operated in batch. demonstrated that cultures of Fomes annosus, Pholiota They found that among the different materials tested, mutabilis, Pleurotus ostreatus,andTrametes versicolor were stainless steel sponge led to the highest laccase activities. stimulated for laccase production by addition of xylidine, Park et al. [79] found that immobilization of the white and in the case of Podospora anserina rather decrease in rot fungus Funalia trogii in Na-alginate beads allowed the activity was observed by xylidine addition [68]. efficient decolouration of dye Acid Black 52. Other factors Enzyme Research 5

Table 1: Production of laccases in diﬀerent cultivation modes. Fungi Type of cultivation Inducer Laccase Activity (U/L) Reference Pycnoporus cinnabarinus Submerged 10 mM Veratryl alcohol (VA) 280 [42] Trametes pubescens Submerged 2 mM Cu2+ 333,000 [43] Neurospora crassa Submerged 1 μM cyclohexamide 10,000 [44] T. versicolor SSF (Immersion, nylon sponge) Tween 80 229 [45] T. versicolor SSF (Immersion, barley bran) Tween 80 600 [45] T. versicolor SSF (Expanded bed, nylon sponge) Tween 80 126 [45] T. versicolor SSF (Expanded bed, barley bran Tween 80 600 [45] T. versicolor SSF (Tray, nylon sponge) Tween 80 343 [45] T. versicolor SSF (Tray, barley bran) Tween 80 3500 [45] T. hirsuta SSF (Tray, grape seeds) — 18,715 [46]

affecting the laccase production is agitation. Hess et al. Table 2: Laccase producing organism and biotechnological appli- [80] found that laccase production by Trametes multicolor cation for use. decreased considerably when the fungus was grown in stirred Laccase Producing Application Reference tank reactor, presumably because of damage to mycelia. Organism Mohorciˇ cˇ et al. [81] found that it was possible to cultivate Trametes versicolor Filtration aid [47] the white rot fungus Bjerkandera adusta in a stirred tank reactor after its immobilization on a plastic net, although Trametes versicolor Wine stabilization [48] Myceliophthora very low activities were attained. Tavares et al. [82]on Dough conditioner [49] contrary observed that agitation did not play an important thermophilia role in laccase production by T. versicolor.Fed-batchmodeof Rhizoctonia praticola Phenolic compound removal [50] ff Trametes versicolor; operation is also shown to be an e ective way of producing Soil decontamination [50] laccase. Galhaup et al. [43] found that operating in fed batch Rhizoctonia praticola increased the laccase production of T. pubescens by twofold Coriolopsis gallica Beer factory waste water [51] and obtained a higher laccase activity. Trametes sp. Distillery waste water [51] Trametes versicolor; Olive Mill wastewaters [51] Pleurotus ostreatus 11. Solid State Fermentation Trametes hirsuta Dough Conditioner [52] Solid state fermentation (SSF) is defined as fermentation process occurring in absence or near absence of free liquid, employing an inert substrate (synthetic materials) or a for production of laccase by T. hirsuta using grape seeds natural substrate (organic materials) as a solid support [83]. as substrate. Tray configuration gave the best results here SSF is shown to be particularly suitable for the production as well, and in a similar study by Rosales et al. [88]tray of enzymes by filamentous fungi because they mimic the configuration produced higher laccase activity in T. hirsuta conditions under which the fungi grow naturally [83, 84]. cultures raised on orange peels. The use of natural solid substrates, especially lignocellulosic agricultural residues as growth substrates has been studied 12. Applications of Laccase in Food Processing for various enzymes like cellulases [85, 86] including laccases [87]. The presence of lignin and cellulose/hemicellulose act Table 2 shows the multiple applications of laccases in the as natural inducers and most of these residues are rich in food industry. Areas of the food industry that benefit sugar promoting better fungal growth and thus making the from processing with laccase enzymes include baking, juice processmoreeconomical[8]. The major disadvantage with processing, wine stabilization, and bioremediation of waste SSF is lack of any established bioreactor designs. There are water [52]. The use of laccase enzymes allows for the several bioreactor designs that exist in the literature that have improvement of functionality along with sensory properties. addressed the major limitations of heat and mass transfer in Laccase can also be utilized for analytical applications includ- solid media. Nevertheless lot of progress is still to be made. ing biosensors, enzymatic, and immunochemical assays [51]. Different bioreactor configurations have been studied for The baking industry utilizes a variety of enzymes to laccase production. Couto et al. [45] tested three bioreactor improve bread texture, volume, flavor, and freshness along configurations immersion, expanded bed and tray for laccase with improving machinability of dough during processing. production by T. versicolor using, and inert (nylon) and Bt the addition of laccase to dough used for baked products, noninert support (barley bran). They found that the tray the enzyme exhibits an oxidizing effect resulting in improved configuration led to the best laccase production. Couto et strength of gluten structures in dough and baked products. al. [46] also compared tray and immersion configurations It has also been found that the addition of laccase results in 6 Enzyme Research increased volume, improved crumb structure, and softness of wines causing flavor alterations and intensification of color baked products. Machinability of dough was also found to be in red wines. This phenomenon is also known as maderiza- improved due to increased strength and stability along with tion [92]. Multiple methods can be utilized to prevent made- reduced stickiness with the addition of laccase. Improved irization and they include catalytic factors, block oxidizers or bread and dough qualities with the addition of laccase were the removal of polyphenols via proteinaceous, clarification, also seen when used with low quality flours [89]. polyvinylpolypyrrolidone (PVPP) and high doses of sulfur Due to the growing awareness of celiac disease (CD), dioxide [48]. However, research by Minussi et al. [48]found increased interest has focused on the development of gluten- that treatment with laccase for the removal of polyphenols free baked products. CD is an immune-mediated enteropa- should be selective, as indiscriminate removal can result in thy triggered by the ingestion of gluten, contained in many undesirable organoleptic characteristics. Minussi et al. [48] cereal flours including wheat, rye, and barley, by genetically further concluded that treatment of white wines with laccase susceptible individuals. Cereal flours, like oats and starches is feasible and could diminish processing costs and increase such as rice, potato, and corn, have been the focus for storability of white wines over extended periods of time. the development of gluten-free baked products [90]. These The use of laccase for stabilization is not limited to wine; flours and starches lack the protein matrix responsible the beer industry has potential to benefit from laccase treat- for dough formation and physical characteristics found in ment. Classic haze formation in beer is attributed protein wheat-based baked products. Mimicking the protein matrix precipitation stimulated by proanthocyanidins polyphenols, formed by the gluten proteins during dough formation which are naturally present in small quantities [92]. This of wheat flour has become exceedingly complex. Recent complex formed is commonly referred to as chill haze, research has focused on using gluten-free oat flour along which occurs upon cooling of the beer. The complex can with enzymes to produce baked products acceptable for CD be redissolved by warming of the beer to room temperature patients. The addition of laccase and proteolytic to oat flour or above. However, after extended periods of time, protein lead to a significant improvement to texture quality of oat sulphydryl groups replace phenolic rings and lead to per- bread, due to increased loaf specific volume and lowering manent haze that does not redissolve at room temperature crumb hardness and chewiness. Chemical analysis of oat [89]. Traditionally, excess polyphenols are removed via flour batter treated with laccase and proteolytic enzyme were PVPP treatment, however, PVPP is difficult to handle and found to cause a β-glucan depolymerisation and protein creates problems in waste water treatment due to its low polymerization, resulting in improved rheological properties biodegradability. Laccase has been identified as easier to and positively contribute to improved bread making perfor- handle and safer for the oxidation of polyphenols in wort mance by oat flour [49]. [89]. The addition of laccase at the end of processing has Laccase is also commonly used to stabilize fruit juices. the added benefit of the removal of polyphenols and excess Many fruit juices contain naturally occurring phenolics and oxygen present; reduced oxygen content results in a longer their oxidation products, which contribute to color and taste. shelf life of beer [92]. The natural polymerization and cooxidation reactions of Since laccase are capable of degrading phenolic com- phenolics and polyphenols over time results in undesirable pounds, utilization for bioremediation of food industry changes in color and aroma. The color change, referred to wastewaters is vital. Bioremediation includes processes and as enzymatic darkening, increases due to a higher concen- actions used to biotransform an environment altered by tration of polyphenols naturally present in fruit juices [91]. contaminants back to its original status [93]. Many countries Research by Giovanelli and Ravasini [47] utilized laccase in heavily regulate pollutants, including the class of aromatic combination with filtration in the stabilization of apple juice. compounds, which includes phenols and amines [50]. Treatment with laccase caused the removal of phenols with Research by Minussi et al. [48] reported the removal of high efficiency compared to other methods, like activated naturally occurring and xenobiotic aromatic compounds coals. The substrate-enzyme complex is then removed via from aqueous suspensions using immobilized laccase on membrane filtration, a critical treatment process. Color organogel supports. The application laccase for bioremedi- stability was found to be greatly increased after treatment ation of wastewater streams is particularly of interest to beer with laccase and active filtration, although turbidity was factories. Fractions of wastewater released from beer factories present. The phenolic content of juices has been found to contain a large amount of polyphenols and are dark brown be greatly reduced after treatment with laccase along with in color. Research by Yague¨ et al. [94] found laccase produced an increase in color stability [91]. Laccase treatment has also by the white rot fungus Coriolopsis gallica was capable of beenfoundtobemoreeffective for color and flavor stability degrading polyphenols present in wastewater. Other research compared to conventional treatments, such as the addition by Gonzalez´ et al. [95] utilized laccase from Trametes sp. of ascorbic acid and sulphites [89]. for the bioremediation of distillery wastewater generated The high concentration of phenolics and polyphenols from the ethanol production from the fermentation of also come into play during wine production, particularly sugarcane molasses with a high content of organic matter and the crushing and pressing stages. The high concentration of an intense dark-brown color. Bioremediation of olive mill polyphenols from stems, seeds, and skins contribute to color wastewaters via immobilized laccase has also been reported. and astringency and are dependent on grape variety and It has also been found that utilizing olive oil mill wastewaters vinification conditions [48]. The complex sequence of events has been beneficial in the cultivation of fungi for laccase resulting in the oxidation of polyphenols occurs in musts and production [89]. Enzyme Research 7

13. Conclusions [6] C. F. Thurston, “The structure and function of fungal laccases,” Microbiology, vol. 140, no. 1, pp. 19–26, 1994. Laccases are versatile oxidases, and their versatility lies in [7] K. Li, F. Xu, and K.-E. L. Eriksson, “Comparison of fungal lac- the high reduction potential that makes them potential cases and redox mediators in oxidation of a nonphenolic lignin candidate for biotechnological applications, especially for model compound,” Applied and Environmental Microbiology, the food industry. Laccases have the potential to make food vol. 65, no. 6, pp. 2654–2660, 1999. processing more economical and environmental friendly. To [8] S. R. Couto and J. L. Toca-Herrera, “Laccase production at proficiently realize this potential it would require more effi- reactor scale by filamentous fungi,” Biotechnology Advances, cient laccase production systems and better understanding of vol. 25, no. 6, pp. 558–569, 2007. their mode of action. With the use of mediators it is possible [9] R. Bourbonnais and M. G. Paice, “Oxidation of non-phenolic to extend the role of laccase to nonphenolic substrates. substrates. An expended role for laccase in lignin biodegradation,” FEBS Letters, vol. 267, no. 1, pp. 99–102, 1990. Extensive occurrence of laccase in various fungal genera [10] L. Gianfreda, F. Xu, and J.-M. Bollag, “Laccases: a useful group ensures their widespread availability, and especially the wood of oxidoreductive enzymes,” Bioremediation Journal, vol. 3, no. rotting basidiomycetes also referred as white rot fungi are 1, pp. 1–25, 1999. the excellent laccase producers. Overexpression of laccases in [11] V. K. Gochev and A. I. Krastanov, “Fungal laccases,” Bulgarian heterologous systems has been actively pursued to enhance Journal of Agricultural Science, vol. 13, pp. 75–83, 2007. their titers and to improve their catalytic activity. Media [12] O. V. Morozova, G. P. Shumakovich, M. A. Gorbacheva, S. V. optimization and use of appropriate inducers could bring Shleev, and A. I. Yaropolov, ““Blue” laccases,” Biochemistry, additional benefits of higher production with expenditure vol. 72, no. 10, pp. 1136–1150, 2007. of minimum resources. Both submerged and solid state [13]W.A.Edens,T.Q.Goins,D.Dooley,andJ.M.Henson, cultivation techniques have been embraced by the researchers “Purification and characterization of a secreted laccase of for laccase production. Submerged fermentation, though, Gaeumannomyces graminis var. tritici,” Applied and Environ- leads the SSF for industrial production of laccase. Future mental Microbiology, vol. 65, no. 7, pp. 3071–3074, 1999. efforts in improving the SSF bioreactor designs can make SSF [14] G. Iyer and B. B. Chattoo, “Purification and characterization of more potent and competitive. With plethora of applications laccase from the rice blast fungus, Magnaporthe grisea,” FEMS in food processing including baking and role in gluten-free Microbiology Letters, vol. 227, no. 1, pp. 121–126, 2003. breads, beverage (wine, juice and beer) stabilization, and [15] T. Binz and G. Canevascini, “Purification and partial characterization of the extracellular laccase from Ophiostoma novo- bioremediation, laccases certainly have important role to ulmi,” Current Microbiology, vol. 35, no. 5, pp. 278–281, 1997. play in green food processing. [16] H. Palonen, M. Saloheimo, L. Viikari, and K. Kruus, “Purifi- cation, characterization and sequence analysis of a laccase from the ascomycete Mauginiella sp,” Enzyme and Microbial Acknowledgments Technology, vol. 33, no. 6, pp. 854–862, 2003. The authors gratefully acknowledge financial support from [17] L.-L. Kiiskinen, L. Viikari, and K. Kruus, “Purification and department of Grain Science and Industry, Kansas State characterisation of a novel laccase from the ascomycete Melanocarpus albomyces,” Applied Microbiology and Biotech- University. This paper is contribution no. 10-389-J from the nology, vol. 59, no. 2-3, pp. 198–204, 2002. Kansas Agricultural Experiment Station, Manhattan, KS. [18] G. D. Thakker, C. S. Evans, and K. K. Rao, “Purification and characterization of laccase from Monocillium indicum Saxena,” Applied Microbiology and Biotechnology, vol. 37, no. References 3, pp. 321–323, 1992. [1] P. Baldrian, “Fungal laccases-occurrence and properties,” [19] S. C. Froehner and K. E. Eriksson, “Purification and properties FEMS Microbiology Reviews, vol. 30, no. 2, pp. 215–242, 2006. of Neurospora crassa laccase,” Journal of Bacteriology, vol. 120, [2] A. Messerschmidt and R. Huber, “The blue oxidases, ascorbate no. 1, pp. 458–465, 1974. oxidase, laccase and ceruloplasmin. Modelling and structural [20] H. P. Molitoris and K. Esser, “The phenoloxidases of the relationships,” European Journal of Biochemistry, vol. 187, no. ascomycete Podospora anserina. V. Properties of laccase I after 2, pp. 341–352, 1990. further purification,” Archiv fur¨ Mikrobiologie, vol. 72, no. 3, [3] A. Kunamneni, A. Ballesteros, F. J. Plou, and M. Alcade, pp. 267–296, 1970. “Fungal laccases—a versatile enzyme for biotechnological [21] U. C. Banerjee and R. M. Vohra, “Production of laccase by applications,” in Communicating Current Research and Educa- Curvularia sp,” Folia Microbiologica, vol. 36, no. 4, pp. 343– tional Topics and Trends in Applied Microbiology,A.Mendez- 346, 1991. Vilas, Ed., pp. 233–244, Formatex, Badajoz, Spain, 2007. [22] A. Rodr´ıguez, M. A. Falcon,´ A. Carnicero, F. Perestelo, [4] R. Bourbonnais, M. G. Paice, I. D. Reid, P. Lanthier, and G. de la Fuente, and J. Trojanowski, “Laccase activities of M. Yaguchi, “Lignin oxidation by laccase isozymes from Penicillium chrysogenum in relation to lignin degradation,” Trametes versicolor and role of the mediator 2,2’-azinobis(3- Applied Microbiology and Biotechnology, vol. 45, no. 3, pp. 399– ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymeriza- 403, 1996. tion,” Applied and Environmental Microbiology, vol. 61, no. 5, [23] M. Scherer and R. Fischer, “Purification and characterization pp. 1876–1880, 1995. of laccase II of Aspergillus nidulans,” Archives of Microbiology, [5] A. Leontievsky, N. Myasoedova, N. Pozdnyakova, and L. vol. 170, no. 2, pp. 78–84, 1998. Golovleva, “’Yellow’ laccase of Panus tigrinus oxidizes non- [24] A. Abdel-Raheem and C. A. Shearer, “Extracellular enzyme phenolic substrates without electron-transfer mediators,” production by freshwater ascomycetes,” Fungal Diversity, vol. FEBS Letters, vol. 413, no. 3, pp. 446–448, 1997. 11, pp. 1–19, 2002. 8 Enzyme Research

[25] C. Junghanns, M. Moeder, G. Krauss, C. Martin, and D. [40] M. S. Revankar and S. S. Lele, “Enhanced production of Schlosser, “Degradation of the xenoestrogen nonylphenol by laccase using a new isolate of white rot fungus WR-1,” Process aquatic fungi and their laccases,” Microbiology, vol. 151, no. 1, Biochemistry, vol. 41, no. 3, pp. 581–588, 2006. pp. 45–57, 2005. [41] A. R. Abyanova, A. M. Chulkin, E. A. Vavilova et al., “A [26]A.F.D.Vasconcelos,A.M.Barbosa,R.F.H.Dekker,I. heterologous production of the Trametes hirsuta laccase in S. Scarminio, and M. I. Rezende, “Optimization of laccase the fungus Penicillium canescens,” Applied Biochemistry and production by Botryosphaeria sp. in the presence of veratryl Microbiology, vol. 46, no. 3, pp. 313–317, 2010. alcohol by the response-surface method,” Process Biochemistry, [42] K. Schliephake, D. E. Mainwaring, G. T. Lonergan, I. K. vol. 35, no. 10, pp. 1131–1138, 2000. Jones, and W. L. Baker, “Transformation and degradation of [27] A. Assavanig, B. Amornkitticharoen, N. Ekpaisal, V. Meevoo- the disazo dye Chicago Sky Blue by a purified laccase from tisom, and T. W. Flegel, “Isolation, characterization and Pycnoporus cinnabarinus,” Enzyme and Microbial Technology, function of laccase from Trichoderma,” Applied Microbiology vol. 27, no. 1-2, pp. 100–107, 2000. and Biotechnology, vol. 38, no. 2, pp. 198–202, 1992. [43] C. Galhaup, H. Wagner, B. Hinterstoisser, and D. Haltrich, [28]S.B.Pointing,A.L.Pelling,G.J.D.Smith,K.D.Hyde,andC. “Increased production of laccase by the wood-degrading A. Reddy, “Screening of basidiomycetes and xylariaceous fungi basidiomycete Trametes pubescens,” Enzyme and Microbial for lignin peroxidase and laccase gene-specific sequences,” Technology, vol. 30, no. 4, pp. 529–536, 2002. Mycological Research, vol. 109, no. 1, pp. 115–124, 2005. [44] A. K. Luke and S. G. Burton, “A novel application for Neu- ff [29]C.Liers,R.Ullrich,K.T.Steen, A. Hatakka, and M. rospora crassa: progress from batch culture to a membrane 14 Hofrichter, “Mineralization of C-labelled synthetic lignin bioreactor for the bioremediation of phenols,” Enzyme and and extracellular enzyme activities of the wood-colonizing Microbial Technology, vol. 29, no. 6-7, pp. 348–356, 2001. ascomycetes Xylaria hypoxylon and Xylaria polymorpha,” [45] S. R. Couto, D. Moldes, A. Liebanas,´ and A. Sanroman,´ Applied Microbiology and Biotechnology, vol. 69, no. 5, pp. 573– “Investigation of several bioreactor configurations for laccase 579, 2006. production by Trametes versicolor operating in solid-state [30] J. I. Lyons, S. Y. Newell, A. Buchan, and M. A. Moran, “Diver- conditions,” Biochemical Engineering Journal,vol.15,no.1,pp. sity of ascomycete laccase gene sequences in a southeastern 21–26, 2003. US salt marsh,” Microbial Ecology, vol. 45, no. 3, pp. 270–281, [46] S. R. Couto, E. Lopez,´ and M. A. Sanroman,´ “Utilisation of 2003. grape seeds for laccase production in solid-state fermentors,” [31] P. R. Williamson, “Biochemical and molecular characteriza- Journal of Food Engineering, vol. 74, no. 2, pp. 263–267, 2006. tion of the diphenol oxidase of Cryptococcus neoformans: [47] G. Giovanelli and G. Ravasini, “Apple juice stabiliza- identification as a laccase,” Journal of Bacteriology, vol. 176, no. tion by combined enzyme-membrane filtration process,” 3, pp. 656–664, 1994. Lebensmittel-Wissenschaft und-Technologie,vol.26,no.1,pp. [32] T. E. Machonkin, L. Quintanar, A. E. Palmer et al., “Spec- 1–7, 1993. troscopy and reactivity of the type 1 copper site in Fet3p [48] R. C. Minussi, M. Rossi, L. Bologna, D. Rotilio, G. M. Pastore, from Saccharomyces cerevisiae: correlation of structure with and N. Duran,´ “Phenols removal in musts: strategy for wine reactivity in the multicopper oxidases,” Journal of the American stabilization by laccase,” Journal of Molecular Catalysis B, vol. Chemical Society, vol. 123, no. 23, pp. 5507–5517, 2001. 45, no. 3-4, pp. 102–107, 2007. [33] C. Stoj and D. J. Kosman, “Cuprous oxidase activity of yeast [49] S. Renzetti, C. M. Courtin, J. A. Delcour, and E. K. Arendt, Fet3p and human ceruloplasmin: implication for function,” “Oxidative and proteolytic enzyme preparations as promising FEBS Letters, vol. 554, no. 3, pp. 422–426, 2003. improvers for oat bread formulations: rheological, biochem- [34] A. Hatakka, “Biodegradation of lignin,” in Lignin, Humic ical and microstructural background,” Food Chemistry, vol. Substances and Coal, M. Hofrichter and A. Steinbuchel, Eds., 119, no. 4, pp. 1465–1473, 2010. pp. 129–179, Wiley-VCH, Weinheim, Germany, 2001. [35] C. Eggert, U. Temp, and K.-E. L. Eriksson, “The ligninolytic [50] J. Karam and J. A. Nicell, “Potential applications of enzymes system of the white rot fungus Pycnoporus cinnabarinus: in waste treatment,” Journal of Chemical Technology and purification and characterization of the laccase,” Applied and Biotechnology, vol. 69, no. 2, pp. 141–153, 1997. Environmental Microbiology, vol. 62, no. 4, pp. 1151–1158, [51] V. Madhavi and S. S. Lele, “Laccase: properties and applica- 1996. tions,” BioResources, vol. 4, no. 4, pp. 1694–1717, 2009. [36] T. M. D’Souza, K. Boominathan, and C. A. Reddy, “Isolation [52] S. R. Couto and J. L. Toca Herrera, “Industrial and biotech- of laccase gene-specific sequences from white rot and brown nological applications of laccases: a review,” Biotechnology rot fungi by PCR,” Applied and Environmental Microbiology, Advances, vol. 24, no. 5, pp. 500–513, 2006. vol. 62, no. 10, pp. 3739–3744, 1996. [53] M. Lopez,´ O. Loera, M. Guerrero-Olazaran´ et al., “Cell growth [37]K.H.Lee,S.G.Wi,A.P.Singh,andY.S.Kim,“Micromorpho- and Trametes versicolor laccaseproductionintransformed logical characteristics of decayed wood and laccase produced Pichia pastoris cultured by solid-state or submerged fermenta- by the brown-rot fungus Coniophora puteana,” Journal of tions,” Journal of Chemical Technology and Biotechnology, vol. Wood Science, vol. 50, no. 3, pp. 281–284, 2004. 85, no. 4, pp. 435–440, 2010. [38] D. Schlosser and C. Hofer,¨ “Laccase-catalyzed oxidation of [54] P. Sharma, R. Goel, and N. Capalash, “Bacterial laccases,” Mn2+ in the presence of natural Mn3+ chelatorsasanovel World Journal of Microbiology and Biotechnology, vol. 23, no. source of extracellular H2O2 production and its impact on 6, pp. 823–832, 2007. manganese peroxidase,” Applied and Environmental Microbi- [55] H. Claus, “Laccases and their occurrence in prokaryotes,” ology, vol. 68, no. 7, pp. 3514–3521, 2002. Archives of Microbiology, vol. 179, no. 3, pp. 145–150, 2003. [39] J. Wu, K.-S. Kim, J.-H. Lee, and Y.-C. Lee, “Cloning, expres- [56] K. Koschorreck, R. D. Schmid, and V. B. Urlacher, “Improving sion in Escherichia coli, and enzymatic properties of laccase the functional expression of a Bacillus licheniformis laccase by from Aeromonas hydrophila WL-11,” Journal of Environmental random and site-directed mutagenesis,” BMC Biotechnology, Sciences, vol. 22, no. 4, pp. 635–640, 2010. vol. 9, article 12, 2009. Enzyme Research 9

[57] H. Agematu, T. Tsuchida, K. Kominato et al., “Enzymatic [74] S. Romero, P. Blanquez,´ G. Caminal et al., “Different dimerization of penicillin X,” Journal of Antibiotics, vol. 46, no. approaches to improving the textile dye degradation capacity 1, pp. 141–148, 1993. of Trametes versicolor,” Biochemical Engineering Journal, vol. [58] R. Gayazov and J. Rodakiewicz-Nowak, “Semi-continuous 31, no. 1, pp. 42–47, 2006. production of laccase by Phlebia radiata in different culture [75] P. Blanquez,´ G. Caminal, M. Sarra,` and T. Vicent, “The effect media,” Folia Microbiologica, vol. 41, no. 6, pp. 480–484, 1996. of HRT on the decolourisation of the Grey Lanaset G textile [59] P. Keyser, T. K. Kirk, and J. G. Zeikus, “Ligninolytic enzyme dye by Trametes versicolor,” Chemical Engineering Journal, vol. system of Phanerochaete chrysosporium: synthesized in the 126, no. 2-3, pp. 163–169, 2007. absence of lignin in response to nitrogen starvation,” Journal [76] M. R. Sedarati, T. Keshavarz, A. A. Leontievsky, and C. S. of Bacteriology, vol. 135, no. 3, pp. 790–797, 1978. Evans, “Transformation of high concentrations of chlorophe- [60] G. F. Leatham and T. K. Kirk, “Regulation of ligninolytic nols by the white-rot basidiomycete Trametes versicolor activity by nutrient nitrogen in white-rot basidiomycetes,” immobilized on nylon mesh,” Electronic Journal of Biotechnol- FEMS Microbiology Letters, vol. 16, no. 1, pp. 65–67, 1983. ogy, vol. 6, no. 2, pp. 27–37, 2003. [61] M. C. Monteiro and M. E. A. De Carvalho, “Pulp bleaching [77] S. R. Couto, M. A. Sanroman,D.Hofer,andG.M.G´ ubitz,¨ using laccase from Trametes versacolor under high tem- “Production of laccase by Trametes hirsuta grown in an perature and alkaline conditions,” Applied Biochemistry and immersion bioreactor and its application in the decolorization Biotechnology, vol. 70–72, no. 1, pp. 983–993, 1998. ff of dyes from a leather factory,” Engineering in Life Sciences, vol. [62] J. A. Buswell, Y. Cai, and S.-T. Chang, “E ect of nutrient 4, no. 3, pp. 233–238, 2004. nitrogen and manganese on manganese peroxidase and laccase [78] S. R. Couto, M. A. Sanroman,D.Hofer,andG.M.G´ ubitz,¨ production by Lentinula (Lentinus) edodes,” FEMS Microbiol- “Stainless steel sponge: a novel carrier for the immobilisation ogy Letters, vol. 128, no. 1, pp. 81–88, 1995. of the white-rot fungus Trametes hirsuta for decolourization [63] M. Heinzkill, L. Bech, T. Halkier, P. Schneider, and T. Anke, of textile dyes,” Bioresource Technology, vol. 95, no. 1, pp. 67– “Characterization of laccases and peroxidases from wood- 72, 2004. rotting fungi (family Coprinaceae),” Applied and Environmen- tal Microbiology, vol. 64, no. 5, pp. 1601–1606, 1998. [79]C.Park,B.Lee,E.-J.Han,J.Lee,andS.Kim,“Decolorization [64] I.-Y. Lee, K.-H. Jung, C.-H. Lee, and Y.-H. Park, “Enhanced of acid black 52 by fungal immobilization,” Enzyme and production of laccase in Trametes vesicolor by the addition of Microbial Technology, vol. 39, no. 3, pp. 371–374, 2006. ethanol,” Biotechnology Letters, vol. 21, no. 11, pp. 965–968, [80] J. Hess, C. Leitner, C. Galhaup et al., “Enhanced formation of 1999. extracellular laccase activity by the white-rot fungus Trametes [65] A. M. R. B. Xavier, D. V. Evtuguin, R. M. P. Ferreira, and F. multicolor,” Applied Biochemistry and Biotechnology, vol. 98– L. Amado, “Laccase production for lignin oxidase activity,” in 100, no. 1–9, pp. 229–241, 2002. Proceedings of 8th International Conference on Biotechnology, [81] M. Mohorciˇ c,ˇ J. Friedrich, and A. Pavko, “Decoloration of the Helsinki, Finland, 2001. diazo dye reactive black 5 by immobilised Bjerkundera adusta [66] S. X. F. Lu, C. L. Jones, and G. T. Lonergan, “Correlation in a stirred tank bioreactor,” Acta Chimica Slovenica, vol. 51, between fungal morphology and laccase expression under the no. 4, pp. 619–628, 2004. influence of cellobiose,” in Proceedings of the 10th International [82] A. P. M. Tavares, M. A. Z. Coelho, M. S. M. Agapito, J. Biotechnology Symposium, Sydney, Australia, 1996. A. P. Coutinho, and A. M. R. B. Xavier, “Optimization and [67] G. Palmieri, P.Giardina, C. Bianco, A. Scaloni, A. Capasso, and modeling of laccase production by Trametes versicolor in G. Sannia, “A novel white laccase from Pleurotus ostreatus,” a bioreactor using statistical experimental design,” Applied Journal of Biological Chemistry, vol. 272, no. 50, pp. 31301– Biochemistry and Biotechnology, vol. 134, no. 3, pp. 233–248, 31307, 1997. 2006. [68] J.-M. Bollag and A. Leonowicz, “Comparative studies of [83]A.Pandey,P.Selvakumar,C.R.Soccol,andP.Nigam,“Solid extracellular fungal laccases,” Applied and Environmental state fermentation for the production of industrial enzymes,” Microbiology, vol. 48, no. 4, pp. 849–854, 1984. Current Science, vol. 77, no. 1, pp. 149–162, 1999. [69] S. B. Pointing, E. B. G. Jones, and L. L. P. Vrijmoed, “Opti- [84] M. Moo-Young, A. R. Moreira, and R. P. Tengerdy, “Principles mization of laccase production by Pycnoporus sanguineus in of solid sate fermentation,” in The Filamentous Fungi,J.E. submerged liquid culture,” Mycologia, vol. 92, no. 1, pp. 139– Smith, D. R. Berry, and B. Kristiansen, Eds., pp. 117–144, 144, 2000. Edward Arnold, London, UK, 1983. [70] F. Zadrazil, A. Gonser, and E. Lang, “Influence of incuba- [85] K. Brijwani, H. S. Oberoi, and P. V. Vadlani, “Production tion temperature on the secretion of extracellular lignolytic of a cellulolytic enzyme system in mixed-culture solid-state enzymes of Pleurotus and Dichomitus squalus into soil,” in fermentation of soybean hulls supplemented with wheat Proceedings of the Conference on Enzymes in the Environment, bran,” Process Biochemistry, vol. 45, no. 1, pp. 120–128, 2010. Granada, Spain, 1999. [71]J.M.Lema,E.Roca,A.Sanroman,M.J.Nunez,M.T. [86] K. Brijwani and P.V. Vadlani, “Physicochemical characteristics Moreira, and G. Feijoo, “Pulsating bioreactors,” in Multiphase of substrate that direct production of cellulolytic enzyme Bioreactor Design,J.M.S.Cabral,M.Mota,andJ.Tramper, system in solid-state fungal fermentation,” to appear in Process Eds., pp. 309–329, Taylor and Francis, London, UK, 2001. Biochemistry. [72] P. Blanquez,´ N. Casas, X. Font et al., “Mechanism of textile [87] S. R. Couto and MA. A. Sanroman,´ “Application of solid-state metal dye biotransformation by Trametes versicolor,” Water fermentation to ligninolytic enzyme production,” Biochemical Research, vol. 38, no. 8, pp. 2166–2172, 2004. Engineering Journal, vol. 22, no. 3, pp. 211–219, 2005. [73] P. Blanquez,´ M. Sarra,` and M. T. Vicent, “Study of the cellular [88] E. Rosales, S. R. Couto, and M. A. Sanroman,´ “Increased retention time and the partial biomass renovation in a fungal laccase production by Trametes hirsuta grown on ground decolourisation continuous process,” Water Research, vol. 40, orange peelings,” Enzyme and Microbial Technology, vol. 40, no. 8, pp. 1650–1656, 2006. no. 5, pp. 1286–1290, 2007. 10 Enzyme Research

[89] R. C. Minussi, G. M. Pastore, and N. Duran,´ “Potential applications of laccase in the food industry,” Trends in Food Science and Technology, vol. 13, no. 6-7, pp. 205–216, 2002. [90] E. Gallagher, “Improving gluten-free bread quality through the application of enzymes,” Agro Food Industry Hi-Tech, vol. 20, no. 1, pp. 34–37, 2009. [91] D. S. Ribeiro, S. M. B. Henrique, L. S. Oliveira, G. A. Macedo, and L. F. Fleuri, “Enzymes in juice processing: a review,” International Journal of Food Science & Technology, vol. 45, pp. 635–641, 2010. [92] T. E. Mathiasen, “Laccase and beer storage,” PCT Int. Appl. WO 9521240 A2, 1995. [93] P. K. Thassitou and I. S. Arvanitoyannis, “Bioremediation: a novel approach to food waste management,” Trends in Food Science and Technology, vol. 12, no. 5-6, pp. 185–196, 2001. [94] S. Yague,M.C.Terr¨ on,´ T. Gonzalez´ et al., “Biotreatment of tannin-rich beer-factory wastewater with white-rot basidiomycete Coriolopsis gallica monitored by pyrolysis/gas chromatography/mass spectrometry,” Rapid Communications in Mass Spectrometry, vol. 14, no. 10, pp. 905–910, 2000. [95] T. Gonzalez,´ M. C. Terron,´ S. Yague,¨ E. Zapico, G. C. Galletti, andA.E.Gonzalez,´ “Pyrolysis/gas chromatography/mass spectrometry monitoring of fungal- biotreated distillery wastewater using Trametes sp. I-62 (CECT 20197),” Rapid Communications in Mass Spectrometry, vol. 14, no. 15, pp. 1417–1424, 2000. SAGE-Hindawi Access to Research Enzyme Research Volume 2010, Article ID 473137, 16 pages doi:10.4061/2010/473137

Review Article Potential Applications of Immobilized β-Galactosidase in Food Processing Industries

Parmjit S. Panesar, Shweta Kumari, and Reeba Panesar

Biotechnology Research Laboratory, Department of Food Engineering & Technology, Sant Longowal Institute of Engineering and Technology, Longowal, Punjab, 148 106, India

Correspondence should be addressed to Parmjit S. Panesar, [email protected]

Received 16 June 2010; Revised 22 September 2010; Accepted 21 November 2010

Academic Editor: Cristina M. Rosell

Copyright © 2010 Parmjit S. Panesar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The enzyme β-galactosidase can be obtained from a wide variety of sources such as microorganisms, plants, and animals. The use of β-galactosidase for the hydrolysis of lactose in milk and whey is one of the promising enzymatic applications in food and dairy processing industries. The enzyme can be used in either soluble or immobilized forms but the soluble enzyme can be used only for batch processes and the immobilized form has the advantage of being used in batch wise as well as in continuous operation. Immobilization has been found to be convenient method to make enzyme thermostable and to prevent the loss of enzyme activity. This review has been focused on the diﬀerent types of techniques used for the immobilization of β-galactosidase and its potential applications in food industry.

1. Introduction Treatment of milk and milk products with lactase to reduce their lactose content seems to be an appropriate method to The enzyme β-galactosidase (EC.3.2.1.23), most commonly increase their potential uses and to deal with the problems of known as lactase, which hydrolyses lactose into its monomers lactose insolubility and lack of sweetness. Furthermore, this that is glucose and galactose has potential applications in treatment could make milk, a most suitable food, available food processing industry. Because of low levels of the enzyme to a large number of adults and children that are lactose in intestine, large fraction of the population shows lactose intolerant. Moreover, the hydrolysis of whey converts lactose intolerance and they have difficulty in consuming milk and into a very useful product like sweet syrup, which can be dairy products. Lactose has a low relative sweetness and used in various processes of dairy, confectionary, baking, solubility, and excessive lactose in large intestine can lead and soft drink industries [3, 4]. Therefore, lactose hydrolysis to tissue dehydration due to osmotic effects, poor calcium not only allows the milk consumption by lactose intolerant absorption due to low acidity, and fermentation of the lactose population but can also solve the environmental problems by microflora resulting in fermentative diarrhea, bloating, linked with whey disposal [5–7]. flatulence, blanching and cramps, and watery diarrhea [1]. The enzyme β-galactosidase can also be used in transg- Furthermore, lactose is a hygroscopic sugar and has a strong lycosylation of lactose to synthesize galacto-oligosaccharides tendency to absorb flavours and odours and causes many (GOSs). These were widely recognized as the nondigestible defects in refrigerated foods such as crystallization in dairy oligosaccharides, not hydrolyzed or absorbed in the upper foods, development of sandy or gritty texture, and deposit intestinal tract, and they pass onto the colon where they are formation [2]. fermented selectively by beneficial intestinal bacteria. Besides Technologically, lactose gets easily crystallized, which sets their prebiotic effects, these GOSs have low cariogenicity, low the limits of its applications to certain processes in the caloric values, and low sweetness [8, 9]. GOSs occur naturally dairy industry. Cheese manufactured from hydrolyzed milk in trace amounts in breast milk, cow milk, honey, and a ripens more quickly than that made from normal milk. variety of fruits and vegetables [10]. As a result, increased 2 Enzyme Research production of GOS is useful. GOS can be readily manufac- ter has been purified by chromatography through DEAE- tured by enzymatic transgalactosylation of β-galactosidase cellulose [27]. The optimization of the ultrasonication from whey lactose, which is available in abundance as a by- methods for the maximum cell disruption of Escherichia product of cheese industry. coli for the release of β-galactosidase has also been reported Thus, the application of β-galactosidase in the hydrolysis [28]. Lactobacillus delbrueckii subsp. bulgaricus cultures were of lactose in dairy industry has attracted the attention of subjected to treatments using sonication, a high-speed bead researchers. Although most industries still hydrolyze lactose mill, and a high-pressure homogenizer for the release of β- with free enzyme, the immobilization of β-galactosidase is galactosidase [29]. an area of great interest because of its potential benefits β-galactosidase has also been purified from psychotropic [11]. The use of immobilization technology is of signif- Pseudoalteromonas sp. isolated from Antarctica and a high icant importance from economic point of view since it yield of purification has been reported by a rapid purification makes reutilization of the enzyme and continuous operation scheme using extraction in an aqueous two-phase system possible and also precludes the need to separate the cells followed by hydrophobic interaction chromatography and from the whey following processing. It can also help to ultrafilteration techniques [30]. improve the enzyme stability. Nowadays, immobilized β- An intracellular β-galactosidase from a thermoaci- galactosidase is intensively being used in lactose hydrolysis of dophilic Alicyclobacillus acidocaldarius subsp. rittmannii has milk/whey and has been tested for the production of galacto- been purified using precipitation (with ammonium sul- oligosaccharides. phate), gel permeation, ion-exchange, and affinity chromatography and preparative electrophoresis [26]. Further, β 2. Microbial Sources of Enzyme a thermostable -galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. subtilis The enzyme β-galactosidase can be obtained from a wide WB600 and recombinant enzyme has been purified by a variety of sources such as microorganisms, plants, and combination of heat treatment, ammonium sulfate frac- animals; however, according to their sources, their properties tionation, ion exchange, and gel filtration chromatography β differ markedly [11, 12]. Enzymes of plants and animal techniques [31]. The intracellular -galactosidase from ther- ffi origin have little commercial value but several microbial mophile B1.2 was purified by ion-exchange and a nity sources of β-galactosidase are of great technological interest. chromatography with a fold purification of 2.2 and 3.9, Microorganisms offer various advantages over other avail- respectively. The molecular mass of the purified enzyme as able sources such as easy handling, higher multiplication determined by native PAGE was approximately 215 kDa, by rate, and high production yield. As a result of commercial SDS-PAGE was 75 kDa, and by gel filtration was 215 kDa ffi ff interest in β-galactosidase, a large number of microorgan- [32]. The e ciency of di erent cell disruption methods β isms [13–26] have been assessed as potential sources of this on the extraction of intracellular -galactosidase enzyme enzyme (Table 1). from Streptococcus thermophilus and Lactobacillus delbrueckii subsp. thermophilus has been tested [33]. Lysozyme enzyme treatment was determined as the most effective method, 2.1. Production and Purification. Microorganisms are con- which resulted in approximately 1.5 and 10 times higher sidered potential source of β-galactosidase for industrial ff enzyme activity than glass bead and homogenization treat- applications. However, they di er in their optimum con- ment, respectively. ditions for the enzyme application especially pH range. The activity and the stability of the partially purified β- A recovery cost of the enzyme depends on the level of galactosidases from Thermus sp strain T2 and K. fragilis have production and purification. Therefore, there has been been compared [34]. Both enzymes showed a remarkable increasing interest in finding microorganisms with adequate hydrolytic activity and a weak transgalactosilation activity, properties for industrial use, higher production capacity, and even in the presence of high concentrations of lactose. less expensive purification methods of this enzyme. A wide The thermophilic enzyme showed a higher resistance to variety of bacterial, yeast, and fungal cultures have been ff β hydrophobic agents and a higher stability at di erent temper- reported for -galactosidase production. atures, pHs, and chemical conditions. However, the enzyme of Thermus was less stable in the presence of oxygen peroxide, 2.1.1. Bacterial Enzymes. The enzyme β-galactosidase can be showing that some residues important for its stability were produced by a large number of bacteria but Streptococcus affected by oxidation. The enzyme from K. fragilis was thermophilus and Bacillus stearothermophilus are considered strongly inhibited by o-nitrophenol in a acompetitive way as potential bacterial sources. The enzyme from Escherichia but it was weakly and competitively inhibited by galactose. coli serves as a model for understanding the catalytic The thermophilic enzyme was competitively inhibited by mechanism of β-galactosidase action, but it is not considered galactose much strongly than its mesophilic counterpart but suitable for use in foods due to toxicity problems associated the inhibition did not change with the temperature. A novel with the host coliform [11]. Hence, the β-galactosidase from thermostable chimeric β-galactosidase was constructed by E. coli is generally not preferred for use in food industry [13– fusing a poly-His tag to the N-terminal region of the β- 15]. galactosidase from Thermus sp.strainT2tofacilitateits β-galactosidase has been isolated from an extremely overexpression in E. coli and its purification by immobi- thermophilic Gram-negative anaerobe. Thermoanaerobac- lized metal-ion affinity chromatography [35]. To improve Enzyme Research 3

Table 1: Microbial sources of β-galactosidase. of large multimeric proteins via selective adsorption on tailor-made immobilized metal-ion affinity chromatography Source Microorganism (s) supports. Furthermore, β-galactosidase from Thermus sp. Alicyclobacillus acidocaldarius subsp. rittmannii Strain T2 has been purified and immobilized in a single Arthrobacter sp. step, combining the excellent properties of epoxy groups Bacillus acidocaldarius, B. circulans, B. coagulans, B. for enzyme immobilization with the good performance subtilis, B. megaterum, B. stearothermophilus of immobilized metal-chelate affinity chromatography for Bacteriodes polypragmatus protein purification [36]. Bifidobacterium bifidum, B. infantis Clostridium acetobutylicum, C. thermosulfurogens 2.1.2. Fungal Enzymes. The optimum pH range for the Corynebacterium murisepticum fungal enzyme is 2.5–5.4, which makes them suitable for Enterobacter agglomerans, E. cloaceae processing of acid whey and its ultrafiltration permeate. Escherichia coli The optimum temperatures for these enzymes are high and can be typically used at temperatures up to 50◦C. The Klebsiella pneumoniae β ff Bacteria purification of -galactosidase from di erent fungal sources Lactobacillus acidophilus, L. bulgaricus,L. helviticus, L. has been carried using a variety of purification techniques. kefiranofaciens, L. lactis, L. sporogenes, L. themophilus, L. β-galactosidase from Aspergillus niger has been purified delbrueckii and resolved into three multiple forms, using molecular siev- Leuconostoc citrovorum ing, ion exchange, and hydrophobic chromatography [37]. Pediococcus acidilacti, P. pento The purification of β-galactosidase has been carried from a Propioionibacterium shermanii cellular extract of Fusarium oxysporum var. lini by heat shock Pseudomonas fluorescens and successive chromatography on DEAE-cellulose DE-52 Pseudoalteromonas haloplanktis and sephadex G-100 [18]. The purification of β-galactosidase Streptococcus cremoris, S. lactis, S. thermophius by ammonium sulphate precipitation and CM-sephadex chromatography from cell-free extracts of fungus Beauveria Sulfolobus solfatarius bassiana has also been reported [38]. An extracellular β- Thermoanaerobacter sp. galactosidase from a themophilic fungus Rhizomucor sp. has Thermus rubus, T. aquaticus been purified by successive DEAE-cellulose chromatography, Trichoderma reesei followed by gel filtration on sephacryl S-300 [39]. The Vibrio cholera precipitation with ammonium sulfate, ion-exchange chro- ffi Xanthomonas campestris matography on DEAE-sephadex, a nity chromatography Alternaria alternate, A. palmi and chromatofocusing has also been used for purification of β-galactosidase from Penicillium chrysogenum NCAIM 00237 Aspergillus foelidis, A. fonsecaeus, A. fonsecaeus, A. Carbonarius, A. Oryzae [40]. β-galactosidase produced by submerged culture of Auerobasidium pullulans Aspergillus japonicus showed 2.95 U mg−1 protein specific Curvularia inaequalis activities with an approximate molecular weight of 27 kDa Fungi Fusarium monilliforme, F. oxysporum [41]. The enzyme was purified 6.43-fold with 24.02% Mucor meihei, M. pusillus yield and a specific activity of 18.96 U mg−1 protein. An Neurospora crassa intracellular β-glycoside hydrolase with β-glucosidase and β β Penicillum canescens, P. chrysogenum, P. expansum -galactosidase activity designated -glucosidase BGL1 has been purified from the thermophilic fungus Talaromyces Saccharopolyspora rectivergula thermophilus. The monomeric enzyme has a molecular mass Scopulariapsis sp of 50 kDa (SDS-PAGE), an isoelectric point of 4.5-4.6. β- Streptomyces violaceus galactosidase activity of β-glucosidase BGL1 is activated by Bullera singularis various mono and divalent cations including Na+,K+,and Candida pseudotropicalis Mg2+, and it is moderately inhibited by its reaction products Yeast Saccharomyces anamensis, S. lactis, S. fragilis that is glucose and galactose [42]. Kluyveromyces bulgaricus, K. fragilis, K. lactis, K. marxianus 2.1.3. Yeast Enzymes. Yeast has been considered as an impor- Source: [12–26]. tant source of β-galactosidase from industrial point of view. With neutral pH optima, these are well suited for hydrolysis of lactose in milk and are widely accepted as safe for use in the enzyme purification a selective one-point adsorption foods. Much work has been carried on the production of was achieved by designing tailor-made low-activated Co- β-galactosidase from different yeast strains for its potential iminodiacetic acid (Co-IDA) or Ni-IDA supports. The new use. The most commercially available yeast β-galactosidase enzyme was not only useful for industrial purposes but also underthetradenameofMaxilact (DSM Food Specialties, has become an excellent model to study the purification Delft, The Netherlands) and Lactase (SNAM Progetti, Italy) 4 Enzyme Research is preparations extracted from K. lactis and Lactozym (Novo, simple to carry out and has little influence on the con- NordiskA/S,Bagsvaerd,Denmark)fromK. fragilis [11, 14, formation of the biocatalyst. However, the disadvantage of 17]. this technique is the relative weakness of the adsorptive Biermann and Glantz [43] first attempted the purifi- binding forces. Different inorganic (alumina, silica, porous cation of β-galactosidase from Sacharomyces lactis by gel glass, ceramics, diatomaceous earth, clay, bentonite, etc.) filtration on sephadex G-100, followed by ion exchange chro- and organic (cellulose, starch, activated carbon and ion- matography on DEAE-sephadex. The homogenizer (800 bar exchange resins, such as Amberlite, Sephadex, Dowex) sup- pressure, pH 7.5) was used for the disruption and extraction port materials can be used for enzyme adsorption. Further of β-galactosidase of K. marxianus [44]. Further, the partial adsorption of enzyme may be stabilized by glutaraldehyde purification of the enzyme from K. marxianus was carried treatment. using DEAE-sepharose column. The studies on the use of fed Immobilization of β-galactosidase on hydrophobic cot- batch culture techniques to achieve high culture productivity ton cloth indicated that the enzyme adsorbed on the cloth in K. fragilis have also been carried out [45]. was about 50% active as free enzymes [87]. The immobiliza- The optimization of β-galactosidase production by K. tion of β-galactosidase active yeast K. fragilis and K. lactis lactis using deprotienized whey as fermentation medium onto chitosan showed an enzyme activity of 0.9–2.2 U/mg has been reported. The optimized condition for the dry cell wt [88]. Enzyme activity of immobilized enzyme enzyme production was reported as follows: temperature from K. fragilis was higher but the operational stability 30.3◦C, pH 4.68, agitation speed 191 rpm, and fermentation of A. oryzae enzyme was 5–14 times higher depending time 18.5 hours [46]. The studies on two commercial β- upon the mode of immobilization [89]. When adsorption galactosidase (Lactozym and Maxilact) preparations indi- method was used, the highest activity was obtained with cated that the enzyme activities of both enzyme preparations yeast enzyme and support Ostsorb-DEAE. The enzyme from present similar behaviour with different pH and temperature A. oryzae immobilized on polyvinyl chloride (PVC) and and similar kinetic parameter values, which suggest that silica gel membrane has been used for the hydrolysis of both enzymes are probably the same [47]. Response surface lactose in skim milk in an axial-annular flow reactor [51]. methodology has also been applied in the production of β- Further, maximum immobilization occurred at pH 5.5 and galactosidase using K. lactis NRRL Y-8279 [48] and maxi- optimal results were obtained with citrate/phosphate buffer mum specific enzyme activity of 4218.4 U g−1was obtained during immobilization of β-galactosidase from E. coli by at the optimum levels of process variables (pH 7.35, agitation physical adsorption on chromosorb-W [51]. A novel reactor speed 179.2 rpm, initial sugar concentration 24.9 g L−1,and consisting of β-galactosidase from B. circulans immobilized incubation time 50.9 hours). on a ribbed membrane composed of PVC and silica has also been used for skim milk lactose hydrolysis [53]. The β β immobilization of partially purified Bullera singularis - 3. Immobilization of -Galactosidase galactosidase in Chitopearl BCW 3510 bead (970 GU/g resin) by simple adsorption has also been carried out Although the enzyme β-galactosidase has numerous appli- [90]. cations in the food and dairy industries, but the moderate The studies on the kinetic behaviour of β-galactosidase stability of enzyme is one of the limitations that hinder from Kluyveromces marxianus (Saccharomyces) lactis, immo- general implementation of biocatalysts at industrial scale. bilized on to different oxide supports, such as alumina, Thus, there is a need to explore their full potential as catalyst silica, and silicated alumina indicated that the immobilized by adopting suitable strategies for enzyme stabilization. enzyme activity strongly depends on the chemical nature The multimeric enzyme can be stabilized by using proper and physical structure of the support [53]. In particular, experimental conditions and genetic tools to cross link when the particle sizes of the support are increased, the or to strengthen the subunit-subunit interaction [49]. The enzymatic activity strongly decreases. Immobilization of β- stability of monomeric or multimeric enzymes can also be galactosidase from Thermus sp. preceded very rapidly onto enhanced by multipoint and multi-subunit covalent immo- PEI-Sepabeads and conventional DEAE-Agarose. However, bilization and enzyme engineering via immobilization [50]. the adsorption strength was much higher in the case of PEI- The enzyme has been immobilized by various methods such Sepabeads [53]. as physical absorption, entrapment, and covalent binding A recombinant thermostable B. stearothermophilus β- method [51–85]ondifferent supports (Table 2). galactosidase was immobilized onto chitosan using Tris (hydroxymethyl) phosphine (THP) and glutaraldehyde, and 3.1. Physical Adsorption. Physical adsorption is considered a packed bed reactor was utilized to hydrolyze lactose in as the simplest method of immobilization in which an milk. The thermostability and enzyme activity of THP- enzyme is immobilized onto a water-insoluble carrier and immobilized β-galactosidase during storage was superior to the biocatalysts are held on the surface of the carri- that of free and glutaraldehyde-immobilized enzymes. The ersbyphysicalforces(vanderwaalsforces).Frequently, THP-immobilized β-galactosidase showed greater relative however, additional forces are involved in the interaction activity in the presence of Ca2+ than the free enzyme and between carrier and biocatalyst principally hydrophobic was stable during the storage at 4◦Cfor6weeks,whereas interactions, hydrogen bridges, and heteropolar (ionic) the free enzyme lost 31% of the initial activity under the bonds [86]. This method has the advantage of being same storage conditions [91]. Response surface methodology Enzyme Research 5

Table 2: Diﬀerent sources of β-galactosidase and methods of immobilization.

Immobilization method Source of β-galactosidase Immobilizing agents References K. fragilis and K. lactis Chitosan [6] A. oryzae Phenol-formaldehyde resin [69] A. oryzae Polyvinyl chloride and Silica gel membrane [51] E. coli Chromosorb-W [52] B. circulans Polyvinyl chloride and Silica [53] B. stearothermophilus Chitosan [54] (1) Physical adsorption A. niger Porous ceramic monolith [70] K. fragilis Chitosan bead [2] K. fragilis Chitosan [71] K. lactis CPC-silica and agarose [72] Thermus sp. T2 PEI- sepabeads, DEAE-agarose [55] K. fragilis Cellulose beads [14] A. oryzae Celite and chitosan [73] Pisum sativum Sephadex G-75 and chitosan beads [56]

K. bulgaricus Alginate using BaCl3 [57] E. coli Polyacrylamide gel [58] A. oryzae Nylon-6 and zeolite [62] (2) Entrapment Thermus aquaticus YT-1 Agarose bead [59] A. oryza Spongy polyvinyl alcohol Cryogel [60] Penicillium expansum F3 Calcium alginate [23] K. lactis, A. oryzae Saccharomyces cerevisiae Poly(vinylalcohol) hydrogel [7] L. bulgaricus Egg shells [61] S. anamensis Calcium alginate [74] E. coli Hen egg white [75] E. coli Polyvinyl alcohol [76]

A. oryzae Silica gel activated with TiCl3 and FeCl3 [77] E. coli (Recombinant β-galactosidase) Cyanuric chloride-activated cellulose [66] K. lactis Corn grits [78] E. coli Gelatin [63] K. lactis Thiosulﬁnate/thiosulfonate [79] (3) Covalent Binding B. circulans Eupergit C (Spherical acrylic polymer) [65] K. fragilis Silica-alumina [64] K. lactis Graphite surface [68] A. oryzae Chitosan bead and nylon membrane [80] A. oryzae Cotton cloth and activated [81] With tosyl chloride A. oryzae Amino-epoxy sepabead [67] K. latis Cotton fabric [82] A. niger Magnetic polysiloxane-polyvinyl alcohol [83] A. oryzae Silica [84] Polyvinylalcoheol hydrogel and magnetic A. oryzae [85] Fe3O4-chitosan as supporting agent

(RSM) and centre composite design (CCD) have been A. oryzae β-galactosidase was immobilized on an used to optimize immobilization of β-galactosidase (BGAL) inexpensive bioaffinity support, concanavalin A-cellulose. from Pisum sativum onto two matrices: Sephadex G-75 and Concanavalin A-cellulose adsorbed and cross-linked β- chitosan beads. The immobilization efficiency of 75.66% galactosidase preparation retained 78% of the initial activity. and 75.19% was achieved with Sephadex G-75 and chitosan, The optimum temperature was increased from 50 to 60◦Cfor respectively [56]. the immobilized β-galactosidase. The cross-linked adsorbed 6 Enzyme Research enzyme retained 93% activity after 1-month storage while Table 3: Cross-linking reagents used in β-galactosidase immobi- the native enzyme showed only 63% activity under similar lization. incubation conditions [92]. Cross-linking reagent References Bis-oxirane [102] 3.2. Entrapment Method. Entrapment method is the phys- Carbodiimide [103] ical enclosure of enzymes in a small space. Matrix and Chromium (III) acetate [63] membrane entrapment (including microcapsulation) are the Glutaraldehyde [14, 20, 69, 70, 104–107] major methods of entrapment. The major advantage of the entrapment technique is the simplicity by which spherical Polyethyleneimine [57, 101, 108] particles can be obtained by dripping a polymer-cell suspen- Sulfate-dextran [109] sion into a medium containing positively charged ions or Transglutaminase [110] through thermal polymerization [86]. Further, beads formed Tris(hydroxymethyl)phosphine [54] particularly from alginate are transparent and generally mechanically stable. The major limitation of this technique for the immobilization of enzymes is the possible slow immobilization of β-galactosidase is the use of liposomes and leakage during continuous use in view of the small molecular in this direction response surface methodology was applied size compared to the cells. However, improvements can be to optimize the entrapment of the enzyme in liposomes by made by using suitable linking procedures. The matrices used dehydration-rehydration vesicle method, which resulted in for the immobilization are usually made up of polymeric an entrapment efficiency of 28% [96]. materials such as Ca-alginate, agar, k-carragenin, polyacry- It has been observed that entrapped cross-linked con- lamide, and collagen. However, some solid matrices such as canavalin A-β-galactosidase complex preparation was more activated carbon, porous ceramic, and diatomaceous earth superior in the continuous hydrolysis of lactose in a batch can also be used for the immobilization. The membranes process as compared to the other entrapped preparations commonly used for the entrapment of enzymes are nylon, because it retained 95% activity after seventh repeated use cellulose, polysulfone, and polyacrylmide. and 93% of its original activity after 2-month storage at Fungal β-galactosidase immobilized in polyvinyl alcohol 4◦C[97]. A. oryzae β-galactosidase was immobilized on gel was more thermostable than free enzyme and retained the surface of a novel bioaffinity support: concanavalin A 70% of activity after 24 h at 50◦C and 5% activity at 60◦C layered calcium alginate-starch beads. The maximum activity [93]. The glutaraldehyde-treated K. bulgaricus cells having β- of the immobilized β-galactosidase has been obtained at ◦ galactosidase were entrapped in alginate using BaCl2 solution 60 C, approximately 10 degrees higher than that of the free [57]. The alginate beads obtained after treatment with enzyme. It has been also observed that the immobilized β- polyethyleneimine followed by glutaraldehyde solution were galactosidase exhibited significantly higher stability to heat, stable. urea, MgCl2,andCaCl2 than the free enzyme [98]. Calcium E. coli β-galactosidase has been immobilized in poly- alginate-entrapped β-galactosidase preparations have been acrylamide gels and through the preparation of cross- used for the hydrolysis of lactose from synthetic solution, linked derivatives of E. coli β-galactosidase by treating the milk,andwheyinbatchprocessesaswellasincontinuous enzyme with bisimidoesters. The combination of three packed bed columns. From the kinetic studies, it was protective agents, namely, bovine serum albumin, cysteine, observed that the Michaelis constant (Km) for the free and and lactose, during immobilization gave an increased yield immobilized β-galactosidase was 2.51 mM and 5.18 mM, V of 190% in the case of dimethyladipimidate (DMA) cross- respectively. Moreover, the max for the soluble and immobi- − − linked preparation [58]. K. marxianus cells having lactase lized enzyme was 4.8×10 4 mol/min and 4.2×10 4 mol/min, activity were entrapped in calcium pectate gel (CPG) and in respectively [99]. calcium alginate gel (CAG) hardened by polyethyleneimine The main problems associated with this type of immo- and glutaraldehyde. Permeabilized cells entrapped in CPG bilization process are desorption of β-galactosidase from hydrolyzed lactose more than 80% in semicontinuous and immobilization matrix and the leakage of the entrapped continuous processes [94]. enzyme due to a small molecular weight compared to pores The comparison of the various methods of immobiliza- of gel in matrices, which can be overcome by cross-linking tion of β-galactosidase from Thermus aquaticus indicated using bifunctional or multifunctional reagents [100]. The that immobilization by cross-linking followed by entrapment conditions for polyethyleimine- (PEI-) coating of agarose β in agarose beads can be beneficial for high enzyme loading supports to achieve a -galactosidase derivative have been with good activity yield [59]. The entrapment of A. oryzae β- optimized that allows a high lactose conversion from whey galactosidase in a spongy polyvinyl alcohol cryogel increased in a steady bed-reactor with no enzyme leakage, together the stability towards temperature, pH, and ionic strength with good elution properties [101]. Various cross-linking β more than the free enzyme [60]. The fibers composed of reagents used for improvement of -galactosidase stability in alginate and gelatin hardened with glutaraldehyde retained immobilized state are described in Table 3 [102–110]. 56% relative activity of β-galactosidase for 35 days without any decrease. Moreover, the optimum conditions were also 3.3. Covalent Binding Method. Covalent binding is the not affected by immobilization [95]. Another approach for retention of enzymes on support surface by covalent bond Enzyme Research 7 formation. Enzyme molecules bind to support material via The comparison of a new and commercially available certain functional groups such as amino, carboxyl, hydroxyl, amino-epoxy support (amino-epoxy-Sepabeads) to conven- and sulfydryl groups. These functional groups must not be tional epoxy supports to immobilize β-galactosidase from A. in the active site. It is often advisable to carry out the immo- oryzae showed that the enzyme stability can be significantly bilization in the presence of its substrate or a competitive improved by the immobilization on this support, suggesting inhibitor so as to protect the active site Functional groups the promotion of some multipoint covalent attachment on support material usually activated by using chemical between the enzyme and the support [67]. The immobiliza- reagents such as cyanogen bromide, carbodimide, and tion of thermophilic β-galactosidase on Sepabeads for lactose glutaraldehyde. hydrolysis showed decrease in product inhibition, which can Eggshells ground into pieces can be good carrier for be helpful in improving the industrial performance of the immobilization of β-galactosidase because of its low cost, enzyme [55]. good mechanical strength, and resistance to microbial attack Alginate-chitosan core-shell microcapsules have also [61]. Fungal enzyme from A. oryzae has been immobilized been used for the immobilization of β-galactosidase [68]. onto powdered nylon-6 and zeolite [62]. Zeolites were non- The rate of 2-nitrophenyl β-galactopyranoside conversion ideal since its coupling yield was low whereas nylon resulted to 2-nitrophenol was faster in the case of calcium alginate- in a stable matrix. The derivatives obtained either by diazo chitosan microcapsules as compared to barium alginate- or by carbodiimide coupling showed the highest activities chitosan microcapsules. Barium alginate-chitosan microcap- during immobilization of the enzyme on glycophase-coated sules, however, did improve the stability of the enzyme at porous glass [103]. 37◦C relative to calcium alginate-chitosan microcapsules or E. coli β-galactosidase immobilized onto gelatin using free enzyme. chromium (III) acetate and glutaraldehyde retained the Among the three different models (without protection relative activities of 25% and 22% for glutaraldehyde and and molecular imprinting technique pretreatment) accom- chromium (III) acetate immobilized enzyme, respectively plished for the encapsulation of β-galactosidase, the highest [64]. The enzyme immobilized on silica-alumina was more enzymatic activity of enzyme was obtained with molecular stable than the free form at acidic pH [65]. The ratio imprinting technique [114]. The free lactase has been of protein to polymer also plays an important role dur- cross-linked into Fe3O4-chitosan magnetic microspheres for ing enzyme immobilization and 100% binding of protein lactulose synthesis by dual-enzymatic method in organic- to polymer can be obtained using optimal conditions aqueous two-phase media using lactose and fructose as the [66]. raw materials [115]. The organic-aqueous media can signifi- The performance of immobilization of the thermostable cantly promote the transglycosidation activity of lactase and β-galactosidase from Thermus sp. strain T2 on a stan- therefore improves the lactulose yield. dard Sepabeads-epoxy support with other Sepabeads-epoxy Immobilization technology has shown promising role in supports partially modified with boronate, iminodiacetic, reducing the product inhibition of β-galactosidase. A. oryzae metal chelates, and ethylenediamine was compared [109]. enzyme immobilized on chitosan beads was more effective Immobilization yields depended on the support, ranging as compared to nylon membranes to reduce the galactose from 95% using Sepabeads-epoxy-chelate, Sepabeads-epoxy- inhibition [116]. Immobilization of the enzyme on hetero- amino, or Sepabeads-epoxy-boronic to 5% using Sepabeads- functional epoxy Sepabeads (boronate-epoxy-Sepabeads and epoxy-IDA. The immobilized β-galactosidase derivatives chelate-epoxy-Sepabeads) has shown considerable results in showedveryimprovedbutdifferent stabilities after favor- reducing the product inhibition [55]. The effect of internal ing multipoint covalent attachment by long-term alkaline mass transfer and product (galactose) inhibition on a incubation, the enzyme immobilized on Sepabeads-epoxy- simulated immobilized enzyme-catalyzed reactor for lactose boronic being the most stable. The optimal derivative was hydrolysis has been studied [104]. A general mathematical very active in lactose hydrolysis even at 70◦C, maintaining its model has been developed for predicting the performance activity after long incubation times under these conditions. and simulation of a packed-bed immobilized enzyme reactor Recently, the cross-linking of β-galactosidase on magnetic performing lactose hydrolysis, which follows Michaelis- beads prepared from different sources (Artemisia seed gum, Menten kinetics with competitive product (galactose) inhi- chitosan, and magnetic fluid) was done in the presence bition. The performance characteristics of a packed-bed- of glutaraldehyde and the effects of various preparation immobilized enzyme reactor have been analyzed taking into conditions on the activity of the immobilized β-galactosidase account the effects of various diffusional phenomena like were studied. The immobilized β-galactosidase resulted in an axial dispersion and internal and external mass transfer increase in enzyme stability [110]. limitations. The effects of intraparticle diffusion resistances, The heat stability of lactase can be increased through external mass transfer, and axial dispersion have been studied immobilization [66, 111, 112]. The effect of temperature and and their effects were shown to reduce internal effective- pH on the catalytic activity of immobilized β-galactosidase ness factor. A. oryzae β-galactosidase was immobilized on from K. lactis indicated that the temperature-activity curves the surface of a novel bioaffinity support: concanavalin are similar for both the free and immobilized enzymes [113]. A layered calcium alginate-starch beads. The immobilized However, the maximum activity of the immobilized enzyme β-galactosidase had a much higher Kiapp value than the was shifted from 40◦Cto50◦C compared with the free free enzyme, which indicated less susceptibility to product enzyme. inhibition by galactose [98]. 8 Enzyme Research

4. Applications of Immobilized β-Galactosidase The β-galactosidase from Bacillus circulans immobilized onto Duolite ES-762 displayed lactose conversion of >70% β Immobilized -galactosidases can be used in a number of in a continuous stirred tank reactor [120]. The immobilized ways to hydrolyze lactose in milk, whey/whey permeate, and β-galactosidase from A. oryzae in a packed bed reactor oligosaccharides synthesis. The choice of process technology displayed 80% of lactose hydrolysis in whey [121]whereas depends on the nature of the substrate, the characteristics of the immobilized β-galactosidase from Saccharomyces fragilis the enzyme, economics of production, and marketing of the resulted in a hydrolytic rate of 50% within 3 h in a recycling product. The primary characteristic, which determines the packed bed reactor [78]. Further, the operational stability choice and application of a given enzyme, is the operational was tested, with the system being used up to 5 times before pH range. Acid-pH enzymes from fungi are suitable for any significant drop in the activity. The addition of Mg2+ processing of acid whey and whey permeate whereas the and Mn2+ enhances the hydrolysis of ONPG and lactose by neutral-pH enzymes from yeasts and bacteria are suitable for β-galactosidase from K. lactis, but the rates of activation by processing of milk and sweet whey. each metal on both substrate were not the same [122]. The immobilized K. lactis β-galactosidase from onto CPC-silica 4.1. Hydrolysis of Milk/Whey. Lactose-hydrolyzed milk has (silanizated and activated with glutaraldehyde) and agarose been used for the preparation of flavoured milk, cheese, (activated with cyanylating agent) displayed 90% lactose and yoghurt. The hydrolysis of lactose in milk for food conversion in packed bed minireactors [72]. β-galactosidase processing also prevents lactose crystallization in frozen and entrapped in a copolymer gel of N-isopropylacrylamide and condensed milk products. Moreover, the use of hydrolyzed acrylamide was effective in hydrolysis of lactose at 5◦Cfor milk in yoghurt and cheese manufacture accelerates the production of low lactose milk. It has been observed that acidification process, because lactose hydrolysis is normally lactose conversion decreased the stability of milk casein the rate-limiting step of the process, which reduces the particles and increased its dispersity [123]. set time of yoghurt and accelerates the development of The kinetic model for the lactose using immobilized β- structure and flavour in cheese [1]. The quality of ice galactosidase from K. fragilis has also been developed. The milk and ice-cream was significantly improved by addition immobilized enzyme was active at a low temperature of 5◦C of lactozyme. It prevents the crystallization of lactose by and it could also be applied for the production of freeze dairy breaking into glucose and galactose and reduces sandiness products to avoid lactose crystallization and to enhance the [117]. digestibilityandflavourofsuchproducts[64]. High concentration of lactose in whey is a major K. fragilis β-galactosidase immobilized on silanized environmental problem since its disposal in local water porous glass modified by glutraldehyde binding retained streams increases the biological oxygen demand manifolds. more than 90% of its activity [124]. A lactose saccharification The hydrolysis of whey lactose is another important appli- of 86%–90% in whey permeate was achieved both in a cation of β-galactosidase in dairy industry. Concentrated batch process and in a recycling packed-bed bioreactor. K. hydrolyzed whey or whey permeates can be used as a lactis β-galactosidase immobilized onto graphite surface and sweetener in products such as canned fruit syrups and soft glutaraldehyde has been used as the cross-linking reagent drinks [1]. Various immobilizing agents employed for the with the specific activity yield of 17% and 25% while the immobilization of β-galactosidase along with hydrolysis of enzyme loading was 1.8 and 1.1 U/cm2 of the graphite exter- lactosehavebeensummarizedinTable4 [118, 119]. nal surface area, respectively. It was observed that specific Fungal β-galactosidase (Miles Chemie) immobilized in activity yield decreased with the increase of the enzyme polyvinyl alcohol gel was found more thermostable than loading [113]. Lactose hydrolysis by a β-galactosidase from soluble enzyme, retaining 70% of the activity after 24 h at Thermus sp. both in solution and immobilized on a com- 50◦C and 5% activity at 60◦C. A lactose hydrolysis of 75% mercial silica-alumina was studied [34]. Both the free and was obtained in 5-6 h and the degree of conversion decreased the immobilized enzymes are competitively inhibited by to 50% after 30 runs [93]. The studies on the hydrolysis of galactose, while glucose inhibited only the action of free lactose using immobilized β-galactosidase (Aspergillus niger) enzyme, in an uncompetitive way. The immobilization step on phenol-formaldehyde resin indicated that the optimal helped to eliminate the inhibition by glucose. Moreover, the temperature was found to be dependent on the operating immobilization reduced to a half the inhibitory action of time but not on the lactose concentration or the conversion galactose. In general, the immobilization reduced the activity [69]. of the enzyme but increased its thermal stability. The immobilized β-galactosidase from A. niger displayed The Lactozym (a commercially available enzyme prepa- 70% hydrolysis in skim milk at 40◦C, with a space time of ration of β-galactosidase obtained from K. fragilis) immo- 10 min [51]. The β-galactosidase enzyme of fungal sources bilized on cellulose beads has been used for the hydrolysis immobilized on hydrogels was used for whey hydrolysis of whey lactose (>90% conversion) and milk lactose (60% and 70%–75% hydrolysis was achieved within 7 h [118]. conversion) in 5 h and the immobilized enzyme could be The immobilization of β-galactosidase from A. oryzae on reused three times without any change in the performance of activated silica gel resulted in the most active immobilized the fluidized bed reactor [14]. The immobilized preparations preparation from TiCl3 and FeCl3-activated silica gel and of β-galactosidase from Thermus sp. resulted in hydrolysis resulted in 81 and 84% hydrolysis, respectively, in 4% lactose yield higher than 99%. These immobilized forms of β- solution [77]. galactosidase could be used in the total hydrolysis of lactose Enzyme Research 9

Table 4: Hydrolysis of lactose with various immobilizing techniques of β-galactosidase.

%Lactose Time of Microbial source Immobilizing agent References hydrolysis hydrolysis Fungal galactosidase (Miles Polyvinyl-alcohol 75% 5-6 h [93] Chemie) E. coli Polyacrylamide gel 47% 6 h [58] K. lactis Thiosulﬁnate/thiosulfonae 85%–90% 2.5 h [79] K. fragilis Cellulose beads >90% 5 h [14] K. lactis Cotton fabric 95% 2 h [82] Fungal β-galactosidase Hydrogels 70%–75% 7 h [116] K. marxianus Calcium alginate 84.8% 2.5 h [117] Concanavalin A layered calcium alginate-starch hybrid A. oryzae 89% 3 h [98] beads Bacillus stearothermophilus Chitosan >80% 2 h [54]

in milk or dairy whey even at 70◦C[55]. The hydrolysis A recombinant thermostable β-galactosidase from Bacil- of lactose by immobilized β-galactosidase has also been lus stearothermophilus immobilized onto chitosan using studied in a continuous flow capillary bed reactor by various Tris (hydroxymethyl) phosphine (THP) and glutaraldehyde temperatures. Based on the observed thermal deactivation resulted in >80% lactose hydrolysis in milk after 2 h of rate constants, at an operating temperature of 40◦C, only operation in a packed bed reactor. Thus, THP-immobilized 10% of the enzyme activity loss could be there in one year recombinant thermostable β-galactosidase from Bacillus [125]. stearothermophilus has the potential application for the pro- The β-galactosidase entrapment in liposomes showed duction of lactose-hydrolyzed milk [54]. Calcium alginate superior thermal stability at various ranges of temperature. entrapped β-galactosidase used for the hydrolysis of lactose Moreover, the proteolytic stability of the β-galactosidase from solution, milk, and whey in batch processes as well was enhanced by encapsulation in liposomes [126]. The as in continuous packed bed column. It was also observed entrapment of β-galactosidase in liposomes by dehydration- that entrapped cross-linked concanavalin A-β-galactosidase rehydration vesicle method has also been used to prevent an was more efficient in the hydrolysis of lactose present in immediate hydrolysis of lactose in milk [96]. A. oryzae β- milk (77%) and whey (86%) in batch processes as compared galactosidase was immobilized by three different techniques: to the entrapped soluble β-galactosidase [93]. Among the adsorption on celite, covalent coupling to chitosan, and two matrices (Sephadex G-75 and chitosan beads) tested aggregation by cross-linking and comparing the yield of for immobilization β-galactosidase (BGAL) from Pisum immobilized preparation, enzymatic characteristics, stability, sativum for lactose hydrolysis, chitosan-PsBGAL displayed and efficiency in oligosaccharide synthesis. Cross-linked higher rate of lactose hydrolysis in milk and whey at room enzyme aggregates of β-galactosidase were found effective temperature and 4◦C than Sephadex-PsBGA and is better in lactose hydrolysis yielding 78% monosaccharide in 12 h suited for industrial application based on its broad pH and [75]. K. lactis β-galactosidase immobilized on cotton fabric temperature optima, high temperature stability, reusability, using glutaraldehyde as the cross-linking reagent was used and so forth [56]. β-galactosidases (from K. lactis and A. for hydrolysis of lactose in whole milk and 95% of lactose oryzae) were also immobilized in poly(vinylalcohol) hydrogel conversion has been observed after 2 h of batch operation lens-shaped capsules LentiKats used for the production of [23]. D-galactose from lactose (200 g L−1) in the batch mode of K. lactis β-galactosidase was covalently immobilized onto a simultaneous saccharification and fermentation process a polysiloxane-polyvinyl alcohol magnet, using glutaralde- [7]. hyde as activating agent that presented a higher operational A. oryzae β-galactosidase was immobilized on an inex- and thermal stability than the soluble enzyme; so this pensive bioaffinity support, and concanavalin A-cellulose immobilized β-galactosidase was also effectively used for was used for the continuous hydrolysis of lactose from the hydrolysis of lactose from milk [83]. A. oryzae β- milk and whey. It was observed that the optimum pH galactosidase was immobilized on silica, the enzyme activity for soluble and immobilized β-galactosidase is 4.8 but the as well as stability has been evaluated, and the best immo- optimum temperature increased from 50 to 60◦C for the bilization results were obtained by using glutaraldehyde immobilized β-galactosidase. The immobilized enzyme had as support’s activator and enzyme stabilizer. Among the higher thermal stability at 60◦C[92]. Recently, a packed bed different treatments (microfiltration, thermal treatment, and reactor together with alginate entrapped permeabilized cells ultrafiltration) of whey, ultrafiltration was the best treatment (K. marxianus) has been used for hydrolysis of milk lactose towards a proper substrate solution for feeding the reactor in a continuous system, which resulted in 87.2% hydrolysis [84]. of milk lactose [127]. 10 Enzyme Research

4.2. Synthesis of Galacto-Oligosaccharides (GOSs). Besides oryzae β-galactosidase gave maximum trisaccharides yield hydrolytic action, β-galactosidase has also transferase activity (17.3% of the total sugar) using 20% (w/v) of lactose, within by which the enzyme produces and hydrolyses a series 2 h as compared to 10% with free enzyme and 4.6% with of oligosaccharides, which have a beneficial effect on the cross-linked aggregates [73]. growth of desirable intestinal microflora [12]. Moreover, the An immobilized-enzyme system using polyethylene- transferase reaction can be used to attach galactose to other imine, glutaraldehyde, and cotton cloth was studied and chemicals, resulting in formation of galacto-oligosaccharides compared the galacto-oligosaccharide production in free- (GOSs), and consequently have potential application in enzyme ultrafiltration and in immobilized-enzyme systems the production of food ingredients, pharmaceuticals, and [135]. In the immobilization process, approximately 50% other biological active compounds. Nowadays, oligosaccha- to approximately 90% enzyme inactivation was found with ride production becomes the interesting subject for the the combination of PEI and GA. Equivalent free- and researchers, because the oligosaccharides have beneficial immobilized-enzyme systems showed very similar maximum effect on human intestinal as “bifidus factor”—promoting GOS production of approximately 22% and approximately growth of desirable intestinal microflora. Oligosaccharides 20% (w/v) at approximately 15 to 17 min, 50% conversion are recognized as useful dietary tools for the modula- for free- and immobilized-systems, respectively. tion of the colonic microflora toward a healthy balance. The synthesis of galacto-oligosaccharides was optimized This usually involves selectively increasing the levels of with respect to lactose concentration and enzyme to sub- gut Bifidobacteria and Lactobacilli at the expense of less- strate ratio using immobilized A. oryzae β-galactosidase desirable organisms such as Escherichia coli, Clostridia,and [136]. In the sequential batch production of galacto- proteolytic bacteroides [128]. The amount and nature of oligosaccharides, biocatalyst efficiency was increased by oligosaccharides formed depend upon the several factors 190% with respect to the free enzyme in solution, and 8500 g including the enzyme source, the concentration and nature of galacto-oligosaccharides per gram of enzyme prepara- of the substrate, and reaction conditions [12, 129, 130]. The tion were produced after 10 batches. The immobilized A. yield of oligosaccharides can be increased by using higher oryzae β-galactosidase enzyme on magnetic Fe3O4–chitosan substrate concentrations or decreasing the water content (Fe3O4–CS) nanoparticles as support resulted in 15.5% [31]. (w/v) maximum yield of galacto-oligosaccharides [137]. β Although -galactosidase catalyzes both hydrolysis and The synthesis of galacto-oligosaccharides (GOSs) using A. transgalactosylation reactions, however, the process condi- oryzae β-galactosidase (free and immobilized) on magnetic ff tions for lactose hydrolysis and GOS synthesis are di erent. polysiloxane-polyvinyl alcohol (mPOS-PVA) has also been The reaction conditions for transgalactosylation should be carried out [138]. A maximum of 26% (w/v) of total sugars high lactose concentration, elevated temperature, and low was achieved at near 55% lactose conversion from 50% water activity in the reaction medium [130]. The tempera- (w/v) lactose solution at pH 4.5 and 40◦C. Trisaccharides ture, concentration of substrate, and enzyme origin play an accounted for more than 81% of the total GOS produced. important role in the enzymatic synthesis of oligosaccharides GOS formation was not considerably affected by pH and [131]. However, the influence of the initial lactose concen- temperature. The concentrations of glucose and galactose tration can be much larger [132, 133]. In general, more and encountered near maximum GOS concentration greatly larger galacto-oligosaccharides (GOSs) can be produced with inhibited the reactions and reduced GOS yield. higher initial lactose concentrations. The higher tempera- The packed bed reactor and a plug-flow reactor have tures can be beneficial in higher oligosaccharide yields. The been successfully used for continuous production of GOS higher yield at higher temperatures is an additional advan- from lactose using immobilized β-galactosidase [63, 90]. tage when operating at high initial lactose concentrations and The selectivity for GOS synthesis can be increased several- consequently elevated temperatures. Hence, immobilized β- fold under microwave irradiation, using immobilized β- galactosidase should be stable at high temperature, low water glucosidase and with added cosolvents such as hexanol [139]. content, and giving high transgalactosylation activity [134]. Recently, a new type of ceramic membrane reactor system Partially purified β-galactosidase from Bullera singularis using immobilized β-galactosidase (Kluyveromyces lactis) ATCC 24193 immobilized in Chitopearl BCW 3510 bead has been proposed for continuous enzymatic production has been used for the production of galacto-oligosaccharides of galactosyl-oligosaccharides (GOSs) from lactose, which (GOSs) from lactose in a packed bed reactor, which resulted resulted in maximum oligosaccharide (38%, w/w) when the in 55% (w/w) oligosaccharides with a productivity of average residence time was 24 min, with an initial 30% (w/w) 4.4 g/(L-h) during a 15-day operation [132]. The enzyme lactose concentration [140]. immobilized on tosylate cotton cloth was used in plug-flow reactor for continuous production of galacto-oligosaccharide from lactose. In general, more and larger GOS can be pro- 5. Scale-Up Issues duced with higher initial lactose concentrations. A maximum GOS production of 27% (w/w) of initial lactose was achieved For the production of lactose-free milk, the enzyme β- at 50% lactose conversion with 500 g/L of initial lactose galactosidase can be added directly to whole milk, but concentration. Tri-saccharides were the major types of GOS after complete lactose hydrolysis at a desired level, the formed, accounting for more than 70% of the total GOSs enzyme can be deactivated by heat treatment. Since the free produced in the reactions. The chitosan-immobilized A. enzyme cannot be reused, thus the resulting operation is Enzyme Research 11 notcosteffective. To overcome this problem, immobilized β- function, reduction of the colon cancer risk, and improved galactosidase is used for the hydrolysis of skim milk. After the intestinal health [146, 147]. Therefore, the public demand desired lactose hydrolysis is achieved, cream is added to the for their production is significantly increased together with hydrolysed milk to adjust its fat content. Although numerous the development of an effective and inexpensive GOS hydrolysis systems have been investigated, only few of them production. Major companies dealing with oligosaccharides have been scaled up and even fewer have been applied at an production (including GOS) are in Japan [148]. Recently, industrial or semi-industrial level. there is also an increasing trend of GOS production in The first company for the commercial hydrolysis of Europe. Besides lactulose and soybean oligosaccharides, all lactose in milk by using immobilized lactase was Centrale oligosaccharides are prepared by transglycosylation from del Latte of Milan, Italy, utilizing the SNAM Progetti mono and disaccharides or by controlled hydrolysis of technology. The process used an immobilized Saccharomyces polysaccharides [147]. (Kluyveromyces) lactis lactase entrapped in cellulose triac- etate fibres. Sumitomo Chemical, Japan, has also developed 6. Conclusions an immobilization process to immobilize β-galactosidase of fungal origin on the rugged surface of an amphoteric ion- β-galactosidase is one of the most important enzymes used exchange resin of phenol formaldehyde polymer and this in food processing, which offers nutritional, technological, technology was used by Drouin Cooperative Butter Factory and environmental applications. Enzyme immobilization for producing market milk and hydrolyzed whey [141]. provides enzyme reutilization and may result in increased Snow Brand’s factory has developed a rotary column activity by providing a more suitable microenvironment for reactor that could be used both as a stirred tank reactor the enzyme. Moreover, immobilized systems can provide and as a packed bed reactor [142].Thereactionratewas better enzyme thermostability and pH tolerance. However, greatly affected by the packing density of immobilized β- major problems associated with the immobilized enzyme galactosidase in the rotary column. This reactor can also system are microbial contamination, protein adherence, and overcome the problem of channeling or severe pressure drop. channeling. The periodic washing and pasteurization and If the hydrolysis of lactose was carried out in horizontal flow direction of feed can solve these problems to great rotary column, 70%–80% lactose hydrolysis was observed extent. The problem of microbial contamination can also and washing of the immobilized enzyme was carried out be solved by exploiting the temperature property of the for 36 cycles, which indicated that horizontal rotary column enzyme. The immobilized enzyme preparations showed up reactor was well suited for hydrolyzing lactose in milk with to 99% hydrolysis, and thus it can be applied successfully fibrous immobilized enzyme. From the pilot plant experi- for the hydrolysis of lactose in milk or whey. The isolation mentations, a commercial plant was set up at Snow Brand’s of pyschrophilic bacteria with cold active β-galactosidase has factory [142]. Although the immobilized β-galactosidase was opened up the possibility of processing of milk and whey washed with phosphate buffer solution and pasteurized with even at low temperatures. On the other side, thermostable Tego-51, the standard plate count of lactose hydrolyzed milk enzymes have the unique ability to retain their activity at increased sharply. higher temperatures for prolonged periods, and the process Thus, immobilized β-galactosidase technology is an is less prone to microbial contamination due to higher effective process for successful hydrolysis of lactose and it operating temperature. Thus, cold active and thermostable can overcome the problems associated with costs of soluble enzymes will have the great potential in the lactose hydrolysis enzyme. However, major problems associated with the and of particular interest to the researchers. Thus, immo- immobilized enzyme system are microbial contamination, bilization enzyme systems will certainly find greater role in protein adherence, and channeling. Therefore, for long-term future times for the hydrolysis of milk, whey, and synthesis operations using immobilized system, periodic washing, of galacto-oligosaccharides. and pasteurization are indispensable processes [143–145]. In immobilized enzyme system, protein adhered to the enzyme can be easily dissolved by using high and low Acknowledgment pH solutions, because the immobilized enzyme has high The authors acknowledge the financial support given by the durability over a wide range of pH. The immobilized enzyme Council for Scientific and Industrial Research (CSIR), New can be pasteurized with benzalkonium chloride (quaternary Delhi, India. ammonium salt) after removing the proteins. The use of acetic acid solution as a cleaning and pasteurizing agent instead of lactic acid can also be effective. The problem of References channeling observed in the packed column system can be overcome by changing the flow direction of feed during the [1] T. P. Shukla and L. E. Wierzbicki, “Beta-galactosidase technology: a solution to the lactose problem,” Food Science and operation [142, 144, 145]. Nutrition, vol. 25, pp. 325–356, 1975. Galacto-oligosaccharides are produced simultaneously [2]C.R.CarraraandA.C.Rubiolo,“Immobilizationofβ- during lactose hydrolysis due to transgalactosylation activity galactosidase on chitosan,” Biotechnology Progress, vol. 10, no. of β-galactosidase. Oligosaccharides/Bifidobacteria provide a 2, pp. 220–224, 1994. wide variety of health benefits, including anticarcinogenic [3]L.S.Tweedie,R.D.MacBean,andT.A.Nickerson,“Present effects, reduction in serum cholesterol, improved liver and potential uses for lactose and some lactose derivative,” 12 Enzyme Research

Food Technology Association of Australia, vol. 30, pp. 57–62, oligosaccharides synthesis,” Biotechnology Techniques, vol. 9, 1978. no. 8, pp. 601–606, 1995. [4]L.F.Pivarnik,A.G.Senecal,andA.G.Rand,“Hydrolyticand [21] A. Hoyoux, I. Jennes, P. Dubois et al., “Cold-adapted transgalactosylic activities of commercial beta-galactosidase β-galactosidase from the antarctic psychrophile Pseudoal- (lactase) in food processing,” Advances in food and nutrition teromonas haloplanktis,” Applied and Environmental Microbi- research, vol. 38, pp. 1–102, 1995. ology, vol. 67, no. 4, pp. 1529–1535, 2001. [5] S. B. Martinez and R. A. Speckman, “b-galactosidase treat- [22] Z. Nagy, T. Kiss, A. Szentirmai, and S. Biro,´ “β-galactosidase ment of frozen dairy product mixes containing whey,” of Penicillium chrysogenum: production, purification, and Journal of Dairy Science, vol. 71, pp. 893–900, 1988. characterization of the enzyme,” Protein Expression and [6] V. Gekas and M. Lopez-Leiva, “Hydrolysis of lactose: a Purification, vol. 21, no. 1, pp. 24–29, 2001. literature review,” Process Biochemistry, vol. 20, pp. 2–12, [23] A. El-Gindy, “Production, partial purification and some 1985. properties of β-galactosidase from Aspergillus carbonarius,” [7]B.Champluvier,B.Kamp,andP.G.Rouxhet,“Immobiliza- Folia Microbiologica, vol. 48, no. 5, pp. 581–584, 2003. tion of β-galactosidase retained in yeast: adhesion of the cells [24] Y. J. Cho, H. J. Shin, and C. Bucke, “Purification and bio- on a support,” Applied Microbiology and Biotechnology, vol. chemical properties of a galactooligosaccharide producing β- 27, no. 5-6, pp. 464–469, 1988. galactosidase from Bullera singularis,” Biotechnology Letters, [8] Z. Grosova,´ M. Rosenberg, M. Gdovin, L. Slavikov´ a,´ and vol. 25, no. 24, pp. 2107–2111, 2003. M. Rebros,ˇ “Production of d-galactose using β-galactosidase [25] Z. Li, M. Xiao, L. Lu, and Y. Li, “Production of non- and Saccharomyces cerevisiae entrapped in poly(vinylalcohol) monosaccharide and high-purity galactooligosaccharides by hydrogel,” Food Chemistry, vol. 116, no. 1, pp. 96–100, 2009. immobilized enzyme catalysis and fermentation with immo- [9] T. Maischberger, T. H. Nguyen, P. Sukyai et al., “Production bilized yeast cells,” Process Biochemistry,vol.43,no.8,pp. of lactose-free galacto-oligosaccharide mixtures: comparison 896–899, 2008. of two cellobiose dehydrogenases for the selective oxidation [26] R. Gul-Guven, K. Guven, A. Poli, and B. Nicolaus, “Purifi- of lactose to lactobionic acid,” Carbohydrate Research, vol. cation and some properties of a β-galactosidase from 343, no. 12, pp. 2140–2147, 2008. the thermoacidophilic Alicyclobacillus acidocaldarius subsp. [10] T. Sako, K. Matsumoto, and R. Tanaka, “Recent progress rittmannii isolated from Antarctica,” Enzyme and Microbial on research and applications of non-digestible galacto- Technology, vol. 40, no. 6, pp. 1570–1577, 2007. oligosaccharides,” International Dairy Journal,vol.9,no.1, [27] D. L. Lind, R. M. Daniel, D. A. Cowan, and H. W. Morgan, pp. 69–80, 1999. “β-Galactosidase from a strain of the anaerobic thermophile, [11] J. H. German, “Applied enzymology of lactose hydrolysis,” Thermoanaerobacter,” Enzyme and Microbial Technology, vol. Milk Powders for the Future, pp. 81–87, 1997. 11, no. 3, pp. 180–186, 1989. [12] R. R. Mahoney, “Galactosyl-oligosaccharide formation dur- [28] J. X. Feliu and A. Villaverde, “An optimized ultrasonication ing lactose hydrolysis: a review,” Food Chemistry, vol. 63, no. protocol for bacterial cell disruption and recovery of beta- 2, pp. 147–154, 1998. galactosidase fusion proteins,” Biotechnology Techniques, vol. [13] T. Finocchiaro, N. F. Olson, and T. Richardson, “Use of 8, no. 7, pp. 509–514, 1994. immobilized lactase in milk systems,” Advances in Biochem- [29]D.Bury,P.Jelen,andM.Kalab,´ “Disruption of Lactobacillus ical Engineering, vol. 15, pp. 71–88, 1980. delbrueckii ssp. bulgaricus 11842 cells for lactose hydrolysis [14] I. Roy and M. N. Gupta, “Lactose hydrolysis by LactozymTM in dairy products: a comparison of sonication, high-pressure immobilized on cellulose beads in batch and fluidized bed homogenization and bead milling,” Innovative Food Science modes,” Process Biochemistry, vol. 39, no. 3, pp. 325–332, and Emerging Technologies, vol. 2, no. 1, pp. 23–29, 2001. 2003. [30] S. Fernandes, B. Geueke, O. Delgado, J. Coleman, and [15] M. S. Joshi, L. R. Gowda, L. C. Katwa, and S. G. Bhat, R. Hatti-Kaul, “β-Galactosidase from a cold-adapted bac- “Permeabilization of yeast cells (Kluyveromyces fragilis)to terium: purification, characterization and application for lactose by digitonin,” Enzyme and Microbial Technology, vol. lactose hydrolysis,” Applied Microbiology and Biotechnology, 11, no. 7, pp. 439–443, 1989. vol. 58, no. 3, pp. 313–321, 2002. [16] M. Stred’ansky, M. Tomaska, E. Sturdik, and L. Krem- [31] W. Chen, H. Chen, Y. Xia, J. Zhao, F. Tian, and H. Zhang, nicky, “Optimization of β-galactosidase extraction from “Production, purification, and characterization of a potential Kluyveromyces marxianus,” Enzyme and Microbial Technol- thermostable galactosidase for milk lactose hydrolysis from ogy, vol. 15, no. 12, pp. 1063–1065, 1993. Bacillus stearothermophilus,” Journal of Dairy Science, vol. 91, [17] R. R. Mahoney, “Enzymes exogenous to milk in dairy, no. 5, pp. 1751–1758, 2008. b-D-galactosidase,” in Encyclopaedia of Dairy Sciences,H. [32] S. Osiriphun and P. Jaturapiree, “Isolation and characteri- Roginski, J. W. Fuquay, and P. F. Fox, Eds., vol. 2, pp. 907– zation of β-galactosidase from the thermophile B1.2,” Asian 914, Academic Press, London, UK, 2003. Journal of Food and Agro-Industry, vol. 1, pp. 5–143, 2009. [18] R. L. Brandao,˜ J. R. Nicoli, and A. F. Figueiredo, “Purification [33]C.Tari,F.I.Ustok,andS.Harsa,“Productionoffood and characterization of a beta-galactosidase from Fusarium grade β-galactosidase from artisanal yogurt strains,” Food oxysporum var. lini,” Journal of Dairy Science,vol.70,no.7, Biotechnology, vol. 24, no. 1, pp. 78–94, 2010. pp. 1331–1337, 1987. [34] M. Ladero, A. Santos, J. L. Garc´ıa,A.V.Carrascosa,B.C. [19] R. M. Adams, S. Yoast, S. E. Mainzer et al., “Characterization C. Pessela, and F. Garc´ıa-Ochoa, “Studies on the activity of two cold-sensitive mutants of the β-galactosidase from and the stability of β-galactosidases from Thermus sp strain Lactobacillus delbruckii subsp. bulgaricus,” Journal of Biolog- T2 and from Kluyveromyces fragilis,” Enzyme and Microbial ical Chemistry, vol. 269, no. 8, pp. 5666–5672, 1994. Technology, vol. 30, no. 3, pp. 392–405, 2002. [20] J. L. Berger, B. H. Lee, and C. Lacroix, “Immobilization [35] B. C. C. Pessela, A. Vian, C. Mateo et al., “Overproduction of of beta-galactosidases from Thermus aquaticus YT-1 for Thermus sp. strain T2 β-galactosidase in Escherichia coli and Enzyme Research 13

preparation by using tailor-made metal chelate supports,” Enzyme and Microbial Technology, vol. 40, no. 6, pp. 1451– Applied and Environmental Microbiology,vol.69,no.4,pp. 1463, 2007. 1967–1972, 2003. [51] A. P. Bakken, C. G. Hill Jr., and C. H. Amundson, “Use [36] B. C. C. Pessela, C. Mateo, A. V. Carrascosa et al., “One-step of novel immobilized β-galactosidase reactor to hydrolyze purification, covalent immobilization, and additional sta- the lactose constituent of skim milk,” Biotechnology and bilization of a thermophilic poly-his-tagged β-galactosidase Bioengineering, vol. 36, no. 3, pp. 293–309, 1990. from Thermus sp. strain T2 by using novel heterofunctional [52] A. Bodalo,´ E. Gomez,´ M. F. Maximo,´ J. L. Gomez,´ and chelate-epoxy sepabeads,” Biomacromolecules,vol.4,no.1, J. Bastida, “Immobilization of β-falactosidase by physical pp. 107–113, 2003. adsorption on chromosorb-W,” Biotechnology Techniques, [37]F.WidmerandJ.L.Leuba,“β-Galactosidase from Aspergillus vol. 5, no. 5, pp. 393–394, 1991. niger. Separation and characterization of three multiple [53] A. P.Bakken, C. G. Hill Jr., and C. H. Amundson, “Hydrolysis forms,” European Journal of Biochemistry, vol. 100, no. 2, pp. of lactose in skim milk by immobilized β-galactosidase 559–567, 1979. (Bacillus circulans),” Biotechnology and Bioengineering, vol. [38] J. M. MacPherson and G. G. Khachatourians, “Partial purifi- 39, no. 4, pp. 408–417, 1992. cation and characterization of β-galactosidase produced by [54]M.DiSerio,C.Maturo,E.DeAlteriis,P.Parascandola,R. Beauveria bassiana,” Biotechnology and Applied Biochemistry, Tesser, and E. Santacesaria, “Lactose hydrolysis by immo- vol. 13, pp. 217–230, 1991. bilized β-galactosidase: the effect of the supports and the [39] S. A. Shaikh, J. M. Khire, and M. I. Khan, “Characterization kinetics,” Catalysis Today, vol. 79-80, pp. 333–339, 2003. of a thermostable extracellular β-galactosidase from a ther- [55]B.C.CH.Pessela,C.Mateo,M.Fuentesetal.,“Theimmobi- mophilic fungus Rhizomucor sp,” Biochimica et Biophysica lization of a thermophilic β-galactosidase on Sepabeads sup- Acta, vol. 1472, no. 1-2, pp. 314–322, 1999. ports decreases product inhibition: complete hydrolysis of [40] Z. Nagy, T. Kiss, A. Szentirmai, and S. Biro,´ “β-galactosidase lactose in dairy products,” Enzyme and Microbial Technology, of Penicillium chrysogenum: production, purification, and vol. 33, no. 2-3, pp. 199–205, 2003. characterization of the enzyme,” Protein Expression and [56] A. Dwevedi and A. M. Kayastha, “Optimal immobilization Purification, vol. 21, no. 1, pp. 24–29, 2001. of β-galactosidase from Pea (PsBGAL) onto Sephadex and [41] R. R. Saad, “Purification and some properties of β- chitosan beads using response surface methodology and its galactosidase from Aspergillus japonicus,” Annals of Microbi- applications,” Bioresource Technology, vol. 100, no. 10, pp. ology, vol. 54, no. 3, pp. 299–306, 2004. 2667–2675, 2009. [42] P. Nakkharat and D. Haltrich, “Purification and charac- [57] M. Decleire, W. De Cat, N. Van Huynh, and J. C. Motte, terisation of an intracellular enzyme with β-glucosidase “Hydrolysis of whey by Kluyveromyces bulgaricus cells immo- and β-galactosidase activity from the thermophilic fungus bilized in stabilized alginate and in chitosan beads,” Acta Talaromyces thermophilus CBS 236.58,” Journal of Biotechnol- Biotechnologica, vol. 7, no. 6, pp. 563–566, 1987. ogy, vol. 123, no. 3, pp. 304–313, 2006. [58] S. K. Khare and M. N. Gupta, “Immobilization of E. coli [43] L. Biermann and M. D. Glantz, “Isolation and charac- β-galactosidase and its derivatives by polyacrylamide gel,” terization of β-galactosidase from Saccharomyces lactis,” Biotechnology and Bioengineering, vol. 31, no. 8, pp. 829–833, Biochimica Biophysisa Acta, vol. 167, no. 2, pp. 373–377, 1968. 1988. [44] M. J. Artolozaga, R. Jones, A. L. Schneider, S. A. Furlan, and [59] J. L. Berger, B. H. Lee, and C. Lacroix, “Immobilization M. F. Carvallo-Jones, “One step partial purification of b-D- of beta-galactosidases from Thermus aquaticus YT-1 for galactosidase from Kluyveromyces marxianus CDB002 using oligosaccharides synthesis,” Biotechnology Techniques, vol. 9, streamline-DEAE,” Bioseparation, vol. 7, pp. 137–143, 1998. no. 8, pp. 601–606, 1995. [45] Z. M. Nor, M. I. Tamer, M. Mehrvar, J. M. Scharer, M. [60] A. Rossi, A. Morana, I. Di Lernia, A. Ottombrino, and M. De Moo-Young, and E. J. Jervis, “Improvement of intracel- Rosa, “Immobilization of enzymes on spongy polyvinyl alco- lular β-galactosidase production in fed-batch culture of hol cryogels: the example of β-galactosidase from Aspergillus Kluyveromyces fragilis,” Biotechnology Letters, vol. 23, no. 11, oryzae,” Italian Journal of Biochemistry, vol. 48, no. 2, pp. 91– pp. 845–849, 2001. 97, 1999. [46] A. O. Ram´ırez Matheus and N. Rivas, “Production and partial [61] H. P. S. Makkar and O. P. Sharma, “Eggshell as a carrier for characterization of β-galactosidase from Kluyveromyces lactis enzyme immobilization,” Biotechnology and Bioengineering, grown in deproteinized whey,” Archivos Latinoamericanos de vol. 25, pp. 592–597, 1983. Nutricion, vol. 53, no. 2, pp. 194–201, 2003. [62] P. G. Pietta, D. Agnellini, and M. Pace, “Immobilization [47]E.Jurado,F.Camacho,G.Luzon,´ and J. M. Vicaria, “Kinetic of Aspergillus oryzae b-galactosidase on nylon and zeolites: models of activity for β-galactosidases: influence of pH, comparison of different methods,” in Proceedings of the 4th ionic concentration and temperature,” Enzyme and Microbial European Congress on Biotechnology, vol. 2, pp. 591–596, Technology, vol. 34, no. 1, pp. 33–40, 2004. 1989. [48] S. Dagbagli and Y. Goksungur, “Optimization of β- [63] S. Sungur and U. Akbulut, “Immobilisation of β-galacto- galactosidase production using Kluyveromyces lactis NRRL Y- sidase onto gelatin by glutaraldehyde and chromium(III) 8279 by response surface methodology,” Electronic Journal of acetate,” Journal of Chemical Technology and Biotechnology, Biotechnology, vol. 11, no. 4, 2008. vol. 59, no. 3, pp. 303–306, 1994. [49] R. Fernandez-Lafuente, “Stabilization of multimeric en- [64] M. Ladero, A. Santos, and F. Garc´ıa-Ochoa, “Kinetic model- zymes: strategies to prevent subunit dissociation,” Enzyme ing of lactose hydrolysis with an immobilized β- galactosidase and Microbial Technology, vol. 45, no. 6-7, pp. 405–418, 2009. from Kluyveromyces fragilis,” Enzyme and Microbial Technol- [50] C. Mateo, J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan, ogy, vol. 27, no. 8, pp. 583–592, 2000. and R. Fernandez-Lafuente, “Improvement of enzyme activ- [65] M. J. Hernaiz and D. H. G. Crout, “Immobilization/stabiliza- ity, stability and selectivity via immobilization techniques,” tion on Eupergit C of the β-galactosidase from B. circulans 14 Enzyme Research

and an α-galactosidase from Aspergillus oryzae,” Enzyme and immobilized on chitosan and nylon supports,” Enzyme and Microbial Technology, vol. 27, no. 1-2, pp. 26–32, 2000. Microbial Technology, vol. 23, no. 1-2, pp. 101–106, 1998. [66] V. Kery,´ S.ˇ Kucˇar,´ M. Matulova,´ and J. Haplova,´ “Immo- [81] N. Albayrak and S. T. Yang, “Immobilization of Aspergillus bilisation of β-d-galactosidase from Escherichia coli on oryzae β-galactosidase on tosylated cotton cloth,” Enzyme and cellulose beads and its use for the synthesis of disaccharide Microbial Technology, vol. 31, no. 4, pp. 371–383, 2002. derivatives,” Carbohydrate Research, vol. 209, no. C, pp. 83– [82] X. Li, Q. Z. K. Zhou, and X. D. Chen, “Pilot-scale lactose 87, 1991. hydrolysis using β-galactosidase immobilized on cotton [67]R.Torres,C.Mateo,G.Fernandez-Lorente´ et al., “A novel fabric,” Chemical Engineering and Processing: Process Intensi- heterofunctional epoxy-amino sepabeads for a new enzyme fication, vol. 46, no. 5, pp. 497–500, 2007. immobilization protocol: Immobilization-stabilization of β- [83]D.F.M.Neri,V.M.Balcao,˜ M. G. Carneiro-da-Cunha, galactosidase from Aspergillus oryzae,” Biotechnology Progress, L. B. Carvalho, and J. A. Teixeira, “Immobilization of β- vol. 19, no. 3, pp. 1056–1060, 2003. galactosidase from Kluyveromyces lactis onto a polysiloxane- [68] E. Taqieddin and M. Amiji, “Enzyme immobilization in polyvinyl alcohol magnetic (mPOS-PVA) composite for novel alginate-chitosan core-shell microcapsules,” Biomate- lactose hydrolysis,” Catalysis Communications, vol. 9, no. 14, rials, vol. 25, no. 10, pp. 1937–1945, 2004. pp. 2334–2339, 2008. [69] S. T. Yang and M. R. Okos, “Effects of temperature on [84] M. P. Mariotti, H. Yamanaka, A. R. Araujo, and H. C. lactose hydrolysis by immobilized β-galactosidase in plug- Trevisan, “Hydrolysis of whey lactose by immobilized β- flow reactor,” Biotechnology and Bioengineering, vol. 33, no. galactosidase,” Brazilian Archives of Biology and Technology, 7, pp. 873–885, 1989. vol. 51, no. 6, pp. 1233–1240, 2008. [70] N. Papayannakos, G. Markas, and D. Kekos, “Studies on [85] H. Hronska, M. Rosenberg, and Z. Grosova,´ “Milk lactose modelling and simulation of lactose hydrolysis by free and hydrolysis by beta-galactosidase immobilized in polyvinylal- immobilized β-galactosidase from Aspergillus niger,” The cohol hydrogel,” New Biotechnology, vol. 25, pp. 13–16, 2009. Chemical Engineering Journal,vol.52,no.1,pp.B1–B12, [86] W. Hartmeier, Immobilized Biocatalysts: An Introduction, 1993. Springer, Heidelberg, Germany, 1986. [71] C. R. Carrara and A. C. Rubiolo, “A method for evaluating [87] S. Sharma and H. Yamazaki, “Preparation of hydrophobic lactose hydrolysis in a fixed bed reactor with β-galactosidase cotton cloth,” Biotechnology Letters, vol. 6, no. 5, pp. 301–306, immobilized on chitosan,” Chemical Engineering Journal, vol. 1984. 65, no. 2, pp. 93–98, 1997. [88] B. Champluvier, B. Kamp, and P. G. Rouxhet, “Immobiliza- [72] C. Giacomini, A. Villarino, L. Franco-Fraguas, and F. tion of β-galactosidase retained in yeast: adhesion of the cells Batista-Viera, “Immobilization of β-galactosidase from on a support,” Applied Microbiology and Biotechnology, vol. Kluyveromyces lactis on silica and agarose: comparison of dif- 27, no. 5-6, pp. 464–469, 1988. ferent methods,” Journal of Molecular Catalysis - B Enzymatic, [89] M. Kminkova, A. Proskova, and J. Kucera, “Immobilization vol. 4, no. 5-6, pp. 313–327, 1998. of mold b-galactosidase,” Collection of Czechoslovak Chemical [73] R. Gaur, H. Pant, R. Jain, and S. K. Khare, “Galacto- Communications, vol. 53, pp. 3214–3219, 1988. oligosaccharide synthesis by immobilized Aspergillus oryzae [90] H.-J. Shin, J.-M. Park, and J.-W. Yang, “Continuous pro- β-galactosidase,” Food Chemistry, vol. 97, no. 3, pp. 426–430, duction of galacto-oligosaccharides from lactose by Bullera 2006. singularis β-galactosidase immobilized in chitosan beads,” [74] M. Banerjee, A. Chakrabarty, and S. K. Majumdar, “Immobi- Process Biochemistry, vol. 33, no. 8, pp. 787–792, 1998. lization of yeast cells containing β-galactosidase,” Biotechnol- [91] W. Chen, H. Chen, Y. Xia et al., “Immobilization of ogy and Bioengineering, vol. 24, no. 8, pp. 1839–1850, 1982. recombinant thermostable β-galactosidase from Bacillus [75] S. F. D’Souza, R. Kaul, and G. B. Nadkarni, “A method for the stearothermophilus for lactose hydrolysis in milk,” Journal of preparation of hen egg white beads containing immobilized Dairy Science, vol. 92, no. 2, pp. 491–498, 2009. biocatalysts,” Biotechnology Letters, vol. 7, no. 8, pp. 589–592, [92] S. A. Ansari and Q. Husain, “Lactose hydrolysis by β 1985. galactosidase immobilized on concanavalin A-cellulose in [76] O. Ariga, M. Kato, T. Sano, Y. Nakazawa, and Y. Sano, batch and continuous mode,” Journal of Molecular Catalysis “Mechanical and kinetic properties of PVA hydrogen immo- B, vol. 63, no. 1-2, pp. 68–74, 2010. bilizing β-galactosidase,” Journal of Fermentation and Bio- [93] K. Batsalova, K. Kunchev, Y. Popova, A. Kozhukharova, engineering, vol. 76, no. 3, pp. 203–206, 1993. and N. Kirova, “Hydrolysis of lactose by β-galactosidase [77] A. Kozhukharova, N. Kirova, K. Batsolva, S. Gargova, and immobilized in polyvinylalcohol,” Applied Microbiology and A. Dimova, “Immobilization of β-galactosidase on silica gel Biotechnology, vol. 26, no. 3, pp. 227–230, 1987. following a metal chelation method,” Khranit. Promst., vol. [94] M. Tomaska, P. Gemeiner, I. Materlin, E. Sturdik, and G. 40, pp. 43–46, 1991. Handrikova, “Calcium pectate gel beads for cell entrapment: [78] M. I. Gonzalez´ Siso and S. Suarez´ Doval, “Kluyveromyces a study on the stability of Kluyveromyces marxianus whole- lactis immobilization on corn grits for milk whey lactose cell lactase entrapped in hardened calcium pectate and cal- hydrolysis,” Enzyme and Microbial Technology, vol. 16, no. 4, cium alginate gels,” Biotechnology and Applied Biochemistry, pp. 303–310, 1994. vol. 21, no. 3, pp. 347–356, 1995. [79] K. Ovsejevi, V. Grazu,´ and F. Batista-Viera, “β-galactosidase [95] A. Tanriseven and S¸. Dogan,ˇ “A novel method for the from Kluyveromyces lactis immobilized on to thiolsulfinate/ immobilization of β-galactosidase,” Process Biochemistry, vol. thiolsulfonate supports for lactose hydrolysis in milk and 38, no. 1, pp. 27–30, 2002. dairy by-products,” Biotechnology Techniques, vol. 12, no. 2, [96] J. M. Rodr´ıguez-Nogales and A. D. Lopez,´ “A novel approach pp. 143–148, 1998. to develop β-galactosidase entrapped in liposomes in order [80] M. Portaccio, S. Stellato, S. Rossi et al., “Galactose com- to prevent an immediate hydrolysis of lactose in milk,” petitive inhibition of β-galactosidase (Aspergillus oryzae) International Dairy Journal, vol. 16, no. 4, pp. 354–360, 2006. Enzyme Research 15

[97] T. Haider and Q. Husain, “Calcium alginate entrapped containing oligosaccharides,” Process Biochemistry, vol. 38, preparations of Aspergillus oryzae β galactosidase: its stability no. 5, pp. 801–808, 2002. and applications in the hydrolysis of lactose,” International [113] Q. Z. K. Zhou and X. D. Chen, “Effects of temperature Journal of Biological Macromolecules, vol. 41, no. 1, pp. 72– and pH on the catalytic activity of the immobilized β- 80, 2007. galactosidase from Kluyveromyces lactis,” Biochemical Engi- [98] T. Haider and Q. Husain, “Immobilization of β-galactosidase neering Journal, vol. 9, no. 1, pp. 33–40, 2001. by bioaffinity adsorption on concanavalin A layered calcium [114] Z. Wu, M. Dong, M. Lu, and Z. Li, “Encapsulation of β- alginate-starch hybrid beads for the hydrolysis of lactose from galactosidase from Aspergillus oryzae based on “fish-in-net” whey/milk,” International Dairy Journal,vol.19,no.3,pp. approach with molecular imprinting technique,” Journal of 172–177, 2009. Molecular Catalysis B, vol. 63, no. 1-2, pp. 75–80, 2010. [99] T. Haider and Q. Husain, “Hydrolysis of milk/whey lactose by [115] X. Hua, R. Yang, W. Zhang, Y. Fei, Z. Jin, and BO. Jiang, β galactosidase: a comparative study of stirred batch process “Dual-enzymatic synthesis of lactulose in organic-aqueous and packed bed reactor prepared with calcium alginate two-phase media,” Food Research International, vol. 43, no. entrapped enzyme,” Chemical Engineering and Processing: 3, pp. 716–722, 2010. Process Intensification, vol. 48, no. 1, pp. 576–580, 2009. [116] M. Portaccio, S. Stellato, S. Rossi et al., “Galactose com- [100] A. Tanaka and T. Kawamoto, Cell and Enzyme Immobiliza- petitive inhibition of β-galactosidase (Aspergillus oryzae) tion, The American Society for Microbiology, Washington, immobilized on chitosan and nylon supports,” Enzyme and DC, USA, 1999. Microbial Technology, vol. 23, no. 1-2, pp. 101–106, 1998. [101] P. Gonzalez,´ F. Batista-Viera, and B. M. Brena, “Polyethylen- [117] D. K. Stevenson, J. S. Crawford, and J. O. Carroll, “Enzymatic imine coated agarose supports, for the reversible immobilisa- β hydrolysis of lactose in ice-cream,” Journal of Dairy Science, tion of -galactosidase from Aspergillus oryzae,” International vol. 66, pp. 75–78, 1983. Journal of Biotechnology, vol. 6, no. 4, pp. 338–345, 2004. [118] A. Kozhucharova, K. Baztosolova, Y. Papova, N. Kirova, D. [102] J. Rogalski, A. Dawidowicz, and A. Leonowicz, “Lactose Klysursky, and D. Simeonov, “Properties and employment hydrolysis in milk by immobilized β-galactosidase,” Journal of β-galactosidase immobilized on hydrogel from silica,” of Molecular Catalysis, vol. 93, no. 2, pp. 233–245, 1994. Bioteknologiya, vol. 1, pp. 61–64, 1990. [103] E. Dominguez, M. Nilsson, and B. Hahn-Hagerdal, “Car- [119] R. Panesar, P. S. Panesar, R. S. Singh, and M. B. Bera, “Appli- bodiimide coupling of β-galactosidase from Aspergillus cability of alginate entrapped yeast cells for the production of oryzae to alginate,” Enzyme and Microbial Technology, vol. 10, lactose-hydrolyzed milk,” Journal of Food Process Engineering, no. 10, pp. 606–610, 1988. vol. 30, no. 4, pp. 472–484, 2007. [104] A. E. Al-Muftah and I. M. Abu-Reesh, “Effects of internal [120] K. Nakanishi, R. Matsuno, K. Torii, K. Yamamoto, and mass transfer and product inhibition on a simulated immo- β bilized enzyme-catalyzed reactor for lactose hydrolysis,” T. Kamikubo, “Properties of immobilized -d-galactosidase Biochemical Engineering Journal, vol. 23, no. 2, pp. 139–153, from Bacillus circulans,” Enzyme and Microbial Technology, 2005. vol. 5, no. 2, pp. 115–120, 1983. [105] S. Ates¸andU.¨ Mehmetoglu,ˇ “A new method for immobi- [121] H. Plainer, B. G. Sprossler, and H. Uhlig, “Technical applica- lization of β-galactosidase and its utilization in a plug flow tion of immobilized lactase and amino acid acylase,” Hoppe- reactor,” Process Biochemistry, vol. 32, no. 5, pp. 433–436, Seyler’s Zeitschrift fur¨ Physiologische Chemie, vol. 365, pp. 1997. 1043–1047, 1984. [106] J. Szczodrak, “Hydrolysis of lactose in whey permeate by [122] Y.-S. Kim, C.-S. Park, and D.-K. Oh, “Lactulose production β immobilized β-galactosidase from Kluyveromyces fragilis,” from lactose and fructose by a thermostable -galactosidase Journal of Molecular Catalysis B, vol. 10, no. 6, pp. 631–637, from Sulfolobus solfataricus,” Enzyme and Microbial Technol- 2000. ogy, vol. 39, no. 4, pp. 903–908, 2006. [107] A. Tanriseven and S¸. Dogan,ˇ “A novel method for the [123] A Miezeliene, A Zubriene, S Budriene, G Dienys, and J. immobilization of β-galactosidase,” Process Biochemistry, vol. Sereikaite, “Use of native and immobilized β-galactosidase in 38, no. 1, pp. 27–30, 2002. the food industry,” Progress in Biotechnology, vol. 17, pp. 171– [108] N. Albayrak and S. T. Yang, “Immobilisation of beta- 175, 2000. galactosidase on fibrous matrix by polyethyleneimine [124] J. Szczodrak, “Hydrolysis of lactose in whey permeate by for production of galacto-oligosaccharides from lactose,” immobilized β-galactosidase from Kluyveromyces fragilis,” Biotechnology Progress, vol. 18, pp. 240–251, 2002. Journal of Molecular Catalysis B, vol. 10, no. 6, pp. 631–637, [109] B. C. C. Pessela, C. Mateo, M. Fuentes et al., “Stabilization 2000. of a Multimeric β-Galactosidase from Thermus sp. Strain T2 [125] R. S. Peterson, C. G. Hill, and C. H. Amundson, “Effects of by Immobilization on Novel Heterofunctional Epoxy Sup- temperature on the hydrolysis of lactose by immobilized β- ports Plus Aldehyde-Dextran Cross-Linking,” Biotechnology galactosidase in a capillary bed reactor,” Biotechnology and Progress, vol. 20, no. 1, pp. 388–392, 2004. Bioengineering, vol. 34, no. 4, pp. 429–437, 1989. [110] S. Zhang, S. Gao, and G. Gao, “Immobilization of β- [126] J. M. Rodriguez-Nogales and A. Delgadillo, “Stability galactosidase onto magnetic beads,” Applied Biochemistry and and catalytic kinetics of microencapsulated β-galactosidase Biotechnology, vol. 160, no. 5, pp. 1386–1393, 2010. in liposomes prepared by the dehydration-rehydration [111] R. F. H. Dekker, “Immobilization of a lactase onto a mag- method,” Journal of Molecular Catalysis B,vol.33,no.1-2, netic support by covalent attachment to polyethyleneimine- pp. 15–21, 2005. glutaraldehyde- activated magnetite,” Applied Biochemistry [127] R. Panesar, P. S. Panesar, R. S. Singh, and J. F. Kennedy, and Biotechnology, vol. 22, no. 3, pp. 289–310, 1989. “Hydrolysis of milk lactose in a packed bed reactor system [112] C. S. Chen, C. K. Hsu, and B. H. Chiang, “Optimization using immobilized yeast cells,” Journal of Chemical Technol- of the enzymic process for manufacturing low-lactose milk ogy & Biotechnology, vol. 86, no. 1, pp. 42–46, 2011. 16 Enzyme Research

[128] R. Fuller and G. R. Gibson, “Probiotics and prebiotics: column reactor,” Nippon Shokuhin Kogyo Gakkaish, vol. 38, microflora management for improved gut health,” Clinical p. 384, 1991. Microbiology and Infection, vol. 4, no. 9, pp. 477–480, 1998. [143] H. Hirohara, H. Yamamoto, E. Kawano, and T. Nagase, [129] J. E. Prenosil, E. Stuker, and J. R. Bourne, “Formation of “Continuous hydrolysis of lactose in skim milk and acid whey oligosaccharides during enzymatic hydrolysis of lactose— by immobilized lactose of Aspergillus oryzae,” in Proceedings part 1: sate of art,” Biotechnology and Bioengineering, vol. 30, of the 6th International Enzyme Engineering Conference, vol. no. 9, pp. 1019–1025, 1987. 6, pp. 295–297, 1982. [130] S. Zarate and M. H. Lopez-Leiva, “Oligosaccharide formation [144] Y. Honda and S. Takafuji, “Hydrolysis of lactose in recon- during enzymatic lactose hydrolysis: a literature review,” stituted skimmed milk using immobilized β-galactosidase Journal of Food Protection, vol. 53, pp. 262–268, 1990. (immobilized Sumylact). 1. Development of bioreactor,” [131] M. A. Boon, A. E. M. Janssen, and K. Van ’t Riet, “Effect of Report of Research Laboratory (Snow Brand Milk Products temperature and enzyme origin on the enzymatic synthesis Co Ltd). 83: 85, 1987. of oligosaccharides,” Enzyme and Microbial Technology, vol. [145] Y. Honda, K. Murata, S. Takafuji, S. Fujiwara, T. Iwasaki, 26, no. 2–4, pp. 271–281, 2000. and M. Kuwazuru, “Hydrolysis of lactose in reconstituted [132] N. Albayrak and S. T. Yang, “Production of galacto- skimmed milk using immobilized β-galactosidase (immobi- oligosaccharides from lactose by Aspergillus oryzae β- lized Sumylact). 2. Results of long term experiments in a pilot galactosidase immobilized on cotton cloth,” Biotechnology plant,” Report of Research Laboratory (Snow Brand Milk and Bioengineering, vol. 77, no. 1, pp. 8–19, 2002. Products Co Ltd). 85: 33, 1987. [133] S. Chockchaisawasdee, V. I. Athanasopoulos, K. Niranjan, [146] S. M. Hawkins, “Bifidobacteria in dairy products,” Cultured and R. A. Rastall, “Synthesis of galacto-oligosaccharide Dairy Products Journal, vol. 28, pp. 16–20, 1993. from lactose using β-galactosidase from kluyveromyces lactis: [147] R. A. Rastall and V. Maitin, “Prebiotics and synbiotics: studies on batch and continuous UF membrane-fitted biore- towards the next generation,” Current Opinion in Biotechnol- actors,” Biotechnology and Bioengineering,vol.89,no.4,pp. ogy, vol. 13, no. 5, pp. 490–496, 2002. 434–443, 2005. [148] M. Rivero-Urgell and A. Santamaria-Orleans, “Oligosaccha- [134] S. X. Chen, D. Z. Wei, and Z. H. Hu, “Synthesis of galacto- rides: application in infant food,” Early Human Development, oligosaccharides in AOT/isooctane reverse micelles by β- vol. 65, no. 2, pp. S43–S52, 2001. galactosidase,” Journal of Molecular Catalysis B, vol. 16, no. 2, pp. 109–114, 2001. [135] N. J. Matella, K. D. Dolan, and Y. S. Lee, “Comparison of galactooligosaccharide production in free-enzyme ultrafiltration and in immobilized-enzyme systems,” Journal of Food Science, vol. 71, no. 7, pp. C363–C368, 2006. [136] L. M. Huerta, C. Vera, C. Guerrero, L. Wilson, and A. Illanes, “Synthesis of galacto-oligosaccharides at very high lactose concentrations with immobilized β-galactosidases from Aspergillus oryzae,” Process Biochemistry, vol. 46, no. 1, pp. 245–252, 2011. [137] C. Pan, B. Hu, W. Li, YI. Sun, H. Ye, and X. Zeng, “Novel and efficient method for immobilization and stabilization of β-d- galactosidase by covalent attachment onto magnetic Fe3O4- chitosan nanoparticles,” Journal of Molecular Catalysis B, vol. 61, no. 3-4, pp. 208–215, 2009. [138] D. F. M. Neri, V. M. Balcao,˜ R. S. Costa et al., “Galacto- oligosaccharides production during lactose hydrolysis by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol,” Food Chemistry, vol. 115, no. 1, pp. 92–99, 2009. [139] T. Maugard, D. Gaunt, M. D. Legoy, and T. Besson, “Microwave-assisted synthesis of galacto-oligosaccharides from lactose with immobilized β-galactosidase from Kluyveromyces lactis,” Biotechnology Letters, vol. 25, no. 8, pp. 623–629, 2003. [140] M. Ebrahimi, L. Placido, L. Engel, K. S. Ashaghi, and P. Czermak, “A novel ceramic membrane reactor system for the continuous enzymatic synthesis of oligosaccharides,” Desalination, vol. 250, no. 3, pp. 1105–1108, 2010. [141] Y. Honda, M. Kako, K Abiko, and Y. Sogo, “Hydrolysis of lactose in milk,” in Industrial Application of Immobilized Biocatalysts,A.Tanaka,T.Tosa,andT.Kobayashi,Eds.,pp. 209–234, Marcel Dekker, New York, NY, USA, 1993. [142] Y Honda, H Hashiba, K Ahiko, and H. Takahashi, “Hydroly- sis of immobilized β-galactosidase using a horizontal rotary SAGE-Hindawi Access to Research Enzyme Research Volume 2010, Article ID 324184, 7 pages doi:10.4061/2010/324184

Research Article Screen-Printed Carbon Electrodes Modiﬁed by Rhodium Dioxide and Glucose Dehydrogenase

Vojtechˇ Polan, Jan Soukup, and Karel Vytrasˇ

Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentska´ 573, 532 10 Pardubice, Czech Republic

Correspondence should be addressed to Karel Vytras,ˇ [email protected]

Received 3 June 2010; Accepted 15 December 2010

Academic Editor: Raﬀaele Porta

Copyright © 2010 Vojtechˇ Polan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The described glucose biosensor is based on a screen-printed carbon electrode (SPCE) modiﬁed by rhodium dioxide, which functions as a mediator. The electrode is further modiﬁed by the enzyme glucose dehydrogenase, which is immobilized on the electrode’s surface through electropolymerization with m-phenylenediamine. The enzyme biosensor was optimized and tested in model glucose samples. The biosensor showed a linear range of 500–5000 mg L−1 of glucose with a detection limit of 210 mg L−1 (established as 3σ) and response time of 39 s. When compared with similar glucose biosensors based on glucose oxidase, the main advantage is that neither ascorbic and uric acids nor paracetamol interfere measurements with this biosensor at selected potentials.

1. Introduction The most methods often used involve detecting hydrogen peroxide (a product of most oxidases) and nicotinamide ade- There exists today an ever-increasing demand for fast, selec- nine dinucleotide (NADH) (a product of dehydrogenases) tive, reliable, and, above all, inexpensive analytical methods. resulting during the catalytic process. NADH oxidation on For food products, it is necessary to monitor whether or carbon electrodes requires high overvoltage (around 1.0 V). not microbial, or some other form of, contamination has This is a highly unfavorable phenomenon, as the impact occurred. Furthermore, it is necessary to monitor compli- of interferents (e.g., uric acid, ascorbic acid, paracetamol) ance with given technological procedures and whether the that are easily oxidized at a given overvoltage become stated raw materials were used [1].Theserequirementsplace most evident at such potentials. High overvoltage can be very great demands on the analysis of given samples. The suppressed by using a so-called mediator [5–9] that enables ffi analysis itself should be very fast, su ciently sensitive and the transfer of electrons between the enzymes active center, accurate, but also inexpensive. To meet these criteria, an or the product of the enzyme reaction, and the electrodes application of electrochemical biosensors seems to be a good surface. As the mechanism of NADH oxidation has not been alternative. fully explained, we have written it according to the generally Electrochemical biosensors combine two advantages: recognized mechanism [10], as shown in the formula below specificity of the enzyme to the given molecule and transfer (1): of the biochemical signal to an electrochemical signal [2]. − As a result, these biosensors are selective in establishing −e− • −H+ • −e NADH −−→ NADH+ −−→ NAD NAD+ aspecificsubstrate[3, 4]. By using these biosensors, it is (1) possible to determine a large number of substances even in slow medium fast complex matrices. Electrochemical biosensors often use redox enzymes The most important step in preparing a biosensor is that during catalysis of substrate splitting reactions. Most used of enzyme immobilization. Should an inadequate procedure redox enzyme’s are oxidases and dehydrogenases. There are for enzyme entrapment be chosen, its denaturation, indirect several methods for establishing a substrates concentration. inactivation, or washing from the electrode may occur. Many 2 Enzyme Research established immobilization techniques are currently used of buffer, stock, and standard solutions were of analytical that include physical and chemical immobilization. Choice of reagent grade and purchased from Lachema (Brno, Czech enzyme immobilization method depends upon the proper- Republic). Phosphate buffer was prepared by mixing aqueous ties of the enzyme, type of the mediator, conditions in which solutions of sodium dihydrogenphosphate and disodium the biosensor is to work, and, last but not least, the physical hydrogenphosphate (both 0.1 M) to achieve solutions of the properties of the analyte (or possibly the size of the molecules required pH values. The glucose stock solution (2.5 g L−1) to be determined). Due to its simplicity, immobilization was prepared and diluted appropriately. Solutions of ascorbic using electropolymerization [11–14] is one of the most acid and uric acid (both Aldrich, 50 mg L−1) were prepared commonly used techniques. Electropolymerization proceeds immediately before use. in a buffer solution that contains both a certain monomer and the enzyme itself which will be immobilized. A major 2.3. Electrode Preparation. Carbon ink (0.95 g, Gwent advantage of this technique is the possibility to regulate the C50905D1, Pontypool, UK) and corresponding catalyst thickness of the membrane formed. (0.05 g) were thoroughly mixed manually for 5 min and This article describes the preparation, optimization, and subsequently sonicated for 5 min. The resulting mixture analytical properties of an enzyme biosensor prepared using was immediately used for the fabrication of electrodes. the screen-printing technique, modified by rhodium dioxide, The working electrodes were prepared by screen-printing of and containing glucose dehydrogenase immobilized in a modified ink onto an inert laser pre-etched ceramic support layer of m-phenylenediamine (the main reason for this (113 × 166 × 0.635 mm, no. ADS96R, Coors Ceramics, selection was price of the substance when compared with Chattanooga, TN, USA). Thick layers of the modified carbon analogous o- or p-derivatives). The main advantage of this ink were formed by brushing the ink through an etched biosensor is that it works even at low input potentials, stencil (thickness 100 μm, electrode printing area 105 mm2) where contributions from other easily oxidizable or reducible with the aid of the spatula provided with the screen-printing molecules are negligible. device (SP-200, MPM, Franklin, MA, USA and/or UL 1505 A, Tesla, Czech Republic) onto the ceramic substrates. ◦ 2. Experimental The resulting plates were dried at 60 Cfor2h. 2.1. Instrumentation. A modular electrochemical system, 2.4. Enzyme Immobilization. Several types of immobilization AUTOLAB, equipped with modules PGSTAT 30 and ECD methods were tested with glucose oxidase, comprising (Ecochemie, Utrecht, Holand) was used in combination with entrapment in Nafion, cross-linking with glutaraldehyde, corresponding software (GPES, Ecochemie). immobilization using cellulose acetate, and electropolymer- The flow injection system consisted of a peristaltic pump ization of pyrrole or m-phenylenediamine. Subsequently, a (Minipuls 3, Gilson SA., France), a sample injection valve GDH enzyme together with cofactor NAD+ were immobi- (ECOM, Ventil C, Czech Republic), and a self-constructed lized using the best method, in terms of retaining enzyme thin-layer electrochemical flow-through cell. The working activity, response time, sensitivity, and dynamic range of electrode was fixed via rubber gaskets (thickness 0.6 mm) concentrations. directly to the back plate of the thin layer cell. The reference electrode was Ag/AgCl/3M KCl (RE-6, BAS, USA), and the 2.4.1. Entrapment in Nafion. An enzyme (GOx, 1 mg) was stainless steel back plate represented the counter electrode of dissolved in 20 μL of 0.1 M phosphate buffer (pH 7.5) and the cell. The responses were evaluated using the peak heights mixed with an equal amount of 0.05%, 0.5%, or 5% Nafion (differences between background and response current of the solution neutralized with ammonia to pH ∼7. The resulting analyte). Corresponding pH values were measured using a mixture (5 μL) was applied directly onto the active area of the portable pH-meter (CPH 52 model, Elteca, Turnov, Czech SPCE/RhO2 surface and air-dried for 30 min. Republic) equipped with a combined glass pH-sensor (OP- 0808P, Radelkis, Budapest, Hungary). The measuring cell 2.4.2. Immobilization in Cellulose Acetate. An enzyme (GOx, ff was calibrated using bu er solutions of the conventional 1 mg) was dissolved in 40 μL of 0.1 M phosphate buffer (pH activity scale. 7.5), and a volume of 3 μL of this solution was applied onto the active area of the SPCE/RhO2 surface and air-dried. 2.2. Chemicals, Reagents, and Solutions. Glucose oxidase (EC Subsequently, volumes of 3 μL of cellulose acetate solution 1.1.3.4. from Aspergillus niger, specific activity 198 U mg−1; in acetone (0.05%, 0.5%, 1.5%, or 3.0%) were applied onto GOx), glucose dehydrogenase (EC 1.1.1.47 from Pseu- the aforementioned enzyme layer and dried for 5 min. domonas sp., specific activity 277 U mg−1; GDH), Nafion (5% m/m solution in lower aliphatic alcohols), nicoti- 2.4.3. Cross-Linking with Glutaraldehyde. Volumes of 5, 10 namide adenine dinucleotide (NAD+) and its reduced or 20 μL of 5% glutaraldehyde (diluted with 0.1 M phosphate form (NADH), rhodium dioxide, acetate cellulose (M buffer, pH 7.5) were mixed with 1 μLof5%BSAandwith ∼37000 g moL−1), m-phenylenediamine, glutaraldehyde so- 35 μL, 30 μL, or 20 μL of the enzyme solution (1 mg of GOx lution (GA, 50 wt. % in H2O), bovine serum albumin (BSA; in 0.1 M phosphate buffer, pH 7.5). After thorough mixing, 5% solution) and pyrrole (98% solution) were purchased avolumeof3μL was applied onto an SPCE/RhO2 and air- from Aldrich. All other chemicals used for the preparation dried for 30 min. As a variant, cross-linking of the enzyme Enzyme Research 3 was also performed with GA vapor, whereby a volume of 3.5 3 μL of the enzyme solution (1 mg of GOx in 40 μLof0.1M 1 3 phosphate buffer, pH 7.5) was applied onto the SPCE, air- dried (30 min), and then the SCPE/RhO2 so treated was 2.5 enclosed overnight (17 hours) in a vial over 5% GA. 2 2 A) µ (

2.4.4. Electropolymerization with Pyrrole. An enzyme solu- I 1.5 tion (3 μL, 1 mg of GOx in 40 μL of 0.1 M phosphate buffer, 1 pH 7.5) was applied onto an SPCE/RhO2. After drying for 30 min, the electrode was dipped into the 5 mM solution of 0.5 pyrrole in 0.1 mM phosphate buffer, pH 6.0. Electropolymer- ization was performed at +0.75 V versus Ag/AgCl for 0.25, 0 0 200 400 600 800 1000 0.5, 1.0, or 2.5 min, respectively. Finally, the electrode was −1 washed with the phosphate buffer. Concentration (mg L ) Figure 1: Immobilization using Nafion. Measurement condition: 2.4.5. Electropolymerization with m-Phenylenediamine— input potential −0.2 V (versus Ag/AgCl); 0.1 M phosphate buffer GOx. The procedure applied was similar to that described (pH 7.5); measured with SPCE/RhO2/GOx; analysis in a batch in the previous paragraph, but, concerning deposition time, arrangement; concentration of Nafion: 1–0.5%, 2–5%. the electrode was polarized in 5 mM m-phenylenediamine for 0.5, 1.0, 5, 10, or 20 min, respectively. Additionally, electropolymerization was performed at +0.75 V versus Ag/AgCl from 5 mM m-phenylenediamine GOx solution stability and sensitivity to glucose [15]. For immobilizing (10 mL containing 1 mg of GOx) for 5 min. glucose oxidase, the following methods and substances were used: immobilization in polymer—Nafion or cellulose 2.4.6. Electropolymerization with m-Phenylenediamine— acetate; immobilization using cross-linking—glutaraldehyde GDH. Avolumeof3μLofNAD+ solution (3 mg in 40 μL and BSA; electropolymerization—pyrrole or phenylenedi- of 0.1 M pH 7.5 phosphate buffer) was applied onto the amine. The entire study devoted to entrapment of the SPCE/RhO2 electrode surface. After drying, the surface was enzyme was performed in a batch arrangement in a cell with overlayered with GDH solution (3 μL, 1 mg in 40 μLof a volume of 10 mL. Selected key factors were monitored for 0.1 M phosphate buffer) and dried for 45 min. An electrode each system: sensitivity, response time, and dynamic range. was then dipped into the 5 mM solution (10 mL) of m- phenylenediamine in 0.1 mM phosphate buffer (pH 6.0) containing the remaining 37 μLofNAD+ and 37 μLofGDH 3.1.1. Entrapment in Nafion. Figure 1 shows calibration solution, and it was left there for electropolymerization dependences obtained in immobilization of 0.5% GOx (5 min at +0.75 V versus Ag/AgCl). After washing with buffer, and 5% Nafion. The concentration of 0.05% was not ffi the electrode was prepared for measurements. su cient to properly entrap the enzyme and the enzyme was shortly washed into the solution, which prevented further measurements. The dynamic ranges for Nafion 2.5. Procedure. Measurements were performed by DC concentrations of 0.5% and 5% were almost identical. The amperometry using both flow injection and batch arrange- 0.5% Nafion, however, shows greater sensitivity to glucose ments. All operational variables were optimized, that is, and the response time here was the shortest, hovering around applied potential (from +0.6 to −0.3 V versus Ag/AgCl), pH 28 s. of phosphate buffer (5–9), and flow rate (0.1–1.5 mL min−1). Responses were evaluated using the peak heights (differences between background and response current of the analyte). 3.1.2. Immobilization of GOx by Cross-Linking with Glu- Injections of analyte were repeated at least three times. taraldehyde and BSA. This immobilization procedure is very popular and well-proven for the design of enzyme 2.6. Sample Processing. A sample of honey was prepared electrochemical biosensors, and, therefore, it was included by dissolving the given amount of honey (3.4 g or 4.4 g of in this study. Concentrations of 0.625%, 1.25%, and 2.5% forest honey) in 50 mL of 0.1 M phosphate buffer of pH glutaraldehyde were compared here in a mixture with 7.5. Similarly, a sample of syrup was prepared, that is, 2.9 g the enzyme. The possibility for enzyme immobilization of orange-flavored syrup was dissolved in 50 mL of 0.1 M using glutaraldehyde saturated vapors was examined as phosphate buffer of pH 7.5. For analysis, 200 μL of the well (Figure 2). The response time was shortest in the samples thus prepared were always taken. case of enzyme immobilization using saturated vapors— 30 s. While the response sensitivity to glucose decreased 3. Results and Discussion (poorer permeability of the analyte to the enzyme and poorer permeability of the metabolic product to the electrode’s 3.1. Effect of Enzyme Immobilization on Biosensor Response. surface) with increasing thickness of the GA layer, the Glucose oxidase was chosen as a test enzyme because of its dynamic range of the setting increased at the same time. 4 Enzyme Research

2 2.5 1

2 2 1 1.5 3 4 1.5 A) A) µ 1 µ ( ( I I 1 2 0.5 3 0.5 4

0 0 100 200 300 400 500 0 0 500 1000 1500 2000 −1 Concentration (mg L ) Concentration (mg L−1)

Figure 2: Immobilization using glutaraldehyde and BSA. Mea- Figure 4: Immobilization using electropolymerization with pyr- − surement condition: input potential 0.2 V (versus Ag/AgCl); role. Measurement condition: input potential −0.2 V (versus ﬀ 0.1 M phosphate bu er (pH 7.5); measured with SPCE/RhO2/GOx; Ag/AgCl); 0.1 M phosphate buﬀer (pH 7.5); measured with analysis in a batch arrangement; concentration of glutaraldehyde: SPCE/RhO2/GOx; analysis in a batch arrangement; time of elec- 1–0.625%, 2–1.25%, 3–2.5%, 4—immobilization with vapour of tropolymerization 1–0.5 min, 2–1 min, 3–0.25 min, and 4–2.5 min. glutaraldehyde.

2 disadvantage of this method, however, is its relatively long 1.8 response time of 240 s. Another problem is the existence of 1 1.6 a very narrow interval for usable concentrations of cellulose acetate for the enzyme immobilization within a range of 1.4 1% (Figure 3)—compared, for example, to Naﬁon with the 1.2 A)

µ choice of 0.5–5.0%. ( 1 I 0.8 3.1.4. Immobilization by Pyrrole Electropolymerization. Pyr- 2 0.6 role was polymerized on the electrodes surface for periods 0.4 of 0.25, 0.5, 1.0, and 2.5 min (Figure 4). The results show 0.2 that for the period of 2.5 min, a very strong polypyrrole 0 membrane is created which causes a slow transport of 0 200 400 600 800 1000 glucose molecules to the GOx enzyme and subsequently, Concentration (mg L−1) the transport of H2O2 to the electrodes surface. This is Figure 3: Immobilization using cellulose acetate. Measurement evidenced by lower responses to glucose and longer response condition: input potential −0.2 V (versus Ag/AgCl); 0.1 M phos- time. Another situation occurs for the period of 0.25 min. ffi phate buffer (pH 7.5); measured with SPCE/RhO2/GOx; analysis in The enzyme is not su ciently entrapped in this case, and, a batch arrangement; concentration of cellulose acetate in acetone: therefore, it is partially washed into the solution, which 1–1.5% and 2–0.5%. is again shown by very low responses. The best result was achieved using electropolymerization of pyrrole lasting 0.5 min. The responses are the highest here and the response 3.1.3. Immobilization Using Cellulose Acetate. For this study, time of 35 s is also acceptable. solutions at concentration of 0.05%, 0.5%, 1.5%, and 3% of cellulose acetate in acetone were used. The first disadvantage 3.1.5. Immobilization Using Electropolymerization with m- of this method of biosensor preparation is the need to use Phenylenediamine. The m-phenylenediamine was polymer- the relatively volatile acetone, which vaporized very quickly ized onto the electrodes surface for periods of 0.5, 1, 5, 10, while being pipetted and spread onto the electrodes surface. and 20 min. Furthermore, GOx was incorporated directly This resulted in an uneven distribution of the cellulose into the phosphate buffer solution with m-phenylenediamine acetate layer. Acetone further dissolved the binder in carbon and the electropolymerization was performed for 5 min. ink (of a resin type), which caused partial washing of the Figure 5 shows that the best response to glucose was achieved electrode. when the m-phenylenediamine was electropolymerized for Likewise in Nafion, the concentration of 0.05% was not 1 min. Shorter times were insufficient to entrap the enzyme sufficient to entrap properly the enzyme and no response to into the polymeric membrane. Longer times, however, glucose was thus observed. By contrast, at the concentration created a thicker membrane which slowed the processes, of 3% the response to glucose was observed only for low transporting the analyte to the enzyme and the metabolite concentrations of glucose up to 50 mg L−1; there was no to the electrodes surface, which was similar to the situation increase in response above this concentration. The crucial for pyrrole. Enzyme Research 5

2.5 0.7

1 0.6 2 2 0.5 3 1.5 0.4

A) 4 µ A) ( 5 µ 0.3 I (

1 I 0.2 6 0.5 0.1 0 0 −0.4 −0.2 0 0.2 0.4 0.6 0.8 − 0 500 1000 1500 2000 0.1 Potential (V) Concentration (mg L−1) Figure 6: Effect of potential on the biosensor response. Measure- Figure 5: Immobilization using electropolymerization with m- ment condition: glucose concentration 1000 mg L−1;batchvolume phenylenediamine. Measurement condition: input potential −0.2 V 200 μL; pH of the supporting electrolyte 7.5; flow rate 0.2 mL min−1; ff (versus Ag/AgCl); 0.1 M phosphate bu er (pH 7.5); measured measured on SPCE/RhO2/GDH; analysis in a flow arrangement. with SPCE/RhO2/GOx; analysis in a batch arrangement; time of electropolymerization 1–1 min, 2–10 min, 3–5 min, 4–0.5 min, 5– 5 min with addition of 1 mg GOx to electropolymerization mixture, 0.04 and 6–20 min. 1 0.02 2 0 In the case of electropolymerization with m-phenylene- −0.4 −0.2 0 0.2 0.4 0.6 0.8 diamine together with the enzyme directly in a phosphate −0.02 Potential (V) A) µ buffer solution, the sensitivity was almost the lowest and ( I −0.04 the response time was relatively long (100 s). However the dynamic range was greatest in this case −0.06

−0.08 3.1.6. Comparison of the Immobilization Techniques. Table 1 compares the various methods of immobilization. Ideally, a −0.1 biosensor should have the shortest-possible response time, the largest dynamic range of concentrations, and highly Figure 7: Effect of interferents on the biosensor response. Mea- sensitive responses to the given analyte. In practice, however, surement condition: ascorbic acid, and uric acid concentrations it is necessary to compromise and to favour one parameter 10 mg L−1; batch volume 200 μL; pH of the supporting electrolyte −1 over another according to the determination requirements. 7.5; flow rate 0.2 mL min ; measured on SPCE/RhO2/GDH; Since all the immobilizations listed show rather sensitive analysis in a flow arrangement; 1—ascorbic acid, 2—uric acid. responses to glucose, the decisive criteria are response time and dynamic range. The most appropriate method can therefore, be considered the electropolymerization with m- 3.2.1. Effect of the Potential on the Biosensor Response. Input phenylenediamine, which was used for immobilization of the potential is one of the most important parameters in the glucose dehydrogenase enzyme. amperometric determination of analytes since its choice affects the selectivity of the given biosensor. Figure 6 shows 3.2. Determination of Glucose by Glucose Dehydrogenase. the dependence of response on the operating potential From the methods of immobilization examined, that one (dependence of the peak size on the potential was observed − using electropolymerization with m-phenylenediamine was in the range of 0.3to+0.6VversusAg/AgClin0.15V selected for preparation of the given biosensor. When intervals). As is visible there, oxidation starts at around working with dehydrogenases, great emphasis must be given +0.15 V and the response increases with the increasing − to correctly executing the immobilization, because not only potential. Oxidation is also observed in the vicinity of 0.3 V, the enzymes but also their cofactors (NAD+ or NADP+) but this response is very low and, therefore, unsuitable for − are immobilized. These cofactors are soluble in aqueous determination of glucose. In the range of 0.2 V to +0.1 V, solutions and thus they wash rapidly into the solution, and the biosensor records no catalytic activity. As this shows, the especially when using flow analysis. The entire procedure most appropriate area for determination of glucose is in the ff for electrode preparation is described in Section2.4.6 (while range of +0.15 to +0.6 V (taking into consideration the e ect Section 3.1.5 stated that the best response to glucose was of interferents). reached where m-phenylenediamine was electropolymerized for 1 min, an electropolymerization time of 5 min was chosen 3.2.2. Effect of Interferents on the Biosensor Response. There here due to better entrapment of the NAD+ cofactor.) can be many interfering substances in the samples (such as 6 Enzyme Research

Table 1: Comparison of individual immobilization methods and their parameters.

Response∗ Linearity Response time Type of immobilization − (μA) (mg L 1) (s) Naﬁon (0.5%) 1.991 10–200 120 Glutaraldehyde vapors 1.054 10–200 30 Cellulose acetate (1.5%) 1.831 10–200 240 Pyrrole (0.5 min) 1.255 50–250 35 m-phenylenediamine (1 min) 1.260 10–500 25 ∗ Measured at glucose concentration of 200 mg L−1.

Table 2: Determination of glucose in real sample using SPCE/RhO2/GDH.

Proposed method Reference method

Sample n x ± R [%] n x ± R [%] uucrit Honey 4 33.84 ± 5.63 4 33.97 ± 2.16 0.017 0.406 Syrup 4 26.06 ± 4.70 4 24.31 ± 4.74 0.185 0.406 n:numberofmeasurements;x:arithmeticmean;R:range;ucrit and u: critical and calculated values of Lord’s test (selected probability—95%).

0.2 0.04

0.16 0.03

0.12 A) µ A) ( µ

I 0.02 ( 0.08 I

0.01 0.04

0 0 0 0.2 0.4 0.6 0.8 1 1.2 0 1000 2000 3000 4000 5000 6000 − Flow rate (mL min 1) Concentration (mg L−1)

Figure 8: Effect of flow rate on the biosensor response. Mea- Figure 9: Biosensor response to glucose at input potential +0.35 V. surement condition: glucose concentration 1000 mg L−1;batch Measurement condition: batch volume 200 μL; input potential volume 200 μL; input potential 0.45 V (versus Ag/AgCl); pH of the 0.35 V (versus Ag/AgCl); pH of the supporting electrolyte 7.5; −1 supporting electrolyte 7.5; measured on SPCE/RhO2/GDH; analysis flow rate 0.5 mL min ; measured on SPCE/RhO2/GDH; analysis − in a flow arrangement. in a flow arrangement; regression equation: y = 5.00 × 10 6x + 0.0129, R2 = 0.991. blood and food). The most important interferents include is unstable (decrease of the response by 20% over three ascorbic acid, uric acid and paracetamol. It has been determinations). This response instability was probably observed that all of these are electroactive at the applied caused by passivation of the electrode’s surface. For this potential of +0.5 V, but, in the potential window of −0.2 to reason, a flow rate ranging from 0.4 to 0.6 mL min−1 seemed +0.45 V, their responses are negligible (Figure 7). For this ideal. For other measurements, the flow rate of 0.5 mL min−1 reason, potentials in the given range were chosen for further was chosen. That seems to be a good compromise between work. buffer consumption, response stability, and speed of the experiment. 3.2.3. Effect of Flow Rate and pH on the Biosensor Response. Optimization of pH was carried out in the range of 5 to Flow rate also belongs among the very important parameters 9. Stable responses were observed at all measured pH values that must be optimized. It was done in the range of and the highest was achieved at pH 8, where at the same time 0.1 mL min−1 to 1 mL min−1. Figure 8 shows that the size the maximum enzyme activity is seen. For further work, the of the response decreases with an increasing flow rate. This pH of 7.5 was chosen because the given pH is close to the is due to the fact that if the flow rate is too high, the physiological pH and that is optimal for the determination NADH+ on the electrode is not fast enough to react. On of biological substances in food and especially in clinical the other hand, at low flow rates, the biosensors response samples. Enzyme Research 7

3.2.4. Biosensor Response to Glucose. Calibration depen- [3] G. J. Moody, G. S. Sangbera, and J. D. R. Thomas, “Chemically dences were measured at two different potentials (+0.35 V immobilised bi-enzyme electrodes in the redox mediated and +0.45 V). At the potential of +0.35 V, the biosensor mode for the low flow injection analysis of glucose and showed lower responses, but the dynamic range was greater hypoxanthine,” Analyst, vol. 112, no. 1, pp. 65–70, 1987. than at the potential of +0.45 V. A big advantage is that at [4] H. Okuma, H. Takahashi, S. Sekimukai, K. Kawahara, and the input potential of +0.35 V, the effects of interferents are R. Akahoshi, “Mediated amperometric biosensor for hypoxanthine based on a hydroxymethylferrocene-modified carbon much more suppressed. The proposed biosensor retained its paste electrode,” Analytica Chimica Acta, vol. 244, no. 2, pp. activity after more than 50 injections. No loss of the original ◦ 161–164, 1991. signal was achieved after 1 month, when stored at 6 C in the [5] F. Ricci, A. Amine, D. Moscone, and G. Palleschi, “A probe refrigerator. for NADH and HO amperometric detection at low applied potential for oxidase and dehydrogenase based biosensor 3.3. Real Samples. Honey and syrup samples were used applications,” Biosensors and Bioelectronics,vol.22,no.6,pp. as real analytes. Measurement was performed under these 854–862, 2007. optimized conditions: input potential +0.35 V; batch vol- [6]M.C.Rodr´ıguez and G. A. Rivas, “An enzymatic glucose ume 200 μL; 0.1 M phosphate buffer pH 7.5; flow rate biosensor based on the codeposition of rhodium, iridium, and − glucose oxidase onto a glassy carbon transducer,” Analytical 0.5 mL min 1in a three-electrode arrangement in the pres- Letters, vol. 34, no. 11, pp. 1829–1840, 2001. ence of SPCE/RhO2/GDH, where the enzyme was entrapped [7]P.Kotzian,P.Brazdilov´ a,´ K. Kalcher, and K. Vytras,ˇ “Determi- by m-phenylenediamine. The determined concentrations are nation of hydrogen peroxide, glucose and hypoxanthine using shown in Table 2. (bio)sensors based on ruthenium dioxide-modified screen- The amperometric determination with SPCE/RhO2/GOx printed electrodes,” Analytical Letters, vol. 38, no. 7, pp. 1099– was used as a reference method (carbon printed electrode 1113, 2005. modified by glucose oxidase and rhodium oxide—the [8] J. Razumiene, A. Vilkanauskyte, V. Gureviciene et al., “New enzyme immobilized by Nafion). Measurement conditions: bioorganometallic ferrocene derivatives as efficient mediators −0.2 V (versus Ag/AgCl); phosphate buffer pH 7.5; flow rate for glucose and ethanol biosensors on PQQ-dependent dehy- 0.2 mL min−1;batchvolume50μL. drogenases,” Journal of Organometallic Chemistry, vol. 668, no. 1-2, pp. 83–90, 2003. [9] B. Prieto-Simon,´ J. Macanas,´ M. Munoz,˜ and E. Fabregas,` 4. Conclusion “Evaluation of different mediator-modified screen-printed electrodes used in a flow system as amperometric sensors for A biosensor containing rhodium dioxide and glucose dehy- NADH,” Talanta, vol. 71, no. 5, pp. 2102–2107, 2007. drogenase enzyme was prepared using the screen-printing [10] A. CH. Pappas, M. I. Prodromidis, and M. I. Karayannis, technique. Various methods of enzyme immobilization were “Flow monitoring of NADH consumption in bioassays based tested, among which m-phenylenediamine electropolymer- on packed-bed reactors bearing NAD-dependent dehydroge- ization proved the best. It excelled with its response time, nases: determination of acetaldehyde using alcohol dehydro- sensitivity, and signal stability. The enzyme biosensor was genase,” Analytica Chimica Acta, vol. 467, no. 1-2, pp. 225–232, optimized and tested in model glucose samples and also 2002. applied to analyze real samples (honey, syrup). [11] H.-Y Chen and J.-J Xu, “Amperometric enzyme biosensors,” Good results in the determination of glucose in real in Encyclopedia of Sensors,C.A.Grimes,C.E.Dickey,and M. V. Pishko, Eds., vol. 1, pp. 145–167, American Scientific samples indicate, among other things, that the biosensor was ff Publishers, Stevenson Ranch, CA, USA, 2006. not a ected by any complicated sample matrix (ascorbic acid [12]S.A.Rothwell,C.P.McMahon,andR.D.O’Neill,“Effects and other oxidizable substances) and has prospects for use of polymerization potential on the permselectivity of poly(o- also for similar applications in the food industry and clinical phenylenediamine) coatings deposited on Pt-Ir electrodes for practice. biosensor applications,” Electrochimica Acta,vol.55,no.3,pp. 1051–1060, 2010. Acknowledgments [13] Y. Sha, Q. Gao, B. Qi, and X. Yang, “Electropolymerization of azure B on a screen-printed carbon electrode and its This paper was supported by the Ministry of Education, application to the determination of NADH in a flow injection Youth, and Sports of the Czech Republic (project MSM analysis system,” Microchimica Acta, vol. 148, no. 3-4, pp. 335– 341, 2004. 0021627502) and the Czech Science Foundation (project [14] X. G. Li, M. R. Huang, W. Duan, and Y. L. Yang, “Novel mul- 203/08/1536). tifunctional polymers from aromatic diamines by oxidative polymerizations,” Chemical Reviews, vol. 102, no. 9, pp. 2925– References 3030, 2002. [15] P.Kotzian, P.Brazdilov´ a,´ S. Rezkovˇ a,´ K. Kalcher, and K. Vytras,ˇ [1] M. Nistor and E. Csoregi,¨ “Biosensors for food analysis,” “Amperometric glucose biosensor based on rhodium dioxide- in Encyclopedia of Sensors,C.A.Grimes,C.E.Dickey,and modified carbon ink,” Electroanalysis, vol. 18, no. 15, pp. M. V. Pishko, Eds., vol. 1, pp. 353–369, American Scientific 1499–1504, 2006. Publishers, Stevenson Ranch, CA, USA, 2006. [2] A. P. F. Turner, I. Karube, and G. S. Wilson, Biosensors: Fundamentals and Applications, Oxford University Press, New York, NY, USA, 1987. SAGE-Hindawi Access to Research Enzyme Research Volume 2010, Article ID 415949, 5 pages doi:10.4061/2010/415949

Research Article Preparation of Antioxidant Enzymatic Hydrolysates from Honeybee-Collected Pollen Using Plant Enzymes

Margarita D. Marinova and Bozhidar P. Tchorbanov

Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, Building 9, BG-1113 Soﬁa, Bulgaria

Correspondence should be addressed to Margarita D. Marinova, [email protected]

Received 14 June 2010; Accepted 16 December 2010

Academic Editor: A. Pandey

Copyright © 2010 M. D. Marinova and B. P. Tchorbanov. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Enzymatic hydrolysates of honeybee-collected pollen were prepared using food-grade proteinase and aminopeptidases entirely of plant origin. Bromelain from pineapple stem was applied (8 mAU/g substrate) in the first hydrolysis stage. Aminopeptidase (0.05 U/g substrate) and proline iminopeptidase (0.03 U/g substrate) from cabbage leaves (Brassica oleracea var. capitata), and aminopeptidase (0.2 U/g substrate) from chick-pea cotyledons (Cicer arietinum L.) were involved in the additional hydrolysis of the peptide mixtures. The degree of hydrolysis (DH), total phenolic contents, and protein contents of these hydrolysates were as follows: DH (about 20–28%), total phenolics (15.3–27.2 μg/mg sample powder), and proteins (162.7–242.8 μg/mg sample powder), respectively. The hydrolysates possessed high antiradical scavenging activity determined with DPPH (42–46% inhibition). The prepared hydrolysates of bee-collected flower pollen may be regarded as effective natural and functional dietary food supplements due to their remarkable content of polyphenol substances and significant radical-scavenging capacity with special regard to their nutritional-physiological implications.

1. Introduction antioxidative and radical scavenging activity of plant food [5, 6]. An antioxidant defense system protects cells from Natural products and preparations for food and nutritional the injurious effects of free radicals. Furthermore, the supplementation or dietary purposes have gained increased biological, biochemical, physiological, pharmaceutical, and attention in recent years. Among them, honeybee-derived medicinal properties of polyphenol compounds have been apicultural products, such as pollen, have been applied extensively studied and have been reviewed by Rice-Evans for centuries in alternative medicine as well as in food et al. [7] in regard to their free-radical scavenging activity diets and supplementary nutrition due to their nutritional and multiple biological activities including vasodilatory, and physiological properties. Each pollen has its own anticarcinogenic, anti-inflammatory, antibacterial, immune- specificity, mainly linked to the floral species or culti- stimulating, antiallergic, antiviral, and estrogenic effects, as vars. Bee-collected pollens contain nutritionally essential well as being inhibitors of specific enzymes. substances including carbohydrates, proteins, amino acids, On the basis of these reports, we have prepared water- lipids, vitamins, mineral substances, and trace elements, but soluble fractions from honeybee-collected pollen and inves- also significant amounts of polyphenol substances mainly tigated their functional properties. As a result, high free- flavonoids which, furthermore, are regarded as principal radical scavenging activities against the DPPH free radical indicating ingredient substances of pollen and can be were exhibited. These results are comparable to the results used for setting up quality standards in relation to their reported for the antioxidant activities in red grapes (Vitis nutritional-physiological properties and for quality control vinifera, L.) extracts [8, 9]. Moreover, we showed that of commercially distributed pollen preparations [1–4]. It enzymatic hydrolysates from honeybee-collected pollens is well known that polyphenols are responsible for the possessed even higher antioxidative properties. In the present 2 Enzyme Research study, our aim was to prepare enzymatic hydrolysates using bovine serum albumin as standard. The total phenolic from honeybee-collected pollens using plant proteinase and content was determined by the Folin-Ciocalteu colorimetric aminopeptidases and, then, to investigate the antioxidant method using catechin as standard, and the absorbance was activities in these peptide samples. measured at 760 nm [16].

2. Materials and Methods 2.2.3. Radical Scavenging Activity. The antiradical power of bee-collected pollen and pollen hydrolysates was evaluated 2.1. Materials. The pollen loads were collected in 2009 from in terms of the hydrogen-donating or radical-scavenging theAjtosareabyhoneybeecolonies(Apis mellifera) settled in ability by the DPPH method [17], which is related to the hives with bottom-fitted pollen traps. The aminopeptidase inhibition in the initiation step of free radical processes. and proline iminopeptidase from cabbage leaves (Bras- DPPH (2,2-diphenyl-1- picrylhydrazyl) is a stable free radical sica oleracea var. capitata) and the aminopeptidase from that accepts an electron or hydrogen radical to become a chick-pea cotyledons (Cicer arietinum L.) were purified as stable diamagnetic molecule and, accordingly, is reduced in described by Marinova et al. [10, 11]. Bromelain from presence of an antioxidant (AH): pineapple stems (EC 3.4.22.4, 2 mAnsonU/mg), L-amino • • acid p-nitroanilides, Folin-Ciocalteu, and 1,1-diphenyl-2- DPPH +AH−→ DPPH − H+A· (1) picrylhydrazyl (DPPH), were purchased from Sigma-Aldrich Co.(St.Louis,USA). For the evaluation of the antioxidant activity of specific compounds or extracts, they are allowed to react with the stable DPPH radical in a methanol solution. In its radical 2.2. Methods form, DPPH has a characteristic absorbance at 515 nm, which disappears upon reduction by H gained from an 2.2.1. Preparation of Enzymatic Hydrolysates from Honeybee- antioxidant compound. Collected Pollen. Honeybee-collected pollens (28% protein) For the test, appropriate methanol stock solutions of the were added, suspended in 5 volumes of distilled water, and pollen preparations (500 mg/L) and DPPH (6 × 10−5 mol/L) homogenized (Ultra-Turax, IKA-Werke, Germany), and pH were prepared. Immediately after adding 0.3 mL of the of the suspension was adjusted at 7.0 using NH4OH. The pollen extract solution to 2.7 mL of the DPPH solution, the digestion was started by addition of 8 mAU/g bromelain at ◦ reduction of the DPPH-radical was measured by monitoring 37 C. After 4 hours, hydrolysis was stopped by boiling in continuously the decrease of absorption at 515 nm in the a microwave for 2 minutes. The additional hydrolysis was dark until stable absorption values were obtained (30 min). carried out by adding aminopeptidase (0.05 units/g sub- The antiradical activity was determined in terms of PI strate) and proline iminopeptidase (0.03 units/g substrate) values (% inhibition) which was calculated by the ratio of from cabbage leaves as well as aminopeptidase from chick- the decrease of absorption of the DPPH-pollen extract test pea cotyledons (0.2 units/g substrate) and incubating for two ◦ solution after a 30-minute reaction time (stable phase) to the hours at 37 C and pH 7.5 with constant stirring. Hydrolysis absorption value of the reference sample where an equivalent was stopped by boiling in a microwave for 2 minutes. volume of methanol was added, as defined according to the The obtained hydrolysates were centrifuged at 6000 × gfor ◦ formula: 30 min at 5 C (MLW K24 D, Germany) to remove the residue. The supernatant fractions were collected and freeze- At PI (% inhibition) = A0 − × 100, (2) dried. A0 where A is the absorbency of the DPPH-methanol solution 2.2.2. Assays of Enzymes’ Activities, Total Nitrogen, Total 0 (reference) and At is the absorbency of the DPPH-pollen Protein, and Total Phenolic Compounds. Aminopeptidases’ extract solution after 30 min of reaction time. activities were determined using L-leucine-p-nitroanilides as substrate [12]. After incubation for 10 min at 30◦Cin 0.05 M sodium phosphate buffer (pH 7.2–7.5), the liberated 2.2.4. SDS-Polyacrylamide Gel Electrophoresis. Sodium dode- p-nitroaniline was measured at 410 nm on a spectropho- cyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) tometer (UV-VIS Spectrophotometer, Shimadzu 1240). The was performed in 15% polyacrylamide gel using Tris- ff iminopeptidase activity was assayed spectrophotometrically glycine bu er, pH 8.3, according to Laemmli [18]. Rabbit β at 410 nm against L-proline-p-nitroanilide (Pro-p-NA) in muscle myosin (205 kDa), -galactosidase (116 kDa), rabbit 0.1 M Tris/HCl buffer (pH 8.0) for 20 min at 30◦C[13]. One muscle phosphorylase b (97 kDa), bovine serum albu- unit of enzyme activity was defined as the amount of enzyme min (66 kDa), lactate dehydrogenase (36.5 kDa), carbonic releasing 1 μmol of p-nitroaniline per minute. anhydrase (29 kDa), trypsin inhibitor (20 kDa), lysozyme The total protein content of the honeybee-collected (14 kDa), aprotinin (6.1 kDa), insulin a (3.4 kDa), and pollen was determined by the method of Kjeldahl using the insulin b (2.4 kD) were used as molecular weight marker equation: N × 6.25, where N is the total Kjeldahl nitrogen proteins. The gel was visualized by silver staining [19]. multiplied by a factor to arrive at protein content [14]. The protein concentration of the samples after hydrolysis was 2.2.5. Determination of the Degree of Hydrolysis (DH) and measured according to the method of Lowry et al. [15], Amino Acid Composition. Thedegreeofhydrolysiswas Enzyme Research 3

Table 1: The contents of protein and total phenolic components of enzymatic hydrolysates from honeybee-collected pollen.

Sample Protein (μg/mg sample) Total phenols (μg/mg sample) PI-value (%) Bee-pollen 162.7 ± 0.2 15.3 ± 0.3 28 ± 2 1BH 227.1 ± 0.3 21.5 ± 0.6 40 ± 1 2 APH1 238.8 ± 0.5 25.6 ± 0.7 44 ± 2 3 APH2 230.5 ± 0.6 24.1 ± 0.9 42 ± 2 4 APH3 242.8 ± 0.5 27.2 ± 0.6 46 ± 1 1 BH bromelain hydrolysate 2 APH1 cabbage aminopeptidase and proline iminopeptidase hydrolysate 3 APH2 chick-pea aminopeptidase hydrolysate 4 APH3 cabbage and chick-pea aminopeptidases hydrolysate

determined using 2,4,6-trinitrobenzenesulfonic acid [20]. A 220 sample solution (0.25 mL) is mixed with 2.0 mL of 0.2 M 116 sodium phosphate buﬀer (pH 8.2) and 2.0 mL of 0.1% 97 trinitrobenzenesulfonic acid, followed by incubation in the 55 dark for 60 min at 50◦C. The reaction is quenched by 36.5 adding 4.0 mL of 0.1 N HCl, and the absorbance is read at 29 340 nm. A 1.5 mM L-leucine solution is used as the standard. 20 Transformation of the measured leucine amino equivalents 14 to degree of hydrolysis is carried out by means of a standard 6.1 curve for each particular protein substrate. 3.4 The amino acid composition was determined after 2.5 50 min of hydrolysis at 165◦C with 6 N HCl, and the analysis was performed on HPLC Nova-Pak C18 (3.9 × 150 mm, 4 μm, Waters). The mobile phase consisted of eluent A 12 3 4 5 · (prepared from Waters AccQ Tag Eluent A concentrate, by Figure 1: SDS-polyacrylamide gel electrophoresis of molecu- adding 200 mL of concentrate to 2 L of Milli-Q water and lar weight markers and enzymatic hydrolysates from honeybee- mixing), eluent B (acetonitrile, HPLC grade), and eluent C collected pollen. (1) Molecular weight markers; (2) aminopepti- (Milli-Q water). The following conditions were used: linear dases hydrolysate (cabbage and chick-pea); (3) aminopeptidase and gradient of 100–0% eluent A, 0–60% eluent B, and 0–40% proline iminopeptidase hydrolysate (cabbage); (4) aminopeptidase eluent C in 30 min and then isocratic 100% of eluent A for hydrolysate (chick-pea); (5) bromelain hydrolysate. 20 min with a ﬂow rate of 1 mL/min.

3. Results and Discussion (Table 1). It suggests that the protein contents correlated closely with the contents of total phenolic components. 3.1. The Total Phenolic Contents and Protein Contents of Enzymatic Hydrolysates from Bee Pollen. The enzymatic hydrolysates from bee pollen were digested and prepared 3.2. DPPH Radical Scavenging Ability of Enzymatic using plant proteinase bromelain, and aminopeptidases from Hydrolysates from Pollen. Although native bee-collected cabbage leaves and chick-pea cotyledons. SDS-PAGE analysis pollen water extract shows considerable antiradical activity indicated that the pollen was perfectly digested by these (PI = 28% inhibition), the reduction of the DPPH radical enzymes (Figure 1). The degrees of hydrolysis of the bee- was signiﬁcantly increased by applying the obtained pollen pollen hydrolysates were as follows: about 20% for the hydrolysates (PI = 42–46% inhibition) as is shown in bromelain hydrolysate (BH), 26% for the cabbage aminopep- Table 1, which indicate an elevated free-radical scavenging tidase and proline iminopeptidase hydrolysate (APH1), 24% eﬃciency of the pollen hydrolysates. The highest degree for the chick-pea aminopeptidase hydrolysate (APH2), and of radical scavenging capacity was assessed in the APH3 28% for the hydrolysate obtained by the combination (PI = 46% inhibition) which, correspondingly, also has the of aminopeptidases from cabbage and chick-pea (APH3). highest concentration of polyphenol substances (27.2 μg/mg Total phenolic contents of these hydrolysates were as fol- sample powder). For this reason, it can be assumed that lows: 21.5 μg/mg sample (BH), 25.6 μg/mg sample (APH1), there is a general correlation between the content of total 24.1 μg/mg sample (APH2), and 27.2 μg/mg sample (APH3), polyphenolics and the free-radical scavenging capacity of the respectively (Table 1). On the other hand, the protein con- pollen preparations. tents of these hydrolysates were as follows: 227.1 μg/mg sam- Although, in comparison, tests with equivalent amount ple (BH), 238.8 μg/mg sample (APH1), 230.5 μg/mg sam- of the synthetic antioxidant gallic acid shows higher PI values ple (APH2), and 242.8 μg/mg sample (APH3), respectively of approximately 90%, it must be taken into consideration 4 Enzyme Research

Table 2: The amino acids composition of honeybee-collected to digest honeybee-collected pollen with a hard cell wall. In pollen and bee-pollen hydrolysate obtained by the combination of this process, consumer demand of honeybee-collected pollen ∗ aminopeptidases from cabbage and chick-pea ( ). for natural foods with medicinal effects such as antioxidative ∗ activity is increasing. Amino acids C (%) C (%) In conclusion, pollen extracts represent a concentrated Asp 7.5 6.6 nature-derived mixture of different active polyphenol com- Hyp 1.5 1.5 pounds which, according to practical applications as a Glu 8.6 7.8 bioactive diet component, are usually applied and consumed Ser 5.8 5.9 in higher amounts than the pure synthetic antioxidant food Gly 9.8 10.5 additives. His+Thr 5.1 5.0 Ala 8.0 7.9 Acknowledgment Arg 4.6 1.0 The authors thank the National Foundation for Scientific Pro 17.1 22.2 Research for financial support of Project TK-X 1608. Tyr 1.7 1.4 Val 6.7 6.7 References Met 2.2 2.1 Ile 5.3 5.1 [1] B. M. Talpay, Der Pollen, Eigenverlag Institut fur˝ Honig- Leu 7.9 7.5 forschung, Bremen, Germany, 1981. [2] R. G. Stanley and H. F. Linskens, Pollen: Biologie, Biochemie, Lys 3.4 2.6 Gewinnung und Verwendung, Urs Freund, Greifenberg, Ger- Phe 8.1 5.5 many, 1985. [3] G. Kroyer, “Flavonoids and phytosterols as bioactive substances in dietary applied pollen products,” in Proceedings of Euro Food Chem X: Functional Foods—A New Challenge for that pollen extract represents a concentrated nature-derived ff the Food Chemists, pp. 102–108, Publishing Company of TUB, mixture of di erent active polyphenol compounds. Budapest, Hungary, 1999. The results of the amino acids’ composition for the bee- [4] J. Kanner, E. Frankel, R. Granit, B. German, and J. E. pollen extract and the pollen hydrolysate obtained by combi- Kinsella, “Natural antioxidants in grapes and wines,” Journal nation of aminopeptidases from cabbage and chick-pea are of Agricultural and Food Chemistry, vol. 42, no. 1, pp. 64–69, shown in Table 2. Relatively high content of hydrophobic 1994. amino acids Pro, Phe, and Gly is characteristic for the bee- [5] N. Salah, N. J. Miller, G. Paganga, L. Tijburg, G. P. Bolwell, pollen extract and the APH3. The amounts of these amino and C. Rice-Evans, “Polyphenolic flavanols as scavengers of acids in bee-pollen hydrolysate after a 6-hour hydrolysis are aqueous phase radicals and as chain-breaking antioxidants,” approximately 22, 5.5 and 10.5%, respectively of the total Archives of Biochemistry and Biophysics, vol. 322, no. 2, pp. content. 339–346, 1995. [6] J. A. Vinson and B. A. Hontz, “Phenol antioxidant index: The honeybee products are considered to be abundant comparative antioxidant effectiveness of red and white wines,” sources of antioxidants. In honey, royal jelly, propolis, and Journal of Agricultural and Food Chemistry, vol. 43, no. 2, pp. bee pollens high antioxidant activity was found [21]. In 401–403, 1995. bee-collected pollen water extracts high radical-scavenging [7] C. A. Rice-Evans, N. J. Miller, and G. Paganga, “Structure- activity, activity against superoxide anion, and hydroxyl antioxidant activity relationships of flavonoids and phenolic radical-scavenging activity were reported [22–25]. acids,” Free Radical Biology and Medicine, vol. 20, no. 7, pp. Antioxidative ability of pollen seems to be due to 933–956, 1996. phenolic compounds. In the present investigations, a very [8]V.Briedis,V.Povilaityte,S.Kazlauskas,andP.R.Venskutonis, high antioxidant activity, expressed as radical-scavenging “Polyphenols and anthocyanins in fruits, grapes juices and activity corresponded to high levels of total phenols, was wines, and evaluation of their antioxidant activityPolifenoliu found in water-soluble extract and in plant proteinase and ir antocianinu kiekis vynuogese, vynuogiu sultyse ir raudon- uose vynuose bei ju antioksidacinio aktyvumo ivertinimas,” aminopeptidases hydrolysates. Medicina, vol. 39, pp. 104–112, 2003. [9] C. H. Chen, M. C. Wu, C. Y. Hou, C. M. Jiang, C. M. Huang, ff 4. Conclusion and Y. T. Wang, “E ect of phenolic acid on antioxidant activity of wine and inhibition of pectin methyl esterase,” Journal of the The use of honeybee-collected pollens as an alternative Institute of Brewing, vol. 115, no. 4, pp. 328–333, 2009. medicine is increasing due to their biologically active proper- [10] M. Marinova, A. Dolashki, F. Altenberend, S. Stevanovic, W. Voelter, and B. Tchorbanov, “Characterization of an ties that make them attractive as a source of essential amino aminopeptidase and a proline iminopeptidase from cabbage acids, vitamins, minerals, and antioxidants in human diets. leaves,” Zeitschrift fur Naturforschung, vol. 63, no. 1-2, pp. 105– Theusefulcomponentsfromhoneybee-collectedpollencan 112, 2008. be fully digested using the food-grade enzymes such as [11] M. Marinova, A. Dolashki, F. Altenberend, S. Stevanovic, bromelain, cabbage aminopeptidase and proline iminopep- W. Voelter, and B. Tchorbanov, “Purification and character- tidase, and chick-pea aminopeptidase, although it is not easy ization of L-phenylalanine aminopeptidase from chick-pea Enzyme Research 5

cotyledons (Cicer arietinum L.),” Protein and Peptide Letters, vol. 16, no. 2, pp. 207–212, 2009. [12] M. J. Chrispeels and D. Boulter, “Control of storage protein metabolism in the cotyledons of germinating mung beans: role of endopeptidase,” Plant Physiology, vol. 55, pp. 1031–1037, 1975. [13] T. Yoshimoto and D. Tsuru, “Proline iminopeptidase from Bacillus coagulans: purification and enzymatic properties,” Journal of Biochemistry, vol. 97, no. 5, pp. 1477–1485, 1985. [14] P. L. Kirk, “Kjeldahl method for total nitrogen,” Analytical Chemistry, vol. 22, no. 2, pp. 354–358, 1950. [15]O.H.Lowry,N.J.Rosebrough,A.L.Farr,andR.J.Randall, “Protein measurement with the Folin phenol reagent,” The Journal of Biological Chemistry, vol. 193, no. 1, pp. 265–275, 1951. [16] K. Slinkard and V. L. Singleton, “Total phenol analysis,” American Journal of Enology and Viticulture, vol. 28, pp. 49– 55, 1977. [17] W. Brand-Williams, M. E. Cuvelier, and C. Berset, “Use of a free radical method to evaluate antioxidant activity,” Food Science and Technology, vol. 28, no. 1, pp. 25–30, 1995. [18] U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature, vol. 227, no. 5259, pp. 680–685, 1970. [19] D. W. Sammmons, L. D. Adams, and E. E. Nishizawa, “Ultrasensitive silver-based color staining of polypeptides in polyacrylamide gels,” Electrophoresis, vol. 2, pp. 135–141, 1980. [20] J. Adler-Nissen, “Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid,” Journal of Agricultural and Food Chemistry,vol.27,no.6,pp. 1256–1262, 1979. [21] T. Nagai, M. Sakai, R. Inoue, H. Inoue, and N. Suzuki, “Antioxidative activities of some commercially honeys, royal jelly, and propolis,” Food Chemistry, vol. 75, no. 2, pp. 237– 240, 2001. [22] M. G. Campos, R. F. Webby, and K. R. Markham, “The unique occurrence of the flavone aglycone tricetin in Myrtaceae pollen,” Zeitschrift fur Naturforschung C, vol. 57, no. 9-10, pp. 944–946, 2002. [23]M.G.Campos,R.F.Webby,K.R.Markham,K.A.Mitchell, and A. P. Da Cunha, “Age-induced diminution of free radical scavenging capacity in bee pollens and the contribution of constituent flavonoids,” Journal of Agricultural and Food Chemistry, vol. 51, no. 3, pp. 742–745, 2003. [24] M. Leja, A. Mareczek, G. Wyzgolik, J. Klepacz-Baniak, and K. Czekonska,´ “Antioxidative properties of bee pollen in selected plant species,” Food Chemistry, vol. 100, no. 1, pp. 237–240, 2007. [25] T. Nagai, R. Inoue, N. Suzuki, T. Myoda, and T. Nagashima, “Antioxidative ability in a linoleic acid oxidation system and scavenging abilities against active oxygen species of enzymatic hydrolysates from pollen Cistus ladaniferus,” International Journal of Molecular Medicine, vol. 15, no. 2, pp. 259–263, 2005. SAGE-Hindawi Access to Research Enzyme Research Volume 2010, Article ID 517283, 5 pages doi:10.4061/2010/517283

Research Article Characterization of Activity of a Potential Food-Grade Leucine Aminopeptidase from Kiwifruit

A. A. A. Premarathne and David W. M. Leung

School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand

Correspondence should be addressed to David W. M. Leung, [email protected]

Received 14 June 2010; Revised 25 August 2010; Accepted 4 October 2010

Academic Editor: Raﬀaele Porta

Copyright © 2010 A. A. A. Premarathne and D. W. M. Leung. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Aminopeptidase (AP) activity in ripe but firm fruit of Actinidia deliciosa was characterized using L-leucine-p-nitroanilide as a substrate. The enzyme activity was the highest under alkaline conditions and was thermolabile. EDTA, 1,10-phenanthroline, iodoacetamide, and Zn2+ had inhibitory effect while a low concentration of dithiothreitol (DTT) had stimulatory effect on kiwifruit AP activity. However, DTT was not essential for the enzyme activity. The results obtained indicated that the kiwifruit AP was a thiol-dependent metalloprotease. Its activity was the highest in the seeds, followed by the core and pericarp tissues of the fruit. The elution profile of the AP activity from a DEAE-cellulose column suggested that there were at least two AP isozymes in kiwifruit: one unadsorbed and one adsorbed fractions. It is concluded that useful food-grade aminopeptidases from kiwifruit could be revealed using more specific substrates.

1. Introduction consumed fresh such as kiwifruit, which have the merit of already being generally regarded safe for the food processing Kiwifruit (Actinidia spp.) is an important commercial crop industry. in New Zealand. The fruit contains a high level of a cysteine Generally, there are many studies on seed aminopep- endopeptidase called actinidin (E.C. 3.4.22.14) found in the tidases [9–11] but there is a paucity of information on cortex of the fruit [1]. Due to this proteolytic activity of the occurrence and characteristics of AP activities in fruits. kiwifruit, it has been used to tenderize meat and prevent Importantly, since there is no prior study on AP from gelatin-based jelly from setting. kiwifruit, a prerequisite towards the goal of evaluating use Aminopeptidases (APs), particularly those from micro- of APs from this fruit for food processing applications bial sources, are important food processing enzymes and is an investigation into the occurrence and biochemical are widely used to modify proteins in food [2–4]. Animal characteristics of aminopeptidase (AP) activity of kiwifruit. waste products were also investigated as a potential source Here, using L-leucine-p-nitroanilide (L-leu-p-NA) as a sub- of useful APs [5]. It is also possible that APs from plants strate, localization and some basic biochemical character- could be of use in the food processing industry [6]. Recently, istics of AP activity within the fruit of Actinidia deliciosa, it has been demonstrated that APs of cabbage leaves or and an attempt to partially purify the enzyme that is chickpea cotyledons can be used to catalyze the hydrolysis of normally sufficient for food-grade enzymes are reported peptide bonds including those of hydrophobic bitter peptides here. in soy protein hydrolysates, resulting in the less bitter or bland taste products which have food processing applications 2. Materials and Methods [7, 8]. However, for debittering protein hydrolysates or other food processing needs an attractive alternative would be to 2.1. Enzyme Extraction. Ripe but firm kiwifruit (Actinidia use APs from fruits grown commercially that are normally deliciosa cv. Hayward) was obtained from a local supermarket 2 Enzyme Research in Christchurch, New Zealand. Unless indicated otherwise, 2.5. EffectofpHonAPActivity.The effect of pH on AP the whole kiwifruit was peeled and cut into small pieces activity in the crude extracts of fruit was determined by before enzyme extraction. Kiwifruit tissues were ground in replacing the potassium phosphate buffer at pH 8.0 in the a mortar and pestle while adding 0.1 M of potassium phos- assay mixture, with the three buffer mixtures (25.0 mM acetic phate buffer pH 8.0 supplemented with 1% (w/v) insoluble acid, 25.0 mM MES, and 50.0 mM Tris) at different pH values polyvinyl polypyrrolidone (PVPP), 5% (v/v) glycerol and ranging from 6 to 10 as described in [13]. Then AP activity 3mM DTT.The ratio of weight of tissue (g) to volume was determined. of extraction buffer (ml) was 2 : 1. The homogenate was filtered through 2 layers of synthetic cloth and centrifuged at ff ff ◦ 2.6. E ect of Di erent Classes of Proteolytic Enzyme Inhibitors 10,000 × gfor20minat4 C. The supernatant was carefully and Promoters on AP Activity. Crude enzyme extracts were removed and used as crude extract of the whole fruit. The preincubated with 0.45 ml of 0.1 M potassium phosphate extraction process was carried out in a cold room or on an buffer (pH 8) in the presence of different inhibitors or ice bath. activators for 30 min at 37◦C. After pre-incubation, the enzyme reaction was initiated by the addition of the substrate 2.2. Determination of Total Protein Concentration. The pro- solution (L-leu-p-NA) and AP activity was determined. Con- tein concentration in extracts was determined based on the centration of activators in the reaction mixture during pre- Coomassie brilliant blue dye-protein binding principle [12]. incubation was 1.0 or 10.0 mM. The chemicals tested were A protein standard curve was prepared using serial dilutions EDTA, 1, 10-phenanthroline, PMSF, DTT, iodoacetamide, of BSA (bovine serum albumin; BDH, England). and NEM.

2.3. Determination of Aminopeptidase (AP) Activity. Amino- 2.7. Effect of Divalent Cations on AP Activity. The crude ◦ peptidase activity was determined as described below unless enzyme extracts were pre-incubated at 37 Cfor30min indicated otherwise using L-leucine-p-nitroanilide (L-Leu- with 0.45 ml of 0.1 M potassium phosphate buffer in the 2+ 2+ 2+ 2+ 2+ p-NA) as a substrate. The substrate solution was prepared presence of the chlorides of Mn ,Co ,Ni ,Mg ,Ca ,or 2+ by dissolving 20 mg of L-Leu-p-NA (Sigma, St. Louis, USA) Zn . The concentration of divalent cations in the reaction in one ml of dimethyl sulfoxide (Sigma, St. Louis, USA) mixture during pre-incubation was 1.0 or 10.0 mM. After and adjusting the volume to 20 ml with 0.01 M potassium pre-incubation, the substrate solution (L-leu-p-NA) was phosphate buffer (pH 8.0). It was found to be stored better at added to start the enzyme reaction and AP activity was −20◦C for use later if prepared at pH 8.0 than at higher pH. determined. The reaction mixture contained 0.45 ml of 0.1 M potassium phosphate buffer at pH 8.0, 0.45 ml substrate solution, and 2.8. Partial Purification of Aminopeptidase. The whole 150 μl enzyme extract in Eppendorf tubes kept on ice. The kiwifruit (550 g) was cut into small pieces and homogenized control tube contained the same reaction mixture except that in 225 ml of 0.1 M of potassium phosphate buffer (pH the enzyme extract had previously been boiled for 5 min 8.0) supplemented with 1% (w/v) insoluble PVPP, 5% ◦ in a water bath at 100 C and centrifuged afterwards. All (v/v) glycerol, and 3 mM DTT (extraction buffer). The the tubes were vortexed, and incubated for 1 h in a water homogenate was filtered through 2 layers of synthetic cloth. ◦ bath at 37 C. After the incubation period, they were placed The filtrate was centrifuged at 10,000 × gat4◦C for 20 min, ◦ in a water bath at 100 C for 5 min to stop the enzyme and the supernatant was removed and used as crude extract. reaction. After this, 0.45 ml distilled water was added to Solid ammonium sulphate ((NH4)2SO4) was added to the all the tubes, vortexed. and then centrifuged for 10 min at crude extract, and the resulting 25–75% precipitate was 10,000 × gatroomtemperature.Thesupernatantswere dissolved in 7.5 ml of 0.01 M potassium phosphate buffer carefully transferred to the cuvettes and the absorbance containing 10% (v/v) glycerol and 0.2 mM DTT (buffer A). was measured at 410 nm. One unit of enzyme activity is After dialysis of the 25–70% ammonium sulphate fraction defined as a change in one unit of absorbance per h at against buffer A, a DEAE cellulose column (10 × 2cm)was ◦ 37 C. used to separate the fractions. Unbound proteins were eluted with buffer A, and then bound proteins were eluted with ff 2.4. Effect of Temperature on AP Activity. The effect of 100 ml of the bu er A containing a linear gradient of 0.00– temperature on AP activity was determined in three different 1.0 M KCl. experiments. To find the optimum temperature for the enzyme activity, AP activity in crude extracts of the whole 2.9. Statistical Analysis. Statistical analysis of the data was fruit was determined at different incubation temperatures performed using STATISTIX 8.0 software. The comparison ranging from 25◦Cto70◦C for 1 h. In another experiment between treatments was analysed using one-way analysis to investigate thermal stability, 150 μl of the enzyme extracts of variance (ANOVA). Where a statistical significance was were pre-incubated with 0.45 ml of potassium phosphate observed, a Tukey’s Honest Significance Difference (HSD) buffer (pH 8.0) for 30 min at the above testing temperatures. test was performed to determine how significant from the After preincubation, the substrate was added to initiate the appropriate zero the values were. Standard errors were enzyme reaction for AP activity determination at 37◦Cfor calculated and graphically represented as symmetrical error 1h. bars. Enzyme Research 3

120 120

100 100

80 80

60 60

40 40 Relative activity (%) Relative activity (%)

20 20

0 0 6788.59 9.510 30 37 40 45 50 55 60 65 70 pH Temperature ( ◦C)

Figure 1: Effect of pH on aminopeptidase activity in extracts of the Figure 2: Effect of temperature on the aminopeptidase activity in whole fruit of A. deliciosa. The enzyme activity at pH 9 was taken as the crude extracts prepared from the whole fruit of A. deliciosa.The 100%. Mean values of three different extracts ± standard errors are enzyme activity at 37◦C was taken as 100%. Mean values of three presented. different extracts ± standard errors are presented.

120 3. Results and Discussion

3.1. Aminopeptidase (AP) Activity in Different Parts of 100 Actinidia deliciosa Fruit. In preliminary experiments, when crudeextractsfromthewholefruithadbeenpreparedwith 80 sodium phosphate or potassium phosphate buffer (pH 7.0), AP activity was not detectable. Kiwifruit contains more than 80 volatile aroma, and flavour compounds including 60 terpenses, esters, aldehydes, alcohols with varying levels of monoterpenes, and phenolic compounds [14, 15]. These 40 Relative activity (%) compounds could have interfered with aminopeptidase isolation and activity. Here, a reliable protocol (as described in Section 2) for extraction of AP from kiwifruit and 20 determination of its activity using L-leucine-p-nitroanilide (L-leu-p-NA) as a substrate has been established. The present 0 study has established for the first time that kiwifruit has 37 40 45 50 55 60 65 70 AP activity and some useful parameters with respect to its Temperature ( ◦C) extraction, assay, stability, localization and purification. Figure 3: Effect of temperature on the stability of aminopeptidase AP activity was found in all parts of the fruit of A. ff activity in crude extracts prepared from the whole fruit of A. deliciosa at di erent levels. The highest specific (units/mg deliciosa.Theenzymeactivityat40◦C was taken as 100%. Mean soluble protein) and total (units/g fresh weight) AP activity values of three different extracts ± standard errors are presented. was localized in the seed followed by the core, inner and outer pericarp, respectively, (Table 1). In contrast, higher enzyme activities were found in the hypodermis of fully ripe grape ◦ berries than in the seed or flesh [13]. 55 C its activity was reduced to 63% (with the activity at 37◦C designated as 100%) and then to about 20% at 60– 70oC (Figure 2). It was most stable at 37–40◦C (Figure 3) 3.2. Effects of pH and Temperature on Kiwifruit AP Activity. but became unstable as only less than 15% of its activity AP activity in crude extracts of the whole kiwifruit was most remained at temperatures higher than 55◦C (ANOVA, P< active at alkaline pH (Figure 1;ANOVA,P<.05). Similarly, .05). hydrolysis of L-leu-p-NA by crude extracts was most active at a range of alkaline pH values from many different plants including potato [16], Arabidopsis thaliana [17], tomato [18], 3.3. Effects of Protease Inhibitors, Activators, and Metal and daylily flowers [19]. Ions. The presence of 1 mM of 1,10-phenanthroline, EDTA- ◦ ◦ Kiwifruit AP was most active at 37 Cand50C, sug- Na2 and iodoacetamide inhibited kiwifruit aminopeptidase gesting the presence of two aminopeptidase isozymes. At activity (Figure 4). In contrast, 1 mM of DTT had a slight 4 Enzyme Research

Table 1: Aminopeptidase activity in diﬀerent parts of kiwifruita.

Type of tissue Total activity (units/g fresh weight) Speciﬁc activity (units/mg soluble protein) Outer pericarp 0.38 ± 0.15 0.37 ± 0.10 Inner pericarp 0.64 ± 0.19 0.52 ± 0.07 Core 2.91 ± 0.68 3.68 ± 0.96 Seed 60.15 ± 7.99 5.78 ± 0.48 aAminopeptidase (AP) activity was determined in extracts of each tissue from three diﬀerent fruits of A. deliciosa.Meanvalues± standard errors are presented.

160 120 140 120 100 100 80 80 60 40 60 Relative activity (%) 20 40

0 Relative activity (%) EDTA PMSF DTT IODO NEM PHE Chemicals tested 20

1mM 0 10 mM Ca Mg Ni Mn Co Zn Figure 4: Effect of proteolytic enzyme inhibitors and activators Divalent cations on aminopeptidase activity in crude extract of the whole fruit 1mM of A. deliciosa. Enzyme activity in the absence of any chemical 10 mM (control) was taken as 100%. Mean values of three different extracts ± standard errors are presented. Figure 5: Effect of divalent cations on aminopeptidase activity in crude extracts of the whole fruit of A. deliciosa. Enzyme activity in the absence of any cations (control) was taken as 100%. Mean values of three different extracts ± standard errors are presented. stimulatory effect (ANOVA, P<.05). The same concentration(1mM)ofNEMandPMSFhadnoeffect. At 10 mM, 1,10-phenanthroline, NEM, iodoacetamide, and EDTA-Na2 caused more inhibition. But 10 mM of DTT and PMSF had (ANOVA, P<.05) whereas the other metal cations tested had neither stimulatory nor inhibitory effect (ANOVA, P<.05). no significant effect. When the concentration of metal ions The observed inhibition of kiwifruit AP activity by metal was increased to 10 mM, the enzyme activity was strongly chelators such as 1,10-phenanthroline and EDTA suggested inhibited by Zn2+ (ANOVA, P<.05), and inhibited to a the involvement of a metal ion in the active site of the lesser extent by Ni2+,Co2+,andMn2+. At this concentration enzyme. Similar effects were also reported in the studies on Ca2+ and Mg2+ did not have any significant effects. This leucine aminopeptidases of potato [16], tomato, E. coli pep A, suggests that the AP activity might be different from that of and porcine LAPs [18]. Furthermore, DTT (a thiol reducing a previously studied protease in kiwifruit that was inhibited agent) at a lower concentration (1 mM) had a stimulatory by calcium ions [21]. Furthermore, kiwifruit AP activity was effect but an inhibitory effect at a higher concentration on differentfromthatinpotato,Arabidopsis, tomato, porcine kiwifruit AP activity suggesting that it was a thiol-dependent and E. coli pep A as they were highly activated by Mn2+ metalloprotease rather than a cysteine protease [20]. On the and Mg2+ ions but were also inhibited by Zn2+ ions [16– other hand, iodoacetamide (1 mM) and NEM (10 mM), the 18]. The kiwifruit AP activity was also different from that specific inhibitors of cysteine protease, had 60% and 40% of grape berries which was not inhibited by EDTA, 1,10- inhibition of kiwifruit AP activity, respectively, suggesting phenanthroline, or metal ions [13]. that cysteine residues were likely involved in the enzyme conformation rather than catalysis. A serine-type protease might not be a significant contributor to the kiwifruit AP 3.4. Partial Purification of Kiwifruit Aminopeptidase. Two activity as PMSF, a serine protease inhibitor, did not have any major peaks of AP activity were separated using DEAE significant effect on its activity. cellulose column chromatography: the unadsorbed and The effects on kiwifruit AP activity of Ca2+,Mg2+,Co2+, adsorbed fractions (Figure 6), suggesting that there were at Ni2+,Mn2+,andZn2+ with chloride as the counter ion least two isoforms of AP activity in A. deliciosa fruit. In these were studied (Figure 5). At metal ion concentrations of fractions only a few low-molecular weight polypeptides were 1mM,onlyZn2+ significantly inhibited kiwifruit AP activity found to be present following SDS PAGE (data not shown). Enzyme Research 5

0.18 0.5 [7] M. Marinova, A. Dolashki, F. Altenberend, S. Stevanovic, 0.16 0.45 W. Voelter, and B. Tchorbanov, “Characterization of an 0.14 0.4 aminopeptidase and a proline iminopeptidase from cabbage . leaves,” Zeitschrift fur Naturforschung Section C, vol. 63, no. 1- 0.12 0 35 0.3 2, pp. 105–112, 2008. 0.1 0.25 [8] M. Marinova, A. Dolashki, F. Altenberend, S. Stevanovic, 0.08 . W. Voelter, and B. Tchorbanov, “Puriﬁcation and character-

AP activity (units) 0 2 0.06 ization of L-phenylalanine aminopeptidase from chick-pea 0.15 . KCI cotyledons (Cicer arietinum L.),” Protein and Peptide Letters, 0 04 . Absorbance at 280 (nm) 0 1 vol. 16, no. 2, pp. 207–212, 2009. 0.02 . 0 05 [9]D.W.M.LeungandJ.D.Bewley,“Increasedactivityof 0 0 11020304050607080 aminopeptidase in the cotyledons of red light-promoted Fraction number lettuce seeds is controlled by the axis,” Physiologia Plantarum, vol. 59, no. 1, pp. 127–133, 1983. AP activity [10] Y. Yamaoka, M. Takeuchi, and Y. Morohashi, “Purification Proteins and partial characterization of an aminopeptidase from mung bean cotyledons,” Physiologia Plantarum,vol.90,no.4,pp. Figure 6: Elution profile from a DEAE cellulose column of 729–733, 1994. aminopeptidase (AP) activity and protein content in a concentrated fraction of ammonium sulfate precipitation of crude extracts of [11] K. Tishinov, N. Stambolieva, S. Petrova, B. Galunsky, and P. A. deliciosa fruit. The first 30 fractions were eluted with 10 mM Nedkov, “Purification and characterization of the sunflower potassium phosphate buffer (pH 8) supplemented with 10% seed (Helianthus annuus L.) major aminopeptidase,” Acta glycerol. The next fractions were eluted with a linear gradient of Physiologiae Plantarum, vol. 31, no. 1, pp. 199–205, 2009. 0 to 1.0 M KCl in the same buffer. One unit of enzyme activity was [12] M. M. Bradford, “A rapid and sensitive method for the defined as a change in one unit of absorbance at 410 nm per h at quantitation of microgram quantities of protein utilizing the 37◦C. Protein content was measured at 280 nm. principle of protein dye binding,” Analytical Biochemistry, vol. 72, no. 1-2, pp. 248–254, 1976. [13] H.-C. Kang, T.-R. Hahn, I.-S. Chung, and J.-C. Park, “Char- acterization of an aminopeptidase from grapes,” International Journal of Plant Sciences, vol. 160, no. 2, pp. 299–306, 1999. This might be a facile route to obtain a relatively pure food- [14] C. O. Perea, H. Young, and D. J. Beever, “Kiwifruit,” in Tropical grade aminopeptidases from kiwifruit. Further studies, using and Subtropical Fruits, P. E. Shaw, J. Chan, and S. Nagy, Eds., more specific substrates, could lead to some useful food- pp. 336–385, Agscience, Auburndale, Fla, USA, 1998. grade aminopeptidases from kiwifruit. Recombinant DNA [15] A. J. Matich, H. Young, J. M. Allen et al., “Actinidia arguta: techniques could also be applied to mass produce kiwifruit- volatile compounds in fruit and flowers,” Phytochemistry, vol. originated APs. 63, no. 3, pp. 285–301, 2003. [16] K. Herbers, S. Prat, and L. Willmitzer, “Functional analysis of a leucine aminopeptidase from Solanum tuberosum L,” Planta, References vol. 194, no. 2, pp. 230–240, 1994. [1] M. Rassam and W. A. Laing, “Purification and character- [17] D. Bartling and E. W. Weiler, “Leucine aminopeptidase from ization of phytocystatins from kiwifruit cortex and seeds,” Arabidopsis thaliana. Molecular evidence for a phylogeneti- Phytochemistry, vol. 65, no. 1, pp. 19–30, 2004. cally conserved enzyme of protein turnover in higher plants,” [2] N. Izawa, K. Tokuyasu, and K. Hayashi, “Debittering of protein European Journal of Biochemistry, vol. 205, no. 1, pp. 425–431, hydrolysates using Aeromonas caviae aminopeptidase,” Journal 1992. of Agricultural and Food Chemistry, vol. 45, no. 3, pp. 543–545, [18] Y.-Q. Gu, F. M. Holzer, and L. L. Walling, “Overexpression, 1997. purification and biochemical characterization of the wound- [3] R. Raksakulthai and N. F. Haard, “Exopeptidases and their induced leucine aminopeptidase of tomato,” European Journal application to reduce bitterness in food: a review,” Critical of Biochemistry, vol. 263, no. 3, pp. 726–735, 1999. Reviews in Food Science and Nutrition, vol. 43, no. 4, pp. 401– [19]M.G.P.MahagamasekeraandD.W.M.Leung,“Development 445, 2003. of leucine aminopeptidase activity during daylily flower [4] S.-J. Lin, Y.-H. Chen, L.-L. Chen, H.-H. Feng, C.-C. Chen, growth and senescence,” Acta Physiologiae Plantarum, vol. 23, and W.-S. Chu, “Large-scale production and application of no. 2, pp. 181–186, 2001. leucine aminopeptidase produced by Aspergillus oryzae LL1 [20] R. L. Wolz, “Strategies for inhibiting protease of unknown for hydrolysis of chicken breast meat,” European Food Research mechanism,” in Proteolytic Enzymes,E.E.SterchiandW. and Technology, vol. 227, no. 1, pp. 159–165, 2008. Stocker, Eds., pp. 90–106, Springer, Berlin, Germany, 1999. [5] S. Mane, M. Damle, P. Harikumar, S. Jamdar, and W. Gade, [21] N. Cicco, B. Dichio, C. Xiloyannis, A. Sofo, and V. Lattanzio, “Purification and characterization of aminopeptidase N from “Influence of calcium on the activity of enzymes involved chicken intestine with potential application in debittering,” in kiwifruit ripening,” in Proceedings of the International Process Biochemistry, vol. 45, no. 6, pp. 1011–1016, 2010. Symposium on Kiwifruit, vol. 753 of Acta Horticulturae,pp. [6] M. D. Marinova and B. P. Tchorbanov, “Brassicaceae plants as 433–438, 2007. a new source of food grade peptidases,” Bulgarian Chemical Communications, vol. 40, no. 4, pp. 397–400, 2008.