Industrial Enzymes an Update

PRATIMA BAJPAI INDUSTRIAL ENZYMES AN UPDATE

Download free eBooks at bookboon.com 2 Industrial enzymes: An update 1st edition © 2018 Pratima Bajpai & bookboon.com ISBN 978-87-403-2129-6 Peer review by Pramod K. Bajpai, Ph.D. Former Distinguished Professor (Chemical Engg.) and Dean (Research & Sponsored Projects) at Thapar University, Patiala – 147 004, India

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CONTENTS

List of Figures 6

List of Tables 7

Preface 8

1 Introduction 9

2 Historical perspectives 18

3 Production of enzymes 23

4 Industrial application of enzymes 30 4.1 Textiles 30 4.2 Pulp and Paper Industry 35 4.3 Starch Industry 49 4.4 Detergents 53 4.5 Leather industry 57 4.6 Food 62

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4.7 Feed 70 4.8 Organic synthesis 75 4.9 Pharmaceuticals 77 4.10 Personal Care 79 4.11 Biofuel 85 4.12 Processing of oil and fats 91

5 Enzyme Market 111

6 Future perspectives 115

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LIST OF FIGURES

Figure 4.3.1: Use of enzymes in processing of starch Figure 5.1: Global industrial enzyme market 2008–2015

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LIST OF TABLES

Table 1.1: Advantages of using enzymes Table 1.2: Specificity of enzymes Table 1.3: Selection of enzyme types used in industrial processes Table 1.4: Enzyme classes and types of reactions Table 3.1: Enzyme production process Table 3.2: Some of the enzyme manufacturing companies Table 4.1.1: Application of enzymes in textile industry Table 4.2.1: Application of enzymes in the pulp and paper industry Table 4.3.1: Enzymes involved in starch degradation Table 4.4.1: Advantages of using enzymes in detergents Table 4.4.2: Enzymes used in detergents (laundry and dish wash) Table 4.6.1: Application of enzymes used in food industry Table 4.7.1: Types of feed enzymes Table 4.7.2: Pre-requisite of enzymes used in animal nutrition Table 4.7.3: Mode of action of different feed enzymes Table 4.10.1: Use of enzymes in cosmetics Table 4.11.1: First generation and second generation feed stocks for bioethanol Table 4.11.2: Enzymes involved in biofuel production Table 4.12.1: Enzymes used in processing of fats and oils

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PREFACE

The demand for industrial enzymes is increasing. Industrial manufacturers are involved in constant research and development, to optimize their production and minimize resource costs. The sector is poised to benefit from growing environmental concerns and increased government intervention the world over to curb the unchecked use of hazardous and environmentally harmful conventional petro-chemicals. Environmental legislation will continue to remain a major driver for change and will help in widening the use of industrial enzymes. The natural greening process is underway in the manufacturing sector. This is actually driven by sustainable production principles and also augurs well for the future of the market. In the coming years, the incremental use of bioprocesses into every aspect of industrial manufacturing will help turbo-charge growth further. A measure of the untapped potential in store is reflected by the encouraging growth in R&D investments witnessed till date. Several of the R&D projects are currently centered on identifying enzymes from fungi and microbes. The biofuel industry will witness the maximum action in the R&D space in the upcoming years. The development of newer grades of next generation enzymes, such as Psychrozymes, will open up newer application areas for enzymes in addition to its existing use in food, feed, textile, leather, oils and fats, beverage alcohol and biofuel industries. The global market for industrial enzymes was fairly immune to the recent turmoil in the global economy and grew moderately in the last decade. The demand for industrial enzymes in matured economies such as the United States, Canada, Western Europe and Japan, was relatively stable during the recent times, while the developing economies of Asia-Pacific, Eastern Europe, along with Africa, and the Middle East regions emerged as the fastest growing markets for industrial enzymes. The global industrial enzymes market is expected to reach USD 9.63 billion by 2024. This book provides thorough and in depth coverage on the role of enzymes in a broad range of industries – Textile Industry, Pulp & Paper Industry, Starch Industry, Detergent Industry, Leather Industry, Food Industry, Feed Industry, Organic Synthesis, Fine Chemicals, Pharmaceuticals, Personal Care. Biofuel, Processing of Fats and Oils – and what the future holds. It is a valuable reference which every biotechnologist/ enzymologist/biochemist/microbiologist/ biochemical engineer/ chemical technologist/chemical engineer must have access. I hope that readers will find it useful.

Pratima Bajpai Pulp and Paper Consultant Kanpur, India

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1 INTRODUCTION

Enzymes are naturally occurring, protein-based molecules that catalyze the various chemical reactions with great specificity and rate enhancements (Binod et al., 2013; van Beilen and Li, 2002; Godfrey and West, 1996; Panke and Wubbolts, 2002; OECD, 1998; Adrio and Demain, 2014; Schafer et al., 2002; Schmid et al., 2002; Leisola et al., 2002; Gurung et al., 2013; Novozymes, 2011; Choi et al., 2015). These reactions are the basis of the metabolism of all living organisms, and provide tremendous opportunities for industry to carry out elegant, efficient and economical biocatalytic conversions (Kirk et al., 2002).

Enzymes have played an important role in several aspects of life since the dawn of time. These are vitally important to the existence of life itself. Civilizations have used enzymes for thousands of years without understanding how they work and what they were. Over the past several generations, science has unlocked the mystery of enzymes and has applied this knowledge to make better use of these amazing substances in an ever-growing number of applications. Enzymes play a very important role in producing the food we eat, the clothes we wear, even in producing fuel for our automobiles. Enzymes are also important in reducing both energy consumption and environmental pollution.

Enzymes are true catalysts in that they are not consumed in the reaction, and each enzyme molecule can catalyze thousands of reactions per second. Enzymes are very specific to the reaction that they drive; each type of enzyme does one thing only which makes them especially effective tools for achieving specific results in the diverse processes. Enzyme- catalyzed processes are slowly replacing chemical processes in several areas of industry. Many chemical transformation processes used in various industries have inherent disadvantages from an environmental and commercial point of view. Nonspecific reactions may result in poor product yields. High temperatures and/or high pressures needed to drive reactions lead to high energy costs and may require large volumes of cooling water downstream. Harsh and hazardous processes involving high pressures, high temperatures, acidity, or alkalinity require high capital investment, and specially designed equipment and control systems. Unwanted byproducts may prove costly or difficult to dispose of. High consumption of chemicals and energy and also generation of harmful byproducts have a negative impact on the environment. In several cases, some or all of these drawbacks can be almost eliminated by using enzymes. Enzyme reactions may often be carried out under mild conditions, they are highly specific, and involve high reaction rates. Industrial enzymes originate from biological systems; they contribute to sustainable development through being isolated from microorganisms which are fermented using primarily renewable resources.

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The developments in the area of biotechnology, particularly genetics and protein engineering, has opened a new era of enzyme applications in several industrial processes and experiencing major R&D initiatives. This has resulted not only in the development of a number of new products but improvement in the process and performance of several existing processes also.

In the presence of an appropriate enzyme, a chemical reaction occurs at a much higher rate but the enzyme is not consumed by the reaction. Their ability to perform very specific chemical transformations has made them increasingly popular in industries where less specific chemical processes produce unwanted byproducts. Purity and predictability are of particular importance in food manufacture where byproducts may be harmful or affect flavor, and because of their specificity, pharmaceutical companies favor biotransformation in the development of novel therapeutic agents. In addition, enzymes are chiral catalysts which mean that they can be used to produce optically active, homo-chiral compounds of a kind that are often difficult to make using traditional organic chemistry. Recently, a greater awareness of conservation issues have forced industries with a history of polluting to consider alternative, cleaner methods so there is now significant growth of biotechnology outside of the pharmaceutical and food industries.

Enzymes have several distinct advantages for use in industrial processes (Table 1.1). One of the most important properties of enzymes that make them important as diagnostic and research tools is the specificity. A few enzymes show absolute specificity; that is, they will catalyze only one particular reaction. Other enzymes will be specific for a particular type of chemical bond or functional group. In general, there are four distinct types of specificity (Table 1.2). Because of these inherent advantages, many industries are keenly interested in adapting enzymatic methods to the requirements of their processes. Enzymes are useful in various areas of applications like textile industry, pulp and paper industry starch industry, detergent industry, leather industry, food and feed industry, fine chemicals, pharmaceuticals, chiral substances and biofuel production, etc. (Kirk et al., 2002). The use of enzymes in animal nutrition is very important. This area is growing, especially for pig and poultry nutrition. Feed enzymes offer the benefit of degrading specific feed components which are found harmful or of no value to the livestock. In cosmetic products, it is used for skin peeling and future applications may be skin protection. Notable medications of enzymes are as digestive aids, for wound cleaning, lysis of vein thrombosis, acute therapy of myocardial infarction and as support in the therapy of certain types of leukemia. Enzymes can be also used in chemical analysis and as a research tool in the life sciences.

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Table 1.3 presents a small selection of enzyme types currently used in industrial processes listed according to class. The classes are defined in Table 1.4.

1. Laccase enzymes are used in chlorine-free denim bleaching process which also enables a new fashion look. 2. Glucosyl transferase enzyme is used in the food industry for the production of functional sweeteners. 3. Hydrolases are the most widely used class of industrial enzymes. Several applications are described in later sections of the book. 4. An alpha-acetolactate decarboxylase is used for reducing the maturation period after the fermentation process in beer brewing. 5. Gucose isomerase enzyme is used to convert glucose into fructose, which increases the sweetness of the syrup.

Matching an enzyme with a process is the greatest challenge to a research and development program. Often an industrial plant can be modified to accommodate the limitations of an enzyme but this is quite costly and the best approach is to find an enzyme more suited to the existing process. Increasingly, new organisms are being found living in unusual environments and these are proving an excellent source of novel enzymes. Living organisms are now generally divided into three groups: the eukaryotes, the bacteria and the archaebacteria. The eukaryotes, which include all animals and plants, are limited in their ability to withstand hostile conditions such as extreme ranges of temperature or pH. Some worms that can live above 600C have been found living around deep ocean volcanic vents, but these are exceptional. Bacteria and archaebacteria are not so constrained and can thrive in quite unbelievable conditions; from freezing to boiling water and from an acidic pH 2 to alkaline pH 12. Archae Pyrococcus furiosa grows optimally at around 113°C and finds it too cold if temperature falls to 100°C. These are the organisms of the future for biotechnology. Many industrial processes are designed to run at high temperatures where chemical reactions are faster and viscosity is reduced. By using enzymes with optimal activities at these temperatures, changes to existing industrial plants can be reduced. Moreover, problems with contamination are reduced, and less cooling is required where the reactions are exothermic.

Enzymes contribute to clean industrial products and processes. They show several advantages over chemicals. Enzymes can be produced from renewable resources and are in turn degraded by microbes in nature. Various industries have replaced old processes using chemicals that cause detrimental effects on the environment and equipment with new processes that use biodegradable enzymes under less corrosive conditions. Currently, industrial enzymes are manufactured by three major suppliers, Novozymes A/S (headquartered in Denmark), Genencor International Inc. (headquartered in the U.S.), and DSM N.V. (headquartered in the Netherlands).

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Presently, almost 4000 enzymes are known, and of these, approximately 200 microbial original types are used commercially. However, only about 20 enzymes are produced on truly industrial scale (Li et al., 2012). With the improved understanding of the enzyme production, biochemistry, fermentation processes, and recovery methods, an increasing number of industrial enzymes can be foreseeable. The world enzyme demand is satisfied by about 12 major producers and 400 minor suppliers. Nearly 75% of the total enzymes are produced by three top enzyme companies, i.e. Denmark-based Novozymes A/S, US-based DuPont (through the May 2011 acquisition of Denmark-based Danisco) and Switzerland-based Roche. The market is highly competitive, has small profit margins and is technologically intensive.

The total market for industrial enzymes reached $3.3 billion in 2010 (BBC Research, 2011; Global Industrial Enzymes Market Research News, 2011; Sarrouh et al., 2012). Of these, industrial enzymes are typically used as bulk enzymes in the detergent, textile, pulp and paper industries, and in the biofuels industry, among others. Usage for leather and bioethanol is responsible for the highest sales figures. Technical enzymes had revenue of nearly $1.2 billion in 2011. The highest sales are expected to be in the biofuels (bioethanol) market (Freedonia group, 2011).

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References Choi JM, Han SS and Kim HS (2015) Industrial applications of enzyme biocatalysis: current status and future aspect. Biotechnol Adv 33:1443–1454.

Adrio JL and Demain AL (2014). Microbial enzymes: tools for biotechnological processes. Biomolecules. 4:117–139.

BBC Research Report BIO030 F (2011). Enzymes in Industrial Applications: Global Markets. BBC Research; Wellesley, MA, USA.

Binod P, Palkhiwala P, Gaikaiwari R, Namppthiri, KM, Duggal, A, Dey K and Pandey A (2013). Industrial enzymes – present status and future perspectives for India. J. Sci. Ind. Res. 72: 271–286.

Freedonia Group; Cleveland, OH, USA: (2011). World Enzymes.

Global Industrial Enzymes Market Research News (2011) In: Report – Global Industrial Enzymes Market: An Analysis.

Godfrey T and West S (1996). Industrial Enzymology. London: Macmillan Press Ltd.

Gurung N, Ray S, Bose S and Rai V (2013). A broader view: microbial enzymes and their relevance in industries, medicine, and beyond. Biomed Res. Int. 2013, 329121.10.1155/2013/329121

Kirk O, Borchert TV and Fuglsang CC (2002). Industrial enzyme applications. Curr. Opin. Biotechnol. 13:345.

Li S, Yang X, Yang S, Zhu M and Wang X (2012). Technology prospecting on enzymes: Application, marketing and engineering, Comput. Struct. Biotechnol. J., 2 p. e201209017.

Leisola M, Jokela J, Pastinen O, Turunen O, and Schoemaker H (2002). Industrial use of enzymes. In: Encyclopedia of Life Support Systems (EOLSS), OOP Hänninen and M Atalay, Eds., pp. 1–25, EOLSS, Oxford, UK, 2002.

Novozymes (2011) Enzymes at work. http://www.novozymes.com/en/about us/brochures/ Documents

OECD (1998). Biotechnology for Clean Industrial Products and Processes. Paris, France: OECD.

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Panke S and Wubbolts MG (2002). Enzyme technology and bioprocess engineering. Curr Opin Biotechnol., 13: 111–116.

Sarrouh B, Santos TM, Miyoshi A, Dias R and Azevedo V (2012). Up-to-date insight on industrial enzymes applications and global market. J Bioprocess Biotechniq S4:002 doi:10.4172/2155-9821.S4-002.

Schafer T, Kirk O, Borchert TV, Fuglsang, CC, Pedersen S, Salmon S, Olsen HS, Deinhammer R and Lund H (2002). Enzymes for technical applications. In Biopolymers; Fahnestock, SR, Steinbü chel, SR, Eds.; Wiley-VCH: Weinheim, Germany, 2002; pp. 377–437.

Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B (2001). Industrial biocatalysis today and tomorrow. Nature 409: 258–268. van Beilen JB and Li Z (2002). Enzyme technology: an overview. Curr Opin Biotechnol 13: 338–344.

Webb EC (1992). Enzyme nomenclature 1992: Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes, Academic Press.

1. They are of natural origin and are nontoxic.

2. They have great specificity of action; hence can bring about reactions not otherwise easily carried out.

3. They work best under mild conditions of moderate temperature and near neutral pH, thus not requiring drastic conditions of high temperature, high pressure, high acidity/ alkaline, which necessitate special expensive equipment.

4. They act rapidly at relatively low concentrations, and the rate of reaction can be readily controlled by adjusting temperature, pH, and amount of enzyme employed.

5. They are easily inactivated when reaction is completed as far as desired.

Table 1.1: Advantages of using enzymes

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1. Absolute specificity – the enzyme will catalyze only one reaction.

2. Group specificity – the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate and methyl groups.

3. Linkage specificity – the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure.

4. Stereochemical specificity – the enzyme will act on a particular steric or optical isomer.

Table 1.2: Specificity of enzymes

EC 1: Oxidoreductases

Catalases 360° Glucose oxidases Laccases EC 2: Transferases 360° thinking. Glucosyltransferases thinking.

360° thinking . 360° thinking.

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EC 3: Hydrolases

Amylases Cellulases Lipases Mannanases Pectinases Phytases Proteases Pullulanases Xylanase

EC 4: Lyases

Pectate lyases alpha-acetolactate decarboxylases

EC 5: Isomerases

Glucose isomerases Epimerases Mutases Lyases Topoisomerases

EC 6: Ligases

Argininosuccinate Glutathione synthase

Table 1.3: Selection of enzyme types used in industrial processesBased on Webb (1992)

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Enzyme commission Class of enzyme Reaction profile number These enzymes catalyze redox reactions, i.e., reactions involving the transfer of electrons from one molecule to another. In biological systems we often see the removal of hydrogen atoms from a substrate. Typical enzymes catalyzing such reactions are called dehydrogenases. For example, alcohol dehydrogenase catalyzes reactions of the type R-CH2OH + A → R-CHO + AH2, EC 1 Oxidoreductases where A is a hydrogen acceptor molecule. Other examples of oxidoreductases are oxidases and laccases, both catalyzing the oxidation of various substrates by dioxygen, and peroxidases, catalyzing oxidations by hydrogen peroxide. Catalases are a special type, catalyzing the disproportionation reaction 2H2O2 → O2 + 2H2O, where hydrogen peroxide is both oxidized and reduced at the same time. Enzymes in this class catalyze the transfer of groups of atoms from one molecule to another or from one position in a molecule to other positions in the same molecule. Common types are EC 2 Transferases acyltransferases and glycosyltransferases. CGTase (cyclodextrin glycosyltransferase) is one such enzyme type, which moves glucose residues within polysaccharide chains in a reaction that forms cyclic glucose oligomers (cyclodextrins). Hydrolases catalyze hydrolysis, the cleavage of substrates by water. The reactions include the cleavage of peptide bonds in proteins by proteases, glycosidic bonds in carbohydrates by a EC 3 Hydrolases variety of carbohydrases, and ester bonds in lipids by lipases. In general, larger molecules are broken down to smaller fragments by hydrolases. Lyases catalyze the addition of groups to double bonds or the formation of double bonds though the removal of groups. Thus bonds are cleaved by a mechanism different from hydrolysis. EC 4. Lyases Pectate lyases, for example, split the glycosidic linkages in pectin in an elimination reaction leaving a glucuronic acid residue with a double bond. Isomerases catalyze rearrangements of atoms within the same EC 5 Isomerases molecule; e.g., glucose isomerase will convert glucose to fructose. Ligases join molecules together with covalent bonds in biosynthetic reactions. Such reactions require the input of energy by the EC 6 Ligases concurrent hydrolysis of a diphosphate bond in ATP, a fact which makes this kind of enzyme difficult to apply commercially.

Table 1.4: Enzyme classes and types of reactions Based on Webb (1992); Novozymes (2011)

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2 HISTORICAL PERSPECTIVES

The term enzyme was introduced by Kuhne in 1878 for the substances in yeast responsible for fermentation (Kuhne, 1877). Louis Pasteur was among the first to study enzyme action. He incorrectly hypothesized that the conversion of sugar into alcohol by yeast was catalyzed by “ferments” that could not be separated from living cells (http://science.jrank. org/pages/2535/Enzyme-Historical-background-enzyme-research.html#ixzz4zdkJPZy2). In 1897 Eduard Buchner (1860–1917), the German biochemist demonstrated the conversion of glucose to ethanol by a cell-free extract from the yeast. In 1833, French chemist Anselme Payen discovered the first enzyme, diastase (Payen and Persoz, 1833) . In 1835, the hydrolysis of starch by diastase was acknowledged as a catalytic reaction by another Swedish scientist Jons Jacob Berzelius (Gurung et al., 2013; Binod et al., 2013).

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The early twentieth century saw dramatic advancement in enzyme studies.

Emil Fischer (1852–1919) recognized the importance of substrate shape for binding by enzymes.

Leonor Michaelis (1875–1949) and Maud Menten (1879–1960) introduced a mathematical approach for quantifying enzyme-catalyzed reactions. They reported that the rate of an enzymatic reaction is proportional to the concentration of enzyme-substrate complex.

James Sumner (1887–1955) and John Northrop (1891–1987) produced highly ordered enzyme crystals and established the protein nature of enzymes.

Buchner brothers in 1897 reported that cell-free extracts from yeast are able to break down glucose into ethanol and carbon dioxide.

Hans Krebs (1900–1981) in 1937 reported a series of enzymatic reactions in the citric acid cycle for the production of Adenosine triphosphate from glucose metabolites.

Today, enzymology is a central part of biochemical study.

Most of the reactions in living organisms are catalyzed by enzymes. Enzymes are the catalytic machinery of living systems. These are highly specific in their action on substrates and often several different enzymes are needed to bring concerted action, the sequence of metabolic reactions performed by the living cell. Almost all enzymes which have been purified are protein in nature, and may or may not possess a non protein prosthetic group for their biological activity. The practical application and industrial use of enzymes to perform certain reactions apart from the cell, dates back many centuries and practiced long before the nature or function of enzymes was completely understood.

Enzymes catalyze fermentation of sugar to ethanol by yeasts. This reaction forms the basis of making beer and wine. Enzymes oxidize ethanol to acetic acid. This reaction has been used in the production of vinegar for thousands of years. Similar reactions of yeasts and acid-forming bacteria are responsible for aroma-forming activities in bread making and for preserving activities in preparation of sauerkraut (Leisola et al., 2002). The fermentative activity of microorganisms was only discovered in the eighteenth century and finally proved by the French scientist Louis Pasteur. The term “enzyme” originates from the Latin with the literal meaning of “in yeast.” This was due to the fact that enzymes were closely associated with yeast activity. The study of enzymes is fairly recent. Enzymes were first clearly recognized in 1833 when scientists discovered that an alcohol precipitate of malt extract contained a thermo-labile substance which converted starch into sugar. The substance was named diastase. This enzyme is now called amylase. The first enzyme was obtained in pure form in 1926, a feat accomplished by James B. Sumner of Cornell University. Sumner in 1947 was able to isolate and crystallize the enzyme urease from the jack bean. He received the Nobel Prize for this work (Nobel prize for Chemistry laureates, 1946). John H. Northrop and Wendell M.

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Stanley shared the 1947 Nobel Prize with Sumner. These researchers discovered a complex method for isolating pepsin. This precipitation method has been used to crystallize several enzymes (Nobel prize for Chemistry laureates, 1946). In 1960, NOVO started producing commercially protease enzyme using Bacillus licheniformis. After 1980, many scientists started developing application of genetic engineering techniques for improving the production of enzymes and also to change the properties of enzymes by protein engineering (Novozymes, 2004; 2011).

Rennin an aspartic protease enzyme coagulates milk protein, has been used for hundreds of years by manufacturers of cheese. In Germany, Röhm Company produced the first commercial enzyme in 1914. This enzyme-trypsin, isolated from animals was found to degrade proteins, and was used as a detergent. It was found to be very effective compared with traditional washing-powders that German housewives’ suspicions were aroused by the small size of the original package, so the product was reformulated and sold in microbial protease enzymes into washing powders. The first bacterial Bacillus protease was sold in 1959, and became big business when Novozymes in Denmark started producing it and major detergent manufacturers started using it around 1965 (Novozyme, 2011).

In 1930, in addition to cheese manufacturers, enzymes were already being used in the food industry for producing the fruit juices. These enzymes-pectinases, clarify the juice and contain several different enzyme activities. The major use of microbial enzymes started in the 1960s in the starch industry. The traditional method for hydrolysis of starch was acid hydrolysis. This method was completely replaced by alpha-amylase enzymes and glucoamylases that could almost completely convert starch to glucose. After detergent manufacture, the starch industry became the second largest user of enzymes.

The industrial enzyme manufactures are selling enzymes for a wide variety of applications. Detergents (37%), textiles (12%), starch (11%), baking (8%), and animal feed (6%) are the main industries, and use about 75% of industrially-produced enzymes (Leisola et al., 2002). Enzymes are also indirectly used in biocatalytic processes involving living or dead and permeabilized microorganisms.

Other important contributors to the development of enzyme chemistry include K. Linderstrøm- Lang and M. Ottesen, who were the first to isolate and characterize a subtilisin, a type of alkaline protease produced by bacteria.

Enzymatic desizing is one of the oldest nonfood applications of bacterial amylases. In 1950, Novo launched the first fermented enzyme, a bacterial alpha-amylase. The use of enzymes in detergents (largest application of industrial enzymes) started slowly in the early 1930s based on Röhm’s 1913 patent on the use of pancreatic enzymes in presoak solutions. In

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1963, a protease with a low alkaline pH optimum (Alcalase®) was developed, which was a real breakthrough for detergent enzymes. The immobilized glucose isomerase was launched in 1974, which became a breakthrough in the starch industry.

The discovery by Avery in 1944 that genetic information is stored in the chromosome as deoxyribonucleic acid (DNA) was perhaps the first major step towards the now extensive use of genetic engineering and the related technique of protein engineering. Another important research was published in 1953 when Watson, Crick and Franklin proposed the double-helical structure for DNA. Genetic information is stored in this molecule, as a linear sequence written in a four-letter chemical alphabet. Now days, scientists understand most of the significance of the information contained in DNA. For example, the linear message laid down in an individual gene of, say, 1,200 letters can be translated into the chain of 400 amino acids making up a particular enzyme; the genetic code has been broken. The first enzyme expressed in a genetically modified organism was a commercial lipase for detergents called Lipolase®. This enzyme was developed by Novo and introduced in 1988 for immediate incorporation into the Japanese detergent Hi-Top made by the Lion Corporation. Recombinant DNA technology has brought about a revolution in the development of new enzymes.

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References Binod P, Palkhiwala P, Gaikaiwari R, Namppthiri KM, Duggal A, Dey K, and Pandey A (2013). Industrial enzymes – present status and future perspectives for India. J. Sci. Ind. Res. 72: 271–286.

Enzyme – Historical Background Of Enzyme Research – Reactions, Enzymes, Hans, and Catalyzed – JRank Articles http://science.jrank.org/pages/2535/Enzyme-Historical-background- enzyme-research.html#ixzz4zdkJPZy2

Gurung N, Ray S, Bose S, and Rai V (2013). A Broader View: Microbial Enzymes and Their Relevance in Industries, Medicine, and Beyond, BioMed Research International, vol. 2013, Article ID 329121, 18 pages. doi:10.1155/2013/329121

Kuhne W (1877). Uber das Verhalten verschiedener organisirter und sog. Ungeformter Fermente, Verhandlungen des Heidelb. Naturhist.-Med. Vereins, Neue Folge, 1(3): 190–193.

Nobel prize for Chemistry laureates (1946) http://www.nobelprize.org/.

Novozymes (2004). Annual Report for 2003, Novozymes, Copenhagen, 2004.

Novozymes (2011) Enzymes at Work. http://www.novozymes.com/en/about-us/brochures/ Documents

Payen A and Persoz JF (1833). Memoir on diastase, the principal products of its reactions, and their applications to the industrial arts, Annales de Chimie et de Physique, 53: pp. 73–92.

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3 PRODUCTION OF ENZYMES

Enzymes have been used throughout the ages either in the form of vegetables rich in enzymes, or as microorganisms used for several purposes, for example in baking, brewing, and in cheese production (Aehle, 2007; Flickinger and Drew, 1998; Kirk, 2005; Schafer et al., 2002). However, it was only in the 19th century that the various biological conversions were ascribed to the action of enzymes. In the Far East, during the early part of the last century, an age-old tradition involving the use of mould called koji in the production of certain foodstuffs and flavour additives based on soya protein and fermented beverages, formed the basis on which the Japanese scientist Takamine developed a fermentation process for the industrial production of fungal amylase. The process included the culture of Aspergillus oryzae on moist wheat bran or rice, and the product was called ‘Takadiastase’. This is still being used as a digestive aid. A number of companies are competing in the industrial enzymes’ business. The global industrial enzyme market size was estimated to be DKK 25 billion or USD 3.8 billion in 2015, of which Novozymes commands a 48% share.

The technology for producing and using commercially important enzyme products combines the disciplines of microbiology, genetics, biochemistry and engineering which have developed and matured through time both singly and in an interactive manner.

Demand for new enzymes arise from the unsatisfactory performance of known enzymes in the established processes or from the development of new processes. The revolution in gene technology over the last two decades has had a big impact on the enzyme industry. Genetic engineering techniques have enabled enzyme manufacturers to produce sufficient quantities of almost any enzyme no matter what the source, whereas protein engineering allows the properties of the enzymes to be adjusted before production.

Commercial sources of enzymes are obtained from three sources, i.e., animal tissue, plants and microorganisms. These naturally occurring enzymes are quite often not readily available in enough quantities for industrial use or food applications. But, by isolating microorganisms that produce the desired enzyme and optimizing the conditions for growth, commercial quantities can be achieved. This method is called fermentation and is well known for more than 3,000 years. Today, this fermentation process is carried out in a vessel called the fermentor. The microorganisms are destroyed after the completion of the fermentation and the enzymes are isolated and processed further for commercial application.

Commercial enzymes obtained from plant include proteolytic enzymes papain, bromelain, and ficin, and lipoxygenase (speciality enzymes) from soyabeans. Animal-derived enzymes

Download free eBooks at bookboon.com 23 INDUSTRIAL ENZYMES: AN UPDATE Production of enzymes include proteinases like pepsin and rennin. The enzyme production process can be divided into the six phases (Table 3.1).

Following criteria are used in the selection of an industrial enzyme.

¾¾Specificity ¾¾Reaction rate ¾¾pH and temperature optima and stability ¾¾Effect of inhibitors ¾¾Affinity to substrates

Enzymes used in the paper industry for bleaching should not contain any cellulases because it would damage the cellulose fibers. Enzymes used in the animal feed industry should be thermo-tolerant to survive in the hot extrusion process used in manufacture of animal feed. The same enzymes should have maximum activity at the body temperature of the animal. Enzymes used in industrial processes must usually be tolerant of various heavy metals and have no requirement for cofactors. They should already be maximally active in the presence of low substrate concentration so that the desired reaction proceeds to completion in a realistic time frame.

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Among various enzymes produced on a commercial scale are:

¾¾Proteases (subtilisin, rennet) ¾¾Hydrolases (pectinase, lipase, lactase) ¾¾Isomerases (glucose isomerase) ¾¾Oxidases (glucose oxidase)

These enzymes are produced using overproducing strains of certain microorganisms. Separation and purification of an enzyme from an organism require the following steps:

¾¾Disruption of cells ¾¾Removal of cell debris and nucleic acids ¾¾Precipitation of proteins ¾¾Ultrafiltration of the desired enzyme ¾¾Chromatographic separations (optional) ¾¾Crystallization, and drying

The process scheme varies depending on whether the enzyme is extracellular or intracellular. In some cases, it may be more beneficial to use dead or resting cells with the desired enzyme activity in the immobilized form. This strategy avoids costly enzyme separation and purification steps and therefore is economically more feasible.

The first step in the large-scale production of enzymes is to grow the organisms producing the desired enzyme. Enzyme production can be regulated and fermentation conditions can be optimized for overproduction of the enzyme.

¾¾Proteases are produced by using overproducing strains of Bacillus, Aspergillus, Rhizopus, and Mucor. ¾¾Pectinases are produced by Aspergillus niger ; lactases are produced by yeast and Aspergillus. ¾¾Lipases are produced by certain strains of yeasts and fungi. ¾¾Glucose isomerases are produced by Flavobacterium arborescens or Bacillus coagulans.

Production of a new microbial enzyme usually starts with screening of microorganisms for desirable activity by using the proper selection methods. The severe environment to which several enzymes are subjected during process applications has given push to screening of extremophiles for enzymes having desirable stability and activity. The enzyme activity produced by an organism from a natural environment is often low and needs to be increased for industrial production. Increase in enzyme activity is usually obtained by mutation of the organism. Another approach that has gained favor is production of the enzyme in a

Download free eBooks at bookboon.com 25 INDUSTRIAL ENZYMES: AN UPDATE Production of enzymes recombinant organism of choice whose growth conditions are well optimized and whose GRAS status is established. Random or site-directed mutagenesis with the objective of engineering the activity and stability properties of an enzyme before its production is becoming a common practice. The microorganisms used for production of enzyme are grown in fermenters using an optimized growth medium. Both solid state- and submerged fermentation are used commercially, but the latter is preferred because of a better handle on asceptic conditions and process control. The submerged culture is the preferred fermentation process for growing enzyme producing microorganisms. The microbial cells are maintained in suspension through continuous agitation and under controlled growing conditions – pH, temperature, nutrients, dissolved oxygen concentration, among others. The medium is an aqueous solution of substances readily available in large quantities at reduced cost. Raw material costs will be related closely to the value of the final product, mainly in the case of the enzymes, which are generally low volume and medium cost products such as starch hydrolysate, corn steep liquor, molasses, whey and many cereals. After the fermentation is completed, the enzyme may be present within the microorganism or released into the medium. When the enzyme is present within the microorganism, then the suspension is centrifuged or filtered and the supernatant or filtrate is discharged and the cell material is collected; otherwise the cell material is discharged and the liquid phase is collected.

Both fed-batch and continuous fermentation processes are in common use. In the fed-batch fermentation process, sterilized nutrients are added to the fermenter during the growth of the biomass. In the continuous process, sterilized liquid nutrients are fed into the fermenter at the same flow rate as the fermentation broth leaving the system, thus achieving steady-state production. Operational parameters like temperature, pH, feed rate, oxygen consumption, and carbon dioxide formation are generally measured and controlled carefully for optimizing the fermentation process.

Downstream processing of enzyme from the raw material constitutes the subsequent major stage in the production process. The purification level depends on the final application of the enzyme product. The industrial bulk enzymes are relatively crude formulations whereas specialty enzymes undergo a thorough purification to yield a homogeneous product. A conventional downstream processing scheme involves stages of clarification for enzyme separation from the solids comprising the raw material, concentration to reduce the process volumes, and purification to separate it from other soluble contaminants. In case of the intracellular enzymes, disruption of cells or tissue for releasing the product is among the primary separation steps. There is a choice of different separation techniques for each process stage. Chromatography is the main technique for high-resolution purification of enzymes.

Some separation techniques allow integration of the downstream processing stages needed for purification thus reducing the number of steps and hence the production costs. The enzyme

Download free eBooks at bookboon.com 26 INDUSTRIAL ENZYMES: AN UPDATE Production of enzymes is finally formulated as a liquid or solid product. In either case, stabilizers are added for imparting long shelf life to the product. Some enzymes are immobilized to solid supports or enzyme crystals are cross-linked to render them insoluble and stable for repeated or long term use in a process application. Large scale production of enzymes has to comply with the standards set by International Organization of Standardization for ensuring production efficiency and quality and also environmental management control, whenever applicable.

Intracellular enzymes can be released by increasing the permeability of the cell membrane. For this purpose, certain salts, such as calcium chloride, and other chemicals, such as dimethylsulfoxide (DMSO), and pH shift may be used. If enzyme release is not complete, then cell disruption may be necessary.

The industrial or bulk enzymes include amylases, lipases, proteases, etc. which are required in large volumes. These enzymes have an inherently low unit value so that they demand significantly reduced manufacturing costs. On the other end of the scale is the therapeutics sector with products such as urokinase, which are produced in lower volumes and at inherently higher manufacturing cost. In between these two, lie the diagnostic enzymes.

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Table 3.2 lists some of the companies, which are producers of enzymes belonging to the different categories.

References Aehle, W (2007). Enzymes in Industry – Production and Applications, 3rd ed., Wiley- VCH Verlag.

Flickinger MC and Drew SW (1998). The Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis & Bioseparation, John Wiley & Sons, 1998.

Kirk O (2005). Enzyme Applications, Industrial. Kirk-Othmer Encyclopedia of Chemical Technology, 5th ed., Wiley Interscience, 10:248–317.

Schafer T, Kirk O, Borchert T V, Fuglsang CC, Pedersen S, Salmon S, Olsen HS, Deinhammer R and Lund H (2002). Enzymes for technical applications. In: Biopolymers; Fahnestock, SR, Steinbü chel, SR, Eds Wiley-VCH: Weinheim, Germany, pp. 377−437.

Selection of an enzyme.

Selection of a production strain.

Construction of an overproducing strain by using genetic engineering techniques.

Optimization of culture medium and production conditions.

Optimization of recovery process and purification if required

Formulation of a stable enzyme product

Table 3.1: Enzyme production process

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Novozymes Bagsvaerd Denmark

Genzyme Cambridge MA, USA

New England Biolabs Beverley MA, USA

Prodigene College Station TX, USA

Amano Pharmaceutical Co. Nagoya Japan

BASF Ludwigshafen Germany

Biocon India Bangalore India

Biozyme Laboratories South Wales UK

Danisco Copenhagen Denmark

DSM Delft The Netherlands

Finnzymes Espoo Finland

Gist Brocades Delft, The Netherlands

Rhone Poulenc Cedex, France

Roche Molecular Biochemicals Indianapolis IN

Worthington Biochemical Corporation Lakewood NJ, USA

Table 3.2: Some of the enzyme manufacturing companies

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4 INDUSTRIAL APPLICATION OF ENZYMES

4.1 TEXTILES The use of enzymes in processing of the textiles is gaining worldwide recognition because of their eco-friendly and non-toxic characteristics with the increasing important requirements for textile manufactures to reduce pollution in production of textiles (Mojsov, 2012; Novozymes, 2011). Enzymes have found wide application in the textile industry for improving production methods and fabric finishing. The enzymes used in the textile industry are listed in Table 4.1.1.

Enzymes are emerging as the best alternative to the polluting textile processing methods. Enzymes are not only beneficial from ecological point of view but they are also saving lot of money by reducing water and energy consumption which ultimately reduce the cost of production.

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4.1.1 ENZYMATIC DESIZING

Many different types of compounds have been used for sizing of fabrics over the years but starch has been the most common sizing agent for more than a century and is still being used today. After weaving, the size is removed for preparing the fabric for the finishing steps of bleaching or dyeing. For desizing woven fabrics, starch hydrolyzing enzymes are used. These enzymes are highly efficient and desize the yarn without harming it. Desizing on a jigger is a simple method where the fabric from one roll is processed in a bath and rewound on another roll. The sized fabric is first washed in hot water (80–95 °C) to gelatinize the starch. The pH of the desizing liquor is adjusted to 5.5–7.5 and temperature is adjusted to 60–80 °C depending on the enzyme. The fabric then goes through an impregnation stage before amylase enzyme is added. Degraded starch in the form of dextrins is then removed by washing at 90–95 °C for two minutes. The jigger process is a batch process. In continuous high-speed processes, the reaction time for the enzyme may be as short as 15 seconds. Desizing on pad rolls is continuous in terms of the passage of the fabric. But, a holding time of 2–16 hours at 20–60 °C is required using low-temperature alpha-amylases before the size is removed in washing chambers. Desizing reactions are performed with high-temperature amylases, in steam chambers at 95–100 °C or even higher temperatures, to allow a fully continuous process.

4.1.2 ENZYMES FOR DENIM FINISHING

Denim is heavy grade cotton. In this, dye is mainly adsorbed on the surface of the fibre. That is why fading can be obtained without significant loss of strength. In traditional process, sodium hypochlorite or potassium permanganate was used along with pumice stones (Pedersen and Schneider, 1998). Disadvantages of this method are that pumice stones cause large amount of back-staining and are required in very large amount and cause significant wear and tear of machine. These disadvantages lead to the use of enzymes. Cellulase enzyme is used in denim washing. Cellulase enzymes work by loosening the indigo dye on the denim in a process known as “Bio-stonewashing”. A small quantity of enzyme can replace several kilograms of pumice stones. The use of less pumice stones results in less damage to garment, machine and less pumice dust in the laundry environment. Some researchers have reported that laccase is an effective replacement for stone-washing effects of denim fabric with and without using a mediator (Campos et al., 2001; Pazarloglu et al., 2005).

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4.1.3 BIOSCOURING

Scouring is removal of non-cellulosic material present on the surface of the cotton. In general, cellulase and pectinase emzymes are combined and used for bioscouring. The pectinase enzyme destroys the structure of cotton cuticle by digesting the pectin and removing the connection between the cuticle and the body of cotton fibre whereas cellulase enzyme destroys cuticle structure by digesting the primary wall of cellulose immediately under the cuticle of cotton. Chemical oxygen demand (COD) and biological oxygen demand (BOD) of the effluents generated by enzymatic scouring process are 20–45% as compared to alkaline scouring (100%). Total dissolved solids (TDS) in the effluents from enzymatic scouring process is 20–50% as compared to alkaline scouring (100%). Handling is very soft in enzymatic scouring compared to harsh feel in alkaline scouring process. With enzymatic scouring, it is possible to effectively scour fabric without negatively affecting the fabric or the environment. It also reduces health risks as the operators are not exposed to aggressive chemicals (Pawar et al., 2002).

4.1.4 ENZYMATIC BLEACHING

The objective of cotton bleaching is to decolorize natural pigments and to confer a pure white appearance to the fibres. Flavonoids are mainly responsible for the color of the cotton (Hedin et al., 1992; Ardon et al., 1996). The most common industrial bleaching agent is hydrogen peroxide. Conventional preparation of cotton requires high amounts of alkaline chemicals and consequently, large quantities of rinse waters are generated. However, radical reactions of bleaching agents with the fibre can lead to a reduction in the degree of polymerization resulting in extensive damage. So, replacement of hydrogen peroxide by an enzymatic bleaching system would not only lead to better product quality because of less fibre damage but also to significant savings on washing water required for the removal of hydrogen peroxide. An alternative to this process is to use a combination of suitable enzyme systems. Pectinases, amyloglucosidases, and glucose oxidases are used. These enzymes are compatible concerning their active pH and temperature range. Tzanov et al. (2003) reported for the first time the enhancement of the bleaching effect achieved on cotton fabrics using laccases in low concentrations. In addition, the short time of the enzymatic pretreatment, sufficient to improve fabric whiteness, makes this bio-process suitable for continuous operations. Laccase from a newly isolated strain of T. hirsuta was found to be responsible for whiteness improvement of cotton most likely due to oxidation of flavonoids (Pereira et al., 2005). Basto et al. (2007) proposed a combined treatment with ultrasound-laccase for bleaching of cotton. The supply of low ultrasound energy (7W) improved the bleaching efficiency of laccase on cotton fabrics. Natural fabrics are normally bleached with hydrogen peroxide before dyeing. Catalase enzyme is used to break down hydrogen peroxide bleaching

Download free eBooks at bookboon.com 32 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes liquor into water molecules and less reactive gaseous oxygen. The enzymatic process results in cleaner waste water or reduced water consumption compared with the traditional clean- up methods.

4.1.5 BIOPOLISHING

Bio-polishing is a finishing process which improves the quality of fabric by reducing the pilling tendency and fuzziness of knitted fabrics. This finishing process applied to cellulose textiles produces permanent effects by the use of enzymes. This process removes protruding fibres and stubs from knitted fabrics, significantly reduces pilling, softens fabric and provides a smooth fabric appearance (Cavaco-Paulo, 1998; Cavaco-Paulo et al., 1996; Cavaco- Paulo and Gübitz, 2003; Lenting and Warmoeskerken, 2001; Steward, 2005). The main characteristics imparted to the fabric during biopolishing treament are – cleaner surface is obtained conferring a cooler feel; lustre is obtained as a side effect; fabric obtains softer feel and tendency of the fabric to pill ends.

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4.1.6 CELLULASES FOR BIOBLASTING

Bioblasting can be used to improve cotton and other natural and man-made cellulosic fibers. The major advantage of bioblasting is the prevention of pilling. A ball of fuzz is called a “pill” in the textile trade. These pills can present a serious quality problem because they result in an unattractive, knotty fabric appearance. Cellulases hydrolyze the microfibrils protruding from the surface of yarn because they are most susceptible to enzymatic attack. These results in weakening of the microfibrils, which tend to break off from the main body of the fibre and leave a smoother yarn surface. After bioblasting, the fabric shows a much lower pilling tendency. Other benefits of removing fuzz are a softer, smoother feel, and superior color brightness. Conventional softeners, tend to be washed out and often result in a greasy feel whereas the softness-enhancing effects of bioblasting are wash-proof and non-greasy.

For cotton fabrics, the use of bioblasting is optional for upgrading the fabric. However, bioblasting is very much required for the new type of regenerated cellulosic fibre lyocell. Lyocell is made from wood pulp and is characterized by a high tendency to fibrillate when wet. In simple terms, fibrils on the surface of the fiber peel up. If they are not removed, finished garments made of lyocell will end up with an unacceptable pilled look. This is the reason why lyocell fabric is treated with cellulases during finishing. Cellulases also improve the attractive, silky appearance of lyocell.

Amylases

Desizing

Cellulases and Hemicellulases

Biostoning of jeans Desizing of CMC Stylish effect on cellulose fibres

Pectinases

Scouring of vegetable as well as bast fibres e.g. cotton, jute

Proteases

Scouring of animal fibres, degumming of silk and modification of wool properties

Lipases

Elimination of fat and waxes

Table 4.1.1: Application of enzymes in textile industry

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4.2 PULP AND PAPER INDUSTRY The last several years showed an increase in activity in the use of enzymes in pulp and paper industry. Suitable biological treatments in combination with less intensive conventional treatment could help solve many of the problems of currently used processes. The use of enzymes in pulp and paper production is making its presence felt in the industry owing to enhanced productivity, reduced environmental damage and less energy requirement (Bajpai, 2015).

There is high scope for enzymes in pulp and paper industry because of their eco friendly nature. Enzymes are extremely attractive “Green Chemicals” that can improve operations in pulp and paper.

In response to environmental concerns and regulations, the industry has greatly reduced the generation of organochlorine compounds that are produced during pulp bleaching. This has been achieved by reducing the amount of residual lignin in pulps and second by using other types of bleaching agents. Xylanase enzymes have helped to achieve this goal by reducing or even eliminating the need for chlorine in the manufacture of elemental chlorine free (ECF) and totally chlorine free (TCF) printing and writing paper grades. Xylanases have saved chemical costs for the industry without interfering with the existing process. This technology has increased the bleaching rate in both TCF and ECF processes. In the case of chlorine dioxide bleaching, the use of enzymes has actually increased the throughput of the plant due to debottlenecking at the chlorine dioxide generator. These developments are viewed very favorably since they enable the industry to make better use of its existing capital equipment. Enzymes have helped meet environmental objectives in other ways also. By reducing costs involved in deinking, enzymes have increased the ability of manufacturers to recycle fiber, thereby placing fewer demands on raw material resources. Enzymes have been used commercially to reduce paper manufacturing costs or improve the product. Lipases are able to control the accumulation of pitch during the production of paper from pulps having high resin content, such as sulfite and mechanical pulps from pine. Enzymes also help removing contaminants in the recycle stream. They can reduce the accumulation of adhesives and pitch residues, called stickies, on machines. They can also facilitate the deinking of recycled paper and improve pulp drainage, which is very much important as the amount of recycled fiber increases in the feedstock stream. Paper machines are able to operate faster, with higher drainage rates, which again save capital costs (Bajpai, 1999; 2012). Several other enzyme applications are also possible. These include eliminating caustic chemicals for cleaning paper machines, improving kraft pulping, reducing refining time, decreasing vessel picking, facilitating retting, selectively removing fiber components, modifying fiber properties, increasing fiber flexibility, and covalently linking side chains or functional groups (Bajpai, 2012). The chemicals used in specialty paper industry have a severe impact on the environment. This can be reduced with the help of enzymatic treatments.

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4.2.1 BLEACHING

Xylanases Bleaching of chemical pulp with xylanase enzyme is the most widely used and best established biotechnical application in pulp bleaching (Bajpai, 2004; 2009; 2015). Xylanase addition to brownstock before bleaching saves on bleaching chemicals. This observation by VTT, scientists (Viikari et al., 1986) lead to the first widespread application of enzymes in the industry. This technology has become one of the solutions considered by the pulp and paper industry to give an innovative, environmentally and economically acceptable answer to the pressures exerted on chlorine bleaching by regulatory authorities in Western countries and by more demanding, environmentally minded consumers. Today about 10% of all kraft pulp is manufactured with xylanase prebleaching. In North America, Iogen Corp, based in Ottawa has established a market leadership position. Globally, other suppliers such as Novozymes, AB Enzymes, Enzymatic Deinking Technologies, Enzyme Development Corporation, DuPont and Diversa, are also selling to this market. In Japan, Oji Paper is unique in manufacturing xylanase on-site at its Yonago mill. The enzyme is produced from a bacterial fermentation of pulp side stream which results in a xylanase/pulp mixture. This mixture is then fed to the main pulp brownstock storage tank. The mechanism of xylanase prebleaching is still a subject of debate. Certainly there is some solubilization of xylan by the enzyme, and this

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-- Reducing AOX discharges, by reducing the use of chlorine gas -- Debottlenecking mills limited by chlorine dioxide generator capacity -- Eliminating the use of chlorine gas for mills at high chlorine dioxide substitution levels -- Increasing the brightness ceiling particularly for mills using ECF and TCF bleaching sequences -- Reducing cost of bleaching chemicals, particularly for mills using large amounts of chlorine dioxide or peroxide.

These benefits are obtained over the long term when the enzymes are selected and applied in the mill in a proper way (Viikari et al., 1994). The ability of xylanases to activate pulps and increase the efficacy of the bleaching chemicals may allow new bleaching technologies to become more effective. For expensive chlorine-free bleaching options such as ozone and hydrogen peroxide, xylanase pretreatment may eventually allow them to become cost effective. Traditional bleaching technologies also stand to benefit from xylanase treatments. Xylanases can be easily used and require essentially no capital expenditure. Because chlorine dioxide doses can be reduced, xylanases may reduce the need for increased chlorine dioxide generation capacity. Similarly, the installation of expensive oxygen delignification facilities may be avoided. The benefit of a xylanase stage can also be taken to shift the degree of substitution towards higher chlorine dioxide charges while maintaining the total dosage of active chlorine. Use of high chlorine dioxide substitution significantly reduces the formation of AOX. In totally chlorine free-bleaching processes, the use of enzymes increases the final brightness, which is an important parameter in marketing chlorine free pulp. In addition, savings in TCF bleaching are important with respect both to strength properties of the pulp and the cost. Several alternative new bleaching methods based on various chemicals such as oxygen, ozone, peroxide and peroxy acids have been developed. In addition, an oxygen delignification stage has already been installed at many kraft mills. In the bleaching sequences in which only oxygen-based chemicals are used, xylanase pretreatment is generally used after oxygen delignification to improve the otherwise lower brightness of the pulp or to reduce bleaching costs. The TCF sequences generally also contain a chelating step in which the amount of interfering metal ions in pulp is reduced. The order of metal removal and enzymatic stages is found to be important for an optimal result. The enzyme stage should be carried out before or simultaneously with the chelating stage for obtaining the maximum benefit of enzymatic treatment in pulp bleaching. In fact, the neutral pH of enzyme treatment is optimum in many cases for chelation of magnesium, iron and manganese ions that must be removed before bleaching with hydrogen peroxide. The TCF technologies are usually based on bleaching of oxygen-delignified pulps with enzymes and hydrogen peroxide (Bajpai, 2012).

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Laccases Certain oxidative enzymes, such as laccases can directly target residual lignin in kraft pulp (Bajpai et al., 2006; Bajpai, 2015; Bourbonnais and Paice, 1996), which is the objective of bleaching. Laccase enzymes are highly specific towards lignin; there is no damage or loss of cellulose and can produce larger chemical savings than xylanase but has yet not been developed to the full scale. It is being studied in several laboratories all over the world. Laccase has however found some applications in textiles and as a hair dye. The problem with laccase is that they require a dedicated bleaching stage, with addition of an oxidant (oxygen or peroxide) and a chemical mediator that can penetrate the fiber cell wall. Laccase has a rather broad range of substrates and may find other applications, such as in extractives removal. Treatment with laccase enzymes results in more removal of lignin as compared to oxygen delignification. This translates into substantial savings of energy and bleaching chemicals which in turn leads to a reduced pollution load.

4.2.2 ENZYMES FOR DEINKING

Recycled fibers are one of the most important fiber sources for tissue, newsprint, and printing paper. Enzymatic deinking represents a very attractive approach to chemical deinking. The most widely used enzyme classes for deinking are cellulases, amylases, and lipases. Cellulases are being used to facilitate deinking of mixed office waste (MOW) (Bajpai and Bajpai, 1998). The company Enzymatic Deinking Technologies has been one of the most active players in this application. Most deinking trials with enzymes are by necessity at neutral or acidic pH which make it difficult to compare with conventional alkaline deinking chemistries. Cellulases appear to be not much effective for ONP deinking (Xia et al., 1996). Ink particles tend to be smaller after treatment of ONP with cellulase, reducing the pulp brightness. With the trend of deinking chemistry moving towards neutral conditions, there may be more opportunity to use enzymes or enzyme/chemical combinations in deinking plants.

The most promising implication of high deinking efficiency from enzyme enhanced deinking is that the dewatering and dispersion steps and also subsequent flotation and washing may not be essential. This would save capital expenses in construction of deinking plants and also reducing consumption of electrical energy for dewatering and dispersion. The requirement of bleach chemicals is usually lower for enzymatic deinking as compared to conventional chemical deinking. Reduced chemical use would result in reduced waste treatment costs while reducing the environmental impact. Reduced bleaching costs and reduced pollution can also be anticipated, since enzymatically deinked pulps have proved to be easier to bleach and require lesser chemicals than pulps deinked by conventional methods. Enzymatically deinked pulps also show improved drainage, higher brightness, superior physical properties

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For deinking of old newsprint (ONP) cellulases and lipases have shown the most promising results. The increase in environmental awareness has resulted in the development of printing inks based on vegetable oils. Use of lipases for deinking of vegetable oil-based newsprint could obtain remarkable ink removal and brightness improvement.

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4.2.3 MODIFICATION OF FIBER PROPERTIES

Enzymatic modification of fibers aims at reduced energy consumption in the production of thermomechanical pulps and increased beatability of chemical pulps or improvement of fiber properties (Bajpai, 1997). In high yield mechanical pulps, most of the lignin and hemicellulose remains in the pulps. According to determinations of the medium pore width and immunolabeling of the untreated wood, it is evident that enzymatic modifications to the composition of mechanical pulps can be obtained only on the outer surface of the fiber. This was verified when xylanases were applied to thermo-mechanical pulp (Jeffries and Lins, 1989). Even when using rather higher enzyme dosages, only about 1% of the pulp was dissolved. When combined with an alkaline pretreatment, the enzymatic treatment was significantly improved, and the amount of energy required for refining the thermomechanical pulp was reduced. In 1942, a patent claimed that microbial hemicellulases from Bacillus and various Aspergillus species could aid refining and hydration of pulp fibers (Diehm, 1942). In 1959, Bolaski et al. patented the use of cellulases from Aspergillus niger to separate and fibrillate pulp. A process patented in 1968 used cellulases from a white rot fungus to reduce beating or refining time (Yerkes, 1968). The desired structural changes in the fiber which are created during beating and refining are external fibrillation and fiber swelling, which improve the flexibility and bonding ability of the fibers. The role of xylans in fiber properties was studied using xylanases from Sporotrichium dimorphosporum in the treatment of fully bleached spruce sulfite and birch kraft pulps. Electron microscopic examination showed external fibrillation and good flexibility of fibers, implying internal modification (Mora et al., 1986). The water retention value, which describes fiber swelling, was significantly increased. The enzymatically treated pulps were comparable with slightly beaten pulps. The beatability was generally improved, and the energy demand was reduced about 3-fold (Noe et al., 1986). Water removal on the paper machine has been shown to improve as a result of limited hydrolysis of the fibers in recycled paper. A mixture of xylanase and cellulase enzymes at low concentrations has been found to markedly increase the freeness of recycled fibers without substantially reducing yield (Fuentus and Robert, 1988). The lower the initial freeness, the greater is the gain following treatment. Many different cellulases and hemicellulases have been found to improve freeness (Bhardwaj et al., 1995, 1997; Pommier et al., 1989, 1990). Freeness shows a rapid initial increase, with over half of the observed effect occurring in the first 30 min. A relatively small amount of the enzyme is required. While the initial effects are largely beneficial, extending the reaction time with large concentrations of enzyme is detrimental. Unfortunately, crude enzyme mixtures also reduce strength properties. Mill trials were performed successfully using a commercial T. reesei enzyme called Pergalase A40 (Pommier et al., 1990). Mixed xylanases and cellulases peel the surface of the fibers. If treatment is limited, the enzymes remove only elements those have a great affinity for water but which contribute little to inter-fiber binding potential. By selectively removing these surface components, pulp water interactions are reduced and drainage increases without noticeable changes in the final mechanical strength properties

Download free eBooks at bookboon.com 40 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes of the pulp. If the treatment is extended, however, fibrillation becomes pronounced and drainage decreases. If large quantities of crude enzymes are used, the average fiber length is reduced, fines disappear, and the strength properties of the fibers are lost. So, an optimum level of enzyme treatment is needed. Drainability of mechanical pulp can also be improved by the addition of hemicellulases (Karsila et al., 1990). Xylanase improves the freeness of deinked recycled pulp while having no negative effect on fiber tensile strength properties. By, comparison, the tear indices of recycled pulps reduced on treatment with cellulase (Karsila et al., 1990). These findings showed that xylanases might be much more effective than cellulases or crude xylanase-cellulase mixtures. Xylanases, however, remove hemicelluloses which promote inter-fiber bonding. This effect can also lead to poor paper properties. The degree of polymerization of pulp treated with cellulase-free xylanase was found to increase, due to the selective removal of xylan, which has a reduced DP (Clark et al., 1989; Puls et al., 1990). But, even the presence of low cellulase activities in the enzyme preparation results in reduced viscosity.

4.2.4 PRODUCTION OF DISSOLVING PULP

Dissolving pulps are used to produce cellulosic materials such as acetates, cellophanes, and rayons (Hinck et al., 1985). Their manufacture is characterized by the derivatization and thus solubilization of highly purified cellulose. Hemicellulosic contaminants lead to color and haze in the product as well as insolubles which hamper the manufacturing process. Their extraction from pulps requires the use of high caustic loadings and appropriate pulping conditions, the latter restricted to sulfite pulping and acid-pretreated kraft pulping. Xylanse enzymes have been studied for the production of dissolving pulps (Christov and Prior, 1994; Christov et al., 1995; Christov and Prior, 1996; Bajpai and Bajpai, 2001; Bajpai et al., 2005). These pulps are used to produce cellulosic materials such as acetate, cellophanes, and rayons. Their manufacture is characterized by the derivatization and thus solubilization of highly purified cellulose. Hemicellulosic contaminants lead to color and haze in the product, as well as to insolubles that hamper the manufacturing process. Their extraction from the pulps requires the use of high caustic loadings and appropriate pulping conditions, the latter restricted to sulfite pulping and acid-pretreated kraft pulping. The use of xylanase for purifying cellulose has been tried. The complete enzymatic hydrolysis of the hemicellulose in the pulp is difficult to achieve. Even with very high enzyme loading, only a relatively small amount of xylan could be removed. However, the inaccessibility of a large portion of the xylan in pulps, has limited the potential of this application. Nevertheless, xylanase treatment may reduce the chemical loading required during caustic extraction or facilitate xylan extraction from kraft pulps.

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4.2.5 REMOVAL OF PITCH

Pitch is composed of fatty acids, resin acids, sterols, glycerol esters of fatty acids, other fats, and waxes and is usually defined empirically as the wood component which is soluble in methylene (Allen, 1975). It is less than 10% of the total weight of wood but causes major problems. Pitch reduction with enzymes is a very efficient biotechnological method (Fischer and Messner, 1992a,b; Fischer et al., 1993; Fujita et al., 1992; Gibson, 1991; Irie and Hata 1990). Different lipases have been used for removal of pitch. Few commercial preparations of lipases for pitch removal are available (Fujita et al., 1992). Enzymatic pitch control helps to reduce pitch-related problems to a satisfactory level. It reduces defects on paper web as well as the frequency of cleaning pitch deposits in the paper machine. At the same time, it also offers other advantages, such as ecofriendly and nontoxic technology, improved pulp and paper quality, reduction in bleaching chemical consumption, reduction of effluent load, and space and cost saving in a mill wood yard by using unseasoned logs. By reducing the outside storage time of logs, this method reduces wood discoloration, wood yield loss, and the natural wood degradation which occurs over longer storage time. With chemical (sulfite) pulps, the applications of lipase improve the properties of resins by lowering their effectiveness. Since 1990, this method has been used commercially (Grant, 1994).

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4.2.6 SLIME CONTROL AND BOIL-OUTS

Slime deposition causes significant operating problems around a paper machine. Slime is the generic name for deposits of microbial origin in a paper mill. It is impractical to run a paper mill as a sterile system. As a result, a vast array of microbes contaminates the mills, and many of the resulting slime compounds have not been characterized. When confronted with slime, often the strategy is to try every available biocide until one is found which targets the microbe and destroys the source of slime. However, in some cases, specific slime compounds have been characterized, and efficient methods for their removal can be used. One such case is with levan, which is a β-2,6-linked polymer of fructose that forms a slime film. This compound is secreted by several species of Bacillus and Pseudomonas bacteria that can grow in the recirculation water around the paper machine, especially for fine paper, where the level of inhibiting compounds is low. The enzyme levan hydrolase can hydrolyze this polymer to low-molecular weight polymers that are water soluble, thereby cleaning the slime out of the system. Commercial levan hydrolase is supplied as the product EDC-1 by Henkel Corporation (Morristown, U.S.A.). The enzyme does not harm cellulose, so it is not harmful to the paper. The enzyme is usually added at the head box of the paper machine, although in some cases it has been added at dryer discharge. The enzyme is effective at pH 4-8 and runs best at pH 5.0. Many enzymatic products are already in industrial use around the world and many additional products are currently being designed and tested for a wide variety of specific applications. Use of enzymes in combination with bio-dispersants appears to be a promising method for slime control.

Use of alpha amylase enzyme in combination with lipase and protease in paper machine boil-out has provided unprecedented results compared to traditional caustic treatment. These enzymes are also effective to remove slime and control the growth of bacteria in paper machine systems. This technology has been well received by the mills; especially those using a starch based coating system.

Enzyme based boil-outs remove the compounds such as starch, slime, pitch, adhesives, latex and other synthetic binders that hold the deposits together. The type of enzyme used and the dispersant depends on the type and amount of deposit present in the system. Starch slurry contains deposits that are microbiological and/or starch protein based. Boil-outs using a product that contains a stabilized protease enzyme are found to be effective in these systems. For the cooked starch system, an alpha amylase product is used to remove deposits comprised mainly of cooked starch. In both cases, a preboil-out system flush is essential. This removes the cooked starch and allows the enzyme to work particularly on deposits. Enzymatic boil-outs are pH neutral and can be dumped directly to the sewer, without neutralizing the solution, and without any upset at the waste treatment facility. Also, it removes the safety concerns associated with working with and around the caustic solution. It eliminates the requirement for excessive rinsing to purge caustic from the system.

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Each of these factors contributes to a reduction in the downtime necessary for a boil-out and maintenance outage, which shows cost savings. Starch based coating systems can be successfully cleaned via a boil-out using the alpha amylase enzyme. The parameters for this boil-out are similar with the enzyme based boil-out product as mentioned above.

4.2.7 REMOVAL OF SHIVES

Shives are small bundles of fibers that have not been separated into individual fibers during the pulping process. They appear as splinters that are darker than the pulp. One of the most important quality criteria for bleached kraft pulp is the shive count. A novel enzyme formulation, Shivex, can be used to increase the efficiency of shive removal by bleaching (Bajpai, 1999; 2015). By treating brownstock with Shivex, mills can increase the degree of shive removal in the subsequent bleaching by 55% (Tolan et al., 1994). Depending upon the shive level in the incoming brown-stock and the desired shive level of the bleaching pulp, this allows a mill to decrease its actual shive count or to increase its margin of safety against shives. The increase in shive removal is accompanied by an increased efficiency in the bleaching of pulp. Therefore, mills can reduce chlorine use in a bleach plant without compromising on shive counts. Shivex is a multicomponent mixture of proteins, some of which are xylanases, but the degree of shive removal by the enzyme is not directly related to the enzymes’ xylanase activity or bleach boosting effectiveness.

4.2.8 DEBARKING

Removal of the bark is the first step in all processing of wood. This step consumes substantial amounts of energy. Extensive debarking is needed for high-quality mechanical and chemical pulp because even small amounts of bark residues cause darkening of the product. In addition to its high energy demand, complete debarking leads to losses of raw material due to prolonged treatment in the mechanical drums. The border between wood and bark is cambium, which consists of only one layer of cells. This living cell layer produces xylem cells toward the inside of the stem and phloem cells toward the outside. In all the wood species studied, common characteristics of the cambium include a high content of pectins and the absence or low content of lignin (Dey and Brenson, 1984; Kato, 1981). The content of pectins in cambium cells varies among the wood species but may be as high as 40% of the dry weight. The content of pectic and hemicellulosic compounds is also high in the phloem (Fu and Timell, 1972). Pectinases are found to be key enzymes in the process, but xylanases may also play a role because of the high hemicellulose content in the phloem of the cambium (Viikari et al., 1989; Bajpai, 2009; 2015). The energy consumed in debarking

Download free eBooks at bookboon.com 44 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes was found to reduce as much as 80% after pretreatment with pectinolytic enzymes (Ratto et al., 1993). One of the major difficulties with enzymatic debarking is the poor infiltration of enzymes in the cambium of whole logs (Viikari et al., 1989; Ratto et al., 1993).

4.2.9 RETTING OF FLAX FIBERS

Enzymes have been used in processing plant fiber sources such as flax and hemp (Sharma, 1987a,b; Gillespie et al., 1990). At present, fiber liberation is affected by retting i.e., the removal of binding material present in plant tissues using enzymes produced in situ by microorganisms. Pectinases are believed to play the main role in this process, but xylanases may also be involved (Sharma, 1987a,b). Replacement of slow natural retting by treatment with artificial mixtures of enzymes could become a new fiber liberation technology (Bajpai, 2009).

4.2.10 REDUCTION OF VESSEL PICKING

The use of tropical hardwoods such as eucalyptus for pulp production has increased in recent years. The trees grow rapidly, so the chip supply is plentiful, and the pulps are

Download free eBooks at bookboon.com Click on the ad to read more 45 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes useful for many applications. The vessel elements of tropical hardwoods are, however, large and hard, and they do not fibrillate during normal beating. As a result, they stick up out of the surface of the paper. During printing, the vessels are torn out, leaving voids. This characteristic reduces the value of tropical hardwood pulps. Although increased beating can eventually increase vessel fibrillation and flexibility, it can also result in poor drainage. A patent of disclosure from Honshu Paper Co. described the use of commercial cellulases to enhance the flexibility of hardwood vessels. Enzyme treatment reduced vessel picking by 85%. At the same time, smoothness and tensile strength increased. Draining time also increased (Jeffries, 1992).

4.2.11 SURFACE SIZING AND COATING

Enzymes have been used for modification of starches for surface sizing and coating for long time (Bajpai, 2008, 2012, 2015; Smook, 1992). Starch imparts many beneficial properties to paper. These include strength, stiffness and erasability. Properly controlled enzymatic modification of starch provides the papermaker ample opportunities to get uniform quality of starch paste, to produce starch paste as per requirement and to produce quality surface sized papers with the minimum cost of the starch component. Alpha amylase enzyme is used for modification of starch. The enzyme modified starch meets almost all the properties required for surface sizing of writing and printing grades of paper (Maurer, 2001a,b; Svenson, 2006; de Souza et al., 2010). The process of enzyme modification can be applied to different types of starches. The operating conditions in terms of enzyme dose and reaction time may vary. Enzyme-modified starch is available from starch producers or can be produced on site at the paper mill using a batch or continuous process. No capital investment is required to switch over from oxidized starch to in situ modification of native starch with enzyme.

Oxidized starch may contain AOX products, which are formed by the reaction of sodium hypochlorite with residual lipids in native starch (Maurer, 2001a,b). The presence of AOX products in starch can affect its use in consumer products. Modification of starch with enzyme does not involve any chemicals; it is totally free from AOX products. Oxidation of native starch with sodium hypochlorite though takes place at relatively lower temperature, it requires longer reaction time. As the reaction is not so selective, it results in significant loss of starch (30–40%) in the form of water soluble material, which goes into the wastewater requiring elaborate treatment. It results in increased cost of oxidized starch. On the other hand, enzymatic reaction is highly selective and hydrolysis can be controlled in order to avoid generation of any soluble material but reduces the viscosity to a desired value. Oxidized starch is produced by chemical modification at the site of starch manufacturers. Therefore, papermaker has no control over its quality in terms of viscosity etc. The cooking of oxidized starch is done at the papermaker’s site for its dispersion and gelation only. The

Download free eBooks at bookboon.com 46 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes enzymatic modification is done by the papermakers at their site where the final viscosity is controlled by the paper mill. The cost of surface sizing using enzymatically modified starch is much lower as compared to that using oxidized starch (Bajpai, 2005). As the native starch contains some residual protein, the brightness of enzymatically modified starch is little lower than the oxidized starch. The optical brightening agents can be used to compensate the brightness. There is also a coloration problem due to the presence of metals in the native starch. Therefore, the native starch used for enzymatic modification should have negligible protein and ash contents. Process conditions (in terms of pH of the starch slurry, temperature time profile, enzyme dose and reaction times) are very sensitive to control the quality of enzymatically modified starch. There is a significant difference in the cost of oxidized starch and enzymatically modified starch. So, the paper mills can realize substantial saving by switching over from oxidized starch to the enzymatically modified starch.

4.2.12 EFFLUENT TREATMENT

Laccase and peroxidase enzymes have been found useful for waste water treatment in the pulp and paper industry. High removal efficiencies were obtained for chlorinated phenols, guaiacols, vanilins and catechols (Forss et al., 1987). The color removal from effluents at neutral pH by low levels of hydrogen peroxide was enhanced by the addition of peroxidase (Paice and Jurasek 1984). No precipitation occurred during the decolorization process. The catalysis with peroxidase (20 mg/l) was observed over a wide range of peroxide concentrations (0.1–800 mM) but the largest effect was between 1 mM and 100 mM. The pH optimum for catalysis was around 5.0.

Field (1986) patented a method for the biological treatment of waste waters containing nondegradable phenolic compounds and degradable non-phenolic compounds. It consisted of an oxidative treatment to reduce or eliminate toxicity of the phenolic compounds followed by an anaerobic purification. This oxidative pretreatment could be performed with laccase enzymes and it was claimed to reduce COD by one thousand fold. Call (1991) patented a process on the use of laccase for waste water treatment. He claimed that waste water from delignification and bleaching could be treated with laccases in the presence of nonaromatic oxidants and reductants and aromatic compounds. Almost complete polymerization of the lignin is obtained which is 20–50% above the values attainable with the addition of laccase alone. About 70–90% lignin is developed into insoluble form, which is removed by flocculation and filtration.

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Amylases

Starch modification Deinking Drainage improvement Boil-outs and slime control

Xylanases

Bleach boosting Refining Drainage Removal of shives Production of dissolving pulp

Cellulases

Deinking Drainage improvement Energy saving Tissue and fiber modification

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Lipases and esterases

Pitch control Stickies control Deinking Cleaning

Pectinases

Refining

Table 4.2.1: Applications of enzymes in the pulp and paper industryBased on Bajpai (2009, 2015)

4.3 STARCH INDUSTRY Starch is the commonest storage carbohydrate in plants. It is used by the plants themselves, by microorganisms and by higher organisms therefore there is a great diversity of enzymes able to catalyze its hydrolysis. Approximately 60 million tons of starch is converted into sweeteners and ingredients per year (https://www.novozymes.com/…/new-enzyme-produces- sweeteners-at-the-lowest-cost- ). These sweeteners are used in popular consumer food products, including soft drinks, confectionery, sauces and canned fruits.

The starch industry became the second largest industry to use enzymes after the detergent industry.

Following three stages are involved in the conversion of starch.

1. Gelatinization: involves the dissolution of the nanogram-sized starch granules to form a viscous suspension 2. Liquefaction: involves the partial hydrolysis of the starch, with concomitant loss in viscosity 3. Saccharification: involves the formation of glucose and maltose by further hydrolysis

Gelatinization is obtained by heating starch with water, and occurs when starchy foods are cooked. Gelatinized starch is rapidly liquefied by partial hydrolysis with enzymes or acids and saccharified by further acidic or enzymatic hydrolysis.

Various manufacturers use different methods for starch liquefaction using alpha-amylases but the principles are the same. Granular starch is slurried at 30–40% (w/w) with cold water, at pH 6.0–6.5, containing 20–80 ppm Ca2+ and the enzyme is added (via a metering pump).

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Calcium ions stabilize and activate the enzyme. The alpha -amylase is usually sold at high activities so that the enzyme dose is 0.5–0.6 kg per ton (about 1500 U/kg dry matter) of starch. When Termamyl (thermostable alpha amylase from Novozymes) is used, the slurry of starch plus enzyme is pumped continuously through a jet cooker, which is heated to 1050C using live steam. Gelatinization takes place very rapidly and the enzymatic activity, combined with the significant shear forces, begins the hydrolysis. The residence time in the jet cooker is few minutes. The partly gelatinized starch is passed into a series of holding tubes maintained at 100–1050C and held for 5 min to complete the gelatinization process. Hydrolysis to the required DE is completed in holding tanks at 90–1000C for 1 to 2 h. These tanks contain baffles to avoid backmixing.

The liquefied starch is usually saccharified but comparatively small amounts are spray-dried for sale as ‘maltodextrins’ to the food industry mainly for use as bulking agents and in baby food. In this case, residual enzymatic activity may be destroyed by reducing the pH towards the end of the heating period.

Figure 4.3.1 shows the use of enzymes in processing of starch and the typical conditions for the same. Table 4.3.1 shows the list of enzymes, involved in starch degradation. Microorganisms are the major source for starch hydrolases, generally called amylases

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(Bisgaard-Frantzen et al., 1999). Amylases are classified according to the specific glucosidic bond they cleave as α-1,4-glucanases or α-1,6 glucanases. Endoglucanases act on interior bonds of starch whereas exoglucanases cleave the bonds successively from nonreducing ends of starch. Activities of amylases result in smaller molecules called dextrins, disaccharides, and monosaccharides. Glycosyl transferases are enzymes that synthesize cyclic molecules from starch.

New alpha amylases with optimized properties, such as improved thermal stability, acid tolerance, and ability to function without the addition of calcium, have been developed (Kirk et al., 2002; Bisgaard-Frantzen et al., 1999; Shaw et al., 1999; Declerck et al., 2000) offering obvious benefits to the industry. Engineering efforts have also been made to develop improved versions of the enzymes used later in the process (i.e. glucoamylase and glucose isomerase) (Sauer et al., 2000; Hartley et al., 2000).

Starch granules

35% in cold water pH 6.5 40 ppm Ca2+

Starch slurry

bacterial α-amylase, 1500 U kg-1 Gelatinisation 105ºC, 5 min

Gelatinised starch (<1 DE)

Liquefaction 95ºC, 2 h

Liqueﬁed starch (l 1 DE) 0.3% D-glucose 2.0% maltose 97.7% oligosaccharides Sacchariﬁcation pH 4.5 pH 5.5 glucoamylase, 150 U kg-1 fungal α-amylase 2000 U kg-1 pullulanase, 100 U kg-1 50 ppm Ca2+ 60ºC, 72 h 55ºC, 48 h

Glucose syrup (99 DE) Maltose syrup (44 DE) 97% D-glucose 4 % D-glucose 1.5% maltose 56% maltose 0.5% isomaltose 28% maltotriose 1.0% other oligosaccharides 12% other oligosaccharides

Figure 4.3.1: Use of enzymes in processing of starch http://www1.lsbu.ac.uk/water/enztech/starch.htm Reproduced with permission

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α -Amylase (1,4- α-D-glucan Glucanohydrolase EC 3.2.1.1)

α-Amylases are α-1,4-endoglucanases, that rapidly decrease starch viscosity, resulting in oligosaccharides. Some of the α-amylases produce higher concentrations of mono- and disaccharides. They are classified as saccharifingα -amylases. α-Amylases, that reduce starch viscosity by producing precursor products for mono- and disaccharides are called liquefying enzymes. Though bond specificity for this enzyme is forα -1,4 linkages, some enzymes acting on α-1,6 linkages of starch molecule have also been reported. End products of the reaction result in oligosaccharides with α-configuration at the first carbon.

Glucoamylase or Amyloglucosidase (1,4- α-D-glucan Glucanohydrolase EC 3.2.1.3)

Glucoamylase catalyzes release of glucose from the non-reducing ends of starch, dextrins, and maltose. Glucoamylases occur widely in microorganisms and plants with filamentous fungi as the major source of the enzyme. Glucoamylases occur as multiple forms in several fungi. Glucoamylases have optimum activity at acidic pH and act at temperatures around 60 °C.

ß-Amylase (1,4-α-D-glucan Maltohydrolase, EC 3.2.1.2)

ß-Amylase hydrolytically cleaves the penultimate α-1,4 bond at the non-reducing ends of starch and causes the production of anomeric ß-maltose. Because the enzyme cannot act on α-1,6 linkages of starch, it also produces ß-limit dextrins.

Isoamylase (Glycogen 6-glucanohydrolase, EC 3.2.1.68)

This enzyme predominantly degrades α-1,6-glucosidic linkages of amylopectin, glycogen, dextrins, and oligosaccharides. Its low affinity to short chains of pullulan makes this substrate less susceptible to the enzyme activity. Isoamylases have been characterized from Bacillus spp.

Pullulanase (α-dextrin 6-glucanohydrolase EC 3.2.1.4)

Very few organisms produce this enzyme that hydrolyse α-1,6 linkages of pullulan. Their molecular mass range from 80-145 kDa.

α-Glucosidase (α-D-glucoside Glucohydrolase EC 3.2.1.20)

α-Glucosidases are exoacting enzymes that catalyze the splitting of α-glucosyl residue from the non-reducing terminals of substrates to liberate α-glucose. Typically they are called maltases, because they hydrolyze maltose to glucose.

Glucose Isomerase or Xylose Isomerase (EC 5.3.1.5)

A range of microorganisms like Streptomyces, Bacillus and Arthrobacter normally produce glucose isomerase or xylose isomerase intracellularly. Glucose isomerase generally acts at 60 °C, isomerizing glucose to fructose. Because its affinity for glucose is low, a concentrated solution of substrate is used for isomerization reaction. The molecular mass of the enzyme ranges from 80 kDa in the case of Actinoplanes missouriensis to 157 kDa in the case of Streptomyces spp. This enzyme is strongly inhibited by Ca2+ and Mn2+. It requires magnesium for activity and cobalt for maintenance of stability.

Table 4.3.1: Enzymes involved in starch degradation

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4.4 DETERGENTS One of the major application fields of enzymes is in laundry and dish-wash detergents (Schafer et al., 2002; Gerhartz, 1990; Bajpai and Tyagi, 2007; Novozymes, 2011; Kirk et al., 2002). This area represents the largest application of industrial enzymes, both in terms of volume and value. Enzymes in detergent industry are the key to cleaning. Enzymes are effective at the moderate temperature and pH values that characterize modern laundering conditions, and in laundering, dishwashing, and industrial and institutional cleaning, they contribute to several advantages (Table 4.4.1).

Use of enzymes in detergent formulations is now common in the developed countries with over half of all detergents presently available are containing enzymes. Detergents represent the largest application of industrial enzymes amounting to 25–30% of the total sales of enzymes and expected to grow faster at a CAGR of about 11.5 % from 2015 to 2020.

Enzyme applications in detergents started in the early 1930s with the use of pancreatic enzymes in presoak solutions. German scientist Otto Rohm first patented the use of pancreatic enzymes in 1913. The enzymes were extracted from the pancreases of slaughtered animals and included proteases (trypsin and chymotrypsin), carboxypeptidases, alpha-amylases, lactases, sucrases,

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Download free eBooks at bookboon.com Click on the ad to read more 53 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes maltases, and lipases. Thus, the foundation was already laid in 1913 for the use of enzymes in detergents. Now days, enzymes are continuously being used in detergent formulations for laundry, automatic dishwashing or cleaning of industrial equipment in the food industry. Soils and stains are removed by mechanical action assisted by enzymes, surfactants, polymers and builders. Surfactants of different kinds help the wash liquor wet fabrics by reducing the surface tension at the interface and help in removing various kinds of soiling. Furthermore, anionic surfactants and polymers increase the repulsive force between the original enzymatically degraded soil and the fabric and thus help to prevent soil re-deposition. Builders act to chelate, precipitate, or ion-exchange calcium and magnesium ions, to provide alkalinity and buffering capacity, and to inhibit corrosion. Enzymes in heavy-duty detergents degrade, and thus help solubilize substrate soils attached to fabrics or hard surfaces (e.g., dishes).

The major component in detergent enzymes is proteases, but other and very different hydrolases are introduced to provide various benefits, such as the efficient removal of specific stains (Table 4.4.2). Constantly, new and improved engineered versions of the ‘traditional’ detergent enzymes, proteases and amylases, are being developed. These new second- and third-generation enzymes are optimized to meet the requirements for performance in detergents, the composition of which is also being continuously developed. The compatibility of enzymes with detergent components is particularly addressed, but their ability to work at lower temperatures has also been amongst the recently reported improvements. To save energy, the temperature used in household laundering and automated dishwashers has been reduced in the recent years. This often results in problems with effective cleaning and stain removal that enzymes can help overcome. Examples of second-generation detergent enzymes include the development of novel amylases having improved activity at lower temperatures and alkaline pH, while maintaining the necessary stability under detergent conditions. These enzymes were developed by the combined use of microbial screening and rational protein engineering methods (Bisgaard-Frantzen et al., 1999). Proteases showing activity at low temperatures have been isolated from nature, but have also been evolved in the laboratory by a directed evolution approach (Wintrode et al., 2000). Moreover, from a starting material of 26 subtilizing proteases, Ness et al. (1999) used one round of DNA shuffling to isolate new proteases with various improved properties. The improvements included characteristics very relevant for detergent proteases (i.e. improved activity and stability at alkaline pH). The introduction of a new enzyme class into a detergent has been the addition of a mannanase – the result of a joint development between Procter and Gamble and Novozymes (McCoy, 2001). This enzyme helps remove various food stains containing guar gum which is a commonly used stabilizer and thickening agent in food products.

Cellulases also clean indirectly by gently hydrolyzing certain glycosidic bonds in cotton fibers. Thus, particulate soils attached to microfibrils are removed. Another desirable effect of cellulases is to achieve greater softness and improved color brightness of worn cotton surfaces. Several detergent brands are based on a blend of two or more, even up to eight

Download free eBooks at bookboon.com 54 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes different enzyme products. One of the driving forces behind the development of new enzymes or the modification of existing ones for detergents is to make enzymes more tolerant to other ingredients, for example builders, surfactants, and bleaching chemicals, and to alkaline solutions. The trend towards lower wash temperatures, at least in Europe, has also increased the requirement for additional and more effective enzymes. Starch and fat stains are relatively easy to remove in hot water, but the additional cleaning power provided by enzymes is required in cooler water. The most widely used detergent enzymes are hydrolases, which remove soils consisting of proteins, lipids, and polysaccharides. Currently, research is being conducted with a view to extending the types of enzymes used in detergents. Many problem stains come from a range of modern food products such as chocolate, ice cream, baby food, desserts, dressings and sauces. To help remove these stains, and classical soiling like blood, grass, egg, and animal and vegetable fat, several different types of hydrolases are added to detergents. The major classes are proteases, lipases, amylases, mannanases, cellulases and pectinases. Historically, proteases were the first of these to be used largely in laundering for increasing the effectiveness of detergents. Cellulases contribute to cleaning and overall fabric care by maintaining, or even rejuvenating, the appearance of washed cotton based garments through selective reactions not available earlier when washing clothes. Some lipases can act as alternatives to current surfactant technology targeting greasy lipid-based stains. Thus lipases are an essential part of enzyme solutions used to replace surfactants.

Often multi-enzyme systems may replace up to 25% of a laundry detergent’s surfactant system without compromising the cleaning effect. This leads to a more sustainable detergent that allows cleaning at low wash temperature. Mannanases and pectinases are used for hard-to-remove stains of salad dressing, ketchup, mayonnaise, ice cream, frozen desserts, milkshakes, body lotions, and toothpaste and also, tangerines, banana, tomatoes, and fruit- containing products like marmalades, juices, drinking yogurts and low-fat dairy products. The obvious advantages of enzymes make them universally acceptable for meeting consumer demands. Due to their catalytic nature, they are ingredients requiring only a small space in the formulation of the overall product. This is of particular value at a time when detergent manufacturers are grouping their products.

In several parts of the world, strongly colored and stubborn stains from serum, blood, food soils, cocoa, and grass are removed with the help of laundry detergent bars. After decades of very little performance enhancement for laundry bars, a new solution that allows the incorporation of enzymes has been developed. A specially formulated protease empowers the producer to produce products that stand out from non-enzymatic laundry detergent bars, offering effective and effortless washing. Stain removal and washing by hand is one of the more time-consuming and physically demanding domestic duties. With the protease product in laundry bars, washing is reduced by at least one rinse and requires much less scrubbing. In addition to obtaining better result, laundry bars containing the enzyme may be formulated to be milder to the hands than old type bars without enzymes.

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Most of the energy spent during a household machine wash is used for heating the water. Thus, for energy saving and thereby helping to reduce carbon dioxide emissions, the most efficient measure is to reduce washing temperatures. Increased use of enzymes combined with a choice of appropriate other ingredients, including surfactants and bleaching systems specifically selected to work at low temperatures, has enabled manufacturers to produce ‘cold water detergents’.

Modern dishwashing detergents face increasing consumer demands for efficient cleaning of tableware. Enzymes are major ingredients for efficiently removing difficult and dried-on soils from dishes and leaving glassware shiny. Enzymes clean well under mild conditions and thereby assist to reduce clouding of glassware. In addition, enzymes also enable environmentally friendly detergents. Phosphates have been used in dishwashing detergents to get dishes clean, but they harm the aquatic environment and are increasingly being banned in detergents around the world. The combination of modifying detergent compositions and using multi-enzyme solutions enables the detergent manufacturers to replace phosphates without compromising the cleaning performance. For removal of starch soils, amylases are used; and proteases are used for removal of protein soil.

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There is a movement in the market towards enzymes in hand dishwashing. Amylases are being used for cleaning dishes from starch containing soils. The amylase removes stubborn starch without scrubbing.

A better cleaning performance in general

Rejuvenation of cotton fabric through the action of cellulases on fibers

Reduced energy consumption by enabling lower washing temperatures

Reduced water consumption through more effective soil release

Minimal environmental impact since they are readily biodegradable

Environmentally friendlier wash water effluents (in particular, phosphate-free and less alkaline) Furthermore, the fact that enzymes are renewable resources also makes them attractive to use from an environmental point of view

Table 4.4.1: Advantages of using enzymes in detergents

Protease enzyme removes the protein based stains like blood, milk, grass, egg, Protease minced meat, etc.

Amylase Amylase hydrolyses starch based products like cereals, pasta, potatoes, rice, etc.

Lipase hydrolyses fatty stains such as lipstick, frying fats, butter, salad oil, sauces Lipase and the tough stains on collars and cuffs containing residues of human sebum

Cellulase imparts biofinishing where in improves the general cleanness and Cellulase whiteness of laundry.

Mannanase Mannan stain removal (reappearing stains)

Table 4.4.2: Enzymes used in detergents (laundry and dish wash) Based on Kirk et al. (2002)

4.5 LEATHER INDUSTRY Leather industry is facing enormous pressure from the various pollution control bodies because of the huge amount of pollution associated with processing. Advancements in processing techniques and adoption of cleaner technologies have enabled the tanners to get rid off pollution from the leather processing. Enzymes in leather industry can solve pollution problem in the leather processing operation. The enzymes are finding application in soaking, unhairing, degreasing and bating of leather processing operations for obtaining better leather qualities.

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One of the oldest applications of enzymes is in the processing of leather hides and skins. The integration of enzymes into the leather industry has offered many benefits to those that use them and can be combined with other methods to reduce emissions as well as increase productivity. China, Italy and India are the largest producers of leather in the industry and therefore consume a lot of enzymes in the process.

4.5.1 SOAKING

In the soaking stage, the enzymes reduce the production times considerably. Enzymes during soaking speeds up the process of removing hyaluronic acid and improve quality through, more effective rehydration of the skin, better removal of proteins or carbohydrates. Both proteases and lipases help soaking processes. They are especially useful when processing fatty raw materials, very dry raw materials, fresh hides without salt, where the removal of non structural proteins and carbohydrates is quite difficult (Kanagaraj, 2009). Removal of dung in the skin/hide is also a problem which can be solved by the use of laccase enzymes in the soaking process. The major components of the composition of dung are lignocellulosic derivatives, cellulose, hemicellulose and lignin. The effects of individual enzymes and enzyme mixtures on dung removal have been examined. The laccase is more effective than individual enzyme treatments. The proposed mechanism for fast dung degradation is based on opening up the structure with lignin and hemicellulose degrading enzymes and breaking down the fibrous structure of cellulose. Enzymatic dung removal is based on solublization of the dung as a whole and not of one or more of the lignocellulosic components (Tozan and Covington, 2002). Sodium chloride up to 3 molar concentrations, enhanced enzyme activity by 20% for cellulose and 100% for xylanase (Auer et al., 1999).

4.5.2 DEPILATION

Depilation is one of the important operations in leather processing where hair is removed by using suitable chemicals. Methods of dehairing include:

¾¾Clipping process ¾¾Scalding process ¾¾Chemical process ¾¾Sweating process ¾¾Enzymatic process

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The most common method is the chemical process which uses lime and sodium sulphide to remove hair follicles. Even though this process is the most efficient, it contributes heavily to pollution. The enzymatic process uses proteases and is a more eco-friendly alternative. It can be used to recover hair stripped during this process as well as when integrated with the chemical process can reduce the sulphide and lime used by up to 40% or decrease the liming time by half. Leather created by using enzymes has also shown more favorable properties. Most importantly, enzymatic dehairing results in a cleaner grain surface and improved softness and area yield. The use of a specific protease also offers tanneries a number of options. For example, the requirements of sulphide and lime can be reduced by as much as 40% while maintaining the same liming time. Alternatively tanners can reduce the liming time by at least half without any loss of quality. Another possibility is to avoid the use of amines, which can be converted into carcinogenic compounds.

Dehairing is the process where enzymatic treatment is the most important factor to expedite the process. Enzymatic dehairing, either in the alkaline range or in the acidic range, has been widely used. Many researchers have carried out enzymatic dehairing with variety of proteolytic enzymes. The experiment carried out with Alcalase, bacterial protease by Novo industry, Denmark, showed that enzyme with proteolytic activity of broad specificity was necessary to bring about depilation (Yates, 1972). Enzymatic dehairing with alkali

Download free eBooks at bookboon.com Click on the ad to read more 59 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes pretreatment is effective in depilation of skin. This includes protease with a narrower range of specificities may be sufficient to induce depilation. Cleavage of proteoglycans and protein denaturation in strongly alkaline conditions would result in the exposure of more peptide bonds, facilitating proteases with narrow ranges of specificities to disrupt the integrity of proteins (Choudhary et al., 2004; Kanagaraj, 2009).

The primary studies on dehairing by Raju et al. (1996) with the extracellular protease secreted by the Bacillus isolate, showed that it has a dual pH maxima at pH 7.5 and 9.0 and the temperature maxima at 37°C. The presence of complex protein substrate in the medium for optimal enzymatic action is required. In enzymatic dehairing, pH and temperature play an important role. At temperatures ranging from 32–37°C, dehairing could be accomplished between 18-24 h. At temperature below 32oC, the duration of enzyme application needs to be increased for complete dehairing. Further, below 25oC no appreciable enzymatic dehairing within a reasonable period is seen. Studies conducted on the temperature stability of the enzyme show that the enzyme is stable in the temperature range from 20 to 50oC. It was also found that although even 2% (w/w) of crude enzyme was sufficient for dehairing, 3% (w/w) of the enzyme was preferred because at this concentration even the tough hair at the neck region was removed completely.

Proteases and amylases enzymes from various sources have been used individually or in combinations to produce effective dehairing of hides and skins (Bienkiewicz, 1983). However, protease enzymes are seen to be more effective and find wider application in enzymatic dehairing than amylase enzymes. Hair gets loosened by the action of autolytic or lysosomal enzymes present in the hides at pH 7.0–8.5 after giving acetic acid treatment or by the autolytic action of protease in the skin or hide.

4.5.3 BATING

Efficient bating relies on the use of enzyme such as proteases, amylases and lipases to clean the hide or skin of degraded hair or epidermis. Pepsin, extracted from pig stomach mucosa is active in acidic condition. It was used during pickling and on chrome tanned hides and skins. It can also be conducted at lower temperature, such as 21–29oC, while in the classical alkaline bating, the activity of enzyme drops rapidly at temperature under 32oC. Pepsin is an enzyme characteristic of the mammalian stomach structure, with molecular weight of 35kDa and a large amount of dicarboxylic, aliphatic and aromatic acids. Also, the enzyme is active in the pH range of 2.0–6.0 and presence of hydrochloric acid. If it is used on chrome tanned hides, after the splitting and shaving operations, the result is higher surface yield, softer leather and more uniform quality of leather (Deselnicu and Bratulesco, 1994). The proteolytic activity of pancreatic bate was determined in media of low ionic strength

Download free eBooks at bookboon.com 60 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes and in the presence of NaCl or NHSO at an ionic strength of 4. A peptide yielding p-nitroaniline as the hydrolytic product was used as the substrate. It has been confirmed that bate concentration of 0.11–3.35 (g/l) of NaCl, NHSO at high concentration (eg. ionic strength 4) subsequently stimulates (60–75%) the protease activity of pancreatic bates (Mozersky et al., 2005).

4.5.4 DEGREASING

The degreasing is mainly done to remove fat and hence it is important to know the fat composition of hide in the animals. The degreasing process takes place in three successive stages:

-- breakdown of the protective membrane of the fat containing sac, -- removal of the fat -- emulsification of the removed fat in water or solubilization in solvent

If one of these steps is carried out incorrectly then the whole degreasing operation will prove to be insufficient. Triglycerol lipase under alkaline condition is able to penetrate fat cell to affect hydrolysis. Results suggest that the lipase can penetrate the adipocyte plasma membrane to affect interfacial catalysis under alkaline condition. Hydrolysis of the intracellular lipid droplet generates fatty acids and intermediate acylglycerols. In the presence of divalent calcium ions, this lipid transported away from the active site favoring the hydrolysis reaction. The depletion of the lipid droplet through the continuous hydrolysis may be responsible for causing a change in the intracellular pressure. This may cause the membrane to collapse spontaneously into the lobules encompassed within the loss of supporting plasma membrane, will also subside and form process around the collapsed plasma membrane. These results show why lipase mediated degreasing is inconsistent within the leather making process (Addy et al., 2001). Degreasing using an unhydroxylated fatty alcohol has been studied on pickled sheep skin. It was found that degreasing effectiveness increased when the pH was increased and when the surfactant concentration increased up to 6% active matter and also with increase of time to 6–8 h (Palop et al., 2000). The use of lipases to degrease hides and pelts have been discussed. A highly synergistic effect in degreasing is achieved when special proteases and emulsifying systems are used at the same time. Protease break down the cell membranes of fat cells in hide and the new lipase reduces the amount of emulsifier required. A better soaking and liming effect is obtained in addition to improved degreasing (Christner, 1992; Marsal et al., 1998). The overall reduction of pollution by using enzyme from soaking to bating has been studied. Pollution reduction from 40 to 90% in different processes was observed (Post, 1997).

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4.5.5 POST TANNING PROCESS

Evaluation and cleaning of chrome tanned stock for dirt, grease, scud and other skins, for the purpose of making more uniformly colored leather has been treated with a lipase and a mild protease. A significant reduction was found in grease stains, neck wrinkle discoloration and other stains. Also, an improvement was observed in the brightness and uniformity of dyeing-backbone to belly and side to side within a mill. This was achieved using very low amounts of a combination of two enzymes selected to be particularly active in the condition of retanning with respect to temperature, pH, running times and presence of other chemicals. Use of acidic lipase at 0.3% and acidic protease at 0.015% in retanning process resulted in more uniform grain and flesh appearance for full grain leathers. Stains from fats and oils were reduced and colors appeared cleaner and brighter (Mitchell and Ouellette, 1998).

4.6 FOOD Applications of enzymes in the food industry are diverse (Table 4.6.1) (Kirk et al., 2002; Schafer et al., 2002; Bhoopathy, 1994. Muir, 1996; Farkye, 1995; Novozymes, 2011; Kuraishi et al., 2001). Enzymes play an important role in the food industry in both traditional and novel products. The ancient processes of brewing and cheese-making relied on enzyme activity

Download free eBooks at bookboon.com Click on the ad to read more 62 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes at various stages of manufacture. The first major breakthrough for microbial enzymes in the food industry came in the early 1960s with the launch of glucoamylase enzyme which was able to completely break starch into glucose. Now almost all glucose production has changed to enzymatic hydrolysis from traditional acid hydrolysis. The enzymatic liquefaction process reduces steam costs by 30%, ash by 50% and by-products by 90%. Since 1973, the starch-processing industry has grown to be one of the largest markets for enzymes.

The use of enzymes such as rennet in cheese making and barley amylases in brewing is as old as the food and beverage industry itself. However, the production of the amylase represents the first example of the industrial production of an enzyme for use in the food industry. The quantity and variety of enzymes used in the food and beverage industry has increased dramatically in the past decade.

Enzymatic hydrolysis is used to form syrups through liquefaction, saccharification, and isomerization. Another big market for enzymes is the baking industry. Supplementary enzymes are added to the dough to ensure high bread quality in terms of volume and a uniform crumb structure. Special enzymes can also increase the shelf life of bread by preserving its freshness longer (Novozymes 2011).

Another application is in the dairy industry to bring about the coagulation of milk as the first step in cheese making. Enzymes from both microbial and animal sources are used.

In many large breweries, industrial enzymes are added to control the brewing process and produce consistent, high-quality beer.

In food processing, animal or vegetable food proteins with better functional and nutritional properties are obtained by the enzymatic hydrolysis of proteins.

In the juice and wine industries, the extraction of plant material using enzymes to break down cell walls gives higher juice yields, improved color and aroma of extracts, and clearer juice.

4.6.1 STARCH MODIFICATION, PRODUCTION OF SWEETENER AND GLUCOSE SYRUPS

Modified starches and syrups of different compositions and physical properties are obtained and used in different types of foodstuffs. By selecting the right enzymes and the proper reaction conditions, valuable enzyme products can be produced to meet virtually any specific need in the food industry (Novozymes, 2011). Several nonfood products obtained by fermentation are obtained from enzymatically modified starch products. For example,

Download free eBooks at bookboon.com 63 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes enzymatically hydrolyzed starches are used in the production of alcohol, ascorbic acid, polyols, enzymes, lysine, and penicillin. The major steps in the conversion of starch are liquefaction, saccharification, and isomerization.

The starch industry started using industrial enzymes at an early date. Special types of syrups which could not be produced using conventional chemical hydrolysis were the first products made using the enzymatic processes (Novozymes, 2011). Several valuable products are derived from starch. There has been intensive development work on application processes. The ability to work under mild conditions, reaction efficiency, specific action, and a high degree of purification and standardization all make enzymes ideal catalysts for the starch industry. The saccharifying enzyme-glucoamylase completely breaks down starch to glucose. The immobilized glucose isomerase was developed in 1973, which made the industrial production of high fructose syrup possible. These sweeteners are used in soft drinks, candies, baking, jams and jellies and many other foods. The environmental benefits are reduced use of strong acids and bases, reduced energy consumption (less greenhouse gas), less corrosive waste, and safer production environment for workers.

Glucose syrups are obtained by complete hydrolysis of the starch. This process cleaves the bonds linking the dextrose units in the starch chain. The method and extent of hydrolysis (conversion) affect the final carbohydrate composition and therefore several functional properties of starch syrups. The degree of hydrolysis is commonly defined as the dextrose equivalent. Originally, acid conversion was used to produce glucose syrups. Today, because of their specificity, enzymes are mostly used to control how the hydrolysis takes place. In this way, tailor-made glucose syrups with well-defined sugar spectra are produced. The sugar spectra are analyzed using high-performance liquid chromatography (HPLC) and gel permeation chromatography (GPC). HPLC and GPC data provide information on the molecular weight distribution and overall carbohydrate composition of the glucose syrups.

Modern enzyme technology is used extensively in the corn wet-milling sector. The enzymatic steps are briefly explained below.

Corn starch is the most widespread raw material used, followed by wheat, tapioca, and potato. As native starch is only slowly degraded using alpha-amylases, a suspension containing 30–40% dry matter needs first to be gelatinized and liquefied to make the starch susceptible to further enzymatic breakdown. This is obtained by adding a heat-stable alpha-amylase to the starch suspension. The mechanical part of the liquefaction process involves the use of stirred tank reactors, continuous stirred tank reactors, or jet cookers. In most plants for sweetener production, starch liquefaction takes place in a single-dose, jet-cooking process. Thermostable alpha-amylase is added to the starch slurry before it is pumped through a jet cooker. Here, live steam is injected to raise the temperature to 105 °C and the slurry’s

Download free eBooks at bookboon.com 64 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes subsequent passage through a series of holding tubes provides the 5-minute residence time necessary to fully gelatinize the starch. The temperature of the partially liquefied starch is then reduced to 90–100 °C by flashing, and the enzyme is allowed to further react at this temperature for one to two hours until the required DE is obtained. The enzyme hydrolyzes the alpha-1,4-glycosidic bonds in the gelatinized starch; the viscosity of the gel rapidly decreases and maltodextrins are produced. The process may be stopped at this point, and the solution purified and dried. Maltodextrins (DE 15–25) are commercially valuable for their rheological properties. They are used as bland-tasting functional ingredients in the food industry as fillers, stabilizers, thickeners, pastes, and glues in dry soup mixes, infant foods, sauces, gravy mixes, etc.

When maltodextrins are saccharified by further hydrolysis using glucoamylase or fungal alpha-amylase, a variety of sweeteners can be produced. These have dextrose equivalents in the ranges 40–45 (maltose), 50–55 (high maltose), and 55–70 (high conversion syrup). By using beta-amylase, glucoamylase, and debranching enzymes, intermediate level conversion syrups with maltose contents of about 80% can be produced. A high yield of 95–97% glucose may be produced from most starchy raw materials. The debranching enzyme most often used is pullulanase (alpha-dextrin endo-1,6-alpha-glucosidase). Glucose can be isomerized to fructose in a reversible reaction. Under industrial conditions, the equilibrium point is

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1349906_A6_4+0.indd 1 22-08-2014 12:56:57 Download free eBooks at bookboon.com Click on the ad to read more 65 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes reached when the level of fructose is 50%. The conversion is normally stopped at a yield of about 45% fructose for avoiding a lengthy reaction time. If an immobilized enzyme system is used, the isomerization reaction in the reactor column is efficient, rapid and economical.

4.6.2 BAKING

Enzymes from malt and fungal alpha-amylases have been used in bread-making for decades. Several new enzymes are now available for the baking industry due to advances in the area of biotechnology. The use of enzymes is expected to increase as consumers demand more natural products free of chemical additives. The dough for bread, rolls, buns, and similar products consists of flour, yeast, water, salt, and other ingredients such as sugar and fat. Flour consists of gluten, starch, nonstarch polysaccharides, lipids, and minerals in trace amounts. As soon as the dough is made, the yeast starts to work on the fermentable sugars and convert them into alcohol and carbon dioxide, which makes the dough rise. The major component of wheat flour is starch. Amylases are able to degrade starch and produce small dextrins for the yeast to act upon. There is also another type of amylase which modifies starch during baking to give a significant antistaling effect. Gluten is a combination of proteins that forms a large network during formation of dough. This network holds the gas in during dough proofing and baking. Therefore, the strength of this network is very important for the quality of all bread raised using yeast. Enzymes such as hemicellulases, xylanases, oxidases and lipases can directly or indirectly improve the strength of the gluten network and improve the quality of the finished bread.

4.6.3 DAIRY PRODUCTS

The use of enzymes for processing milk and particularly rennet for the production of cheese has a long tradition. Rennet, as other enzymatic coagulants, is preparation of proteases with a milk-clotting role and indispensable in cheese production. The rennet contains the enzyme chymosin, and nowadays there are several industrially produced chymosin products or similar proteases available as substitutes. Proteases are also used, to modify the functional properties of cheese, to speed up cheese ripening and to modify milk proteins to reduce allergenic properties of some dairy products. Protein is not the only possible allergen in milk. Many adults are unable to drink milk. Cow’s milk contains 5% lactose, and in order to break it down the enzyme lactase is required. This enzyme is high in humans at birth, but only low levels are found in certain sections of the world’s population during adulthood. Lactase is used to hydrolyze lactose for increasing digestibility or to improve the solubility or sweetness of various dairy products. Finally, lipases are used mainly in cheese ripening.

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4.6.4 BREWING

Traditionally, beer is produced by using the mashing process. Crushed barley malt and hot water in large circular vessels, called mash copper, are mixed. Besides malt, few adjuncts are also added to the mash. After mashing, the mash is filtered and the resulting liquid, known as sweet wort, is then run off to the copper, where it is boiled with hops. The hopped wort is cooled and taken to the fermentation vessels and yeast is added. After fermentation, the green beer is matured before final filtration and bottling. The traditional source of enzymes used for the conversion of cereals into beer is barley malt, one of the key ingredients in brewing. If very little enzyme activity is present in the mash, there will be undesirable results: The extract yield will be quite low, wort separation will take too long, the fermentation process will be very slow, very little alcohol will be produced, the beer filtration rate will be reduced, and the flavor and stability of the beer will be of inferior quality. Industrial enzymes are used to supplement the malt’s own enzymes for preventing these problems. In addition, industrial enzymes can be used to produce low-carbohydrate beer to ensure better adjunct liquefaction, to reduce the beer maturation time, and to produce beer from inexpensive raw materials.

4.6.5 DISTILLING POTABLE ALCOHOL

Fermented alcoholic beverage production from raw materials containing starch has been practiced for centuries. Before the 1960s, the enzymatic degradation of starch to fermentable sugars was achieved by adding malt or koji. Koji is used as an enzyme source for alcohol production in Japan and China. Today, in many countries malt has been completely replaced in distilling operations by industrial enzymes. This offers several advantages. A few liters of enzyme preparation can be used to replace 100 kg of malt, making enzymes much easier to handle and store. When switching to commercial enzymes, savings of 20–30% can be expected on raw material costs. Furthermore, since industrial enzymes have a uniform standardized activity, distilling becomes more predictable with a better chance of obtaining a good yield from each batch of fermentation. The quality of malt, on the other hand, can vary from year to year and from batch to batch, as can koji. Microbial amylases are available with activities covering a broad pH and temperature range, and therefore suitable for the low pH values found in the mash. The commercial enzymes have replaced malt in all but the most conservative parts of the distilling industry. The selection of raw material differs around the world. In the alcohol industry, starch is usually hydrolyzed by enzymes in two stages – liquefaction and saccharification. The yeast can then transform the smaller molecules – mainly glucose – into alcohol.

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Food (including dairy)

Protease Milk clotting, Infant formulas (low allergenic), Flavor

Lipase Cheese flavor

Lactase Lactose removal (milk)

Pectin methyl esterase Firming fruit-based products

Pectinase Fruit-based products

Transglutaminase Modify visco-elastic properties

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Baking

Amylase Bread softness and volume Flour adjustment

Xylanase Dough conditioning

Lipase Dough stability and conditioning (in situ emulsifier)

Phospholipase Dough stability and conditioning (in situ emulsifier)

Glucose oxidase Dough strengthening

Lipoxygenase Dough strengthening Bread whitening

Protease Biscuits, cookies

Transglutaminase Laminated dough strengths

Beverage

Pectinase De-pectinization Mashing

Amylase Juice treatment Low calorie beer

Glucanase Mashing

Acetolactate decarboxylase Maturation (beer)

Laccase Clarification (juice) Flavor (beer) Cork stopper treatment

Table 4.6.1: Application of enzymes used in food industry

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4.7 FEED The increasing economic pressures, currently being placed upon animal producers, demand more-effective utilization of low-grade feedstuffs. Furthermore, consumer awareness and new legislation require that any increase in animal production cannot be obtained via growth- promoting drugs or other chemical substances. One increasingly popular approach to this problem is to supplement animal diets with hydrolytic enzymes in an attempt to help in the digestion and absorption of poorly available nutrients, or to remove antinutritional factors from the diet (Aehle, 2004; Selle and Ravindran, 2007; Bedford and Schulze, 1998; Bedford and Partridge, 2011). Concerns raised by this practice include the ability of such enzymes to survive processing temperatures and even the animals’ digestive tract.

Animal feed is the largest cost item in livestock and poultry production, accounting for 60 to 70% of total expenses. To reduce the costs, many producers supplement feed with enzyme additives, enabling them to produce more meat per animal or to produce the same amount of meat cheaper and faster.

The feed enzymes market was valued at USD 842.9 Million in 2016. It is projected to grow at a CAGR of 9.3% from 2017 to reach 1428.6 Million by 2022.

Some types of feed ingredients are not fully digested by livestock. But, by adding enzymes to feed, the digestibility of the components can be improved. Enzymes are now a successful tool and also well proven that allows feed producers to extend the range of raw materials used in feed, and also to improve the efficacy of existing formulations. Enzymes are added to the feed either directly or as a premix together with vitamins, minerals, and other feed additives. In premixes, the coating of the enzyme granulate protects the enzyme from deactivation by other feed additives such as choline chloride. The coating has another function in the feed mill – to protect the enzyme from the heat treatments sometimes used to destroy Salmonella and other unwanted microorganisms in feed. Enzyme products in a liquid formulation are developed for those cases where the degree of heat treatment (conditioning) for the feed is high enough to cause an unacceptable loss of activity. Thereby addition can be performed accurately after the conditioning with insignificant loss of activity. A wide range of enzyme products for animal feed are now available to degrade substances such as phytate, beta-glucan, starch, protein, pectin-like polysaccharides, xylan, raffinose, and stachyose (Table 4.7.1). Hemicellulose and cellulose can also be degraded. As demonstrated by many feed trials carried out to date, the major benefits of supplementing feed with enzymes are:

¾¾Faster growth of the animal ¾¾Better feed conversion ratio

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¾¾More uniform production ¾¾Better health status ¾¾An improved environment for chickens due to reductions in “sticky droppings”.

Table 4.7.2 shows the pre-requisite of enzymes used in animal nutrition.

4.7.1 PHYTASE ENZYMES

Around 50–80% of the total phosphorus in pig and poultry diets is present in the form of phytate or phytic acid. The phytate-bound phosphorus is largely unavailable to monogastric animals because they do not naturally have the enzyme needed to break it down – phytase. There are two good reasons for supplementing feeds with phytase. One is to reduce the harmful environmental impact of phosphorus from animal manure in areas with intensive livestock production. Phytate or phytic acid in manure is degraded by soil microorganisms, which lead to high levels of free phosphate in the soil and, eventually in surface water. Several studies have found that optimizing phosphorus intake and digestion with phytase reduces the release of phosphorus by around 30%. According to Novozymes, the amount of phosphorus released into the environment would be reduced by 2.5 million tons a year

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4.7.2 NONSTARCH POLYSACCHARIDES (NSP) DEGRADING ENZYMES

Cereals such as barley, wheat and rye are incorporated into animal feeds to provide a major source of energy. However, much of the energy remains unavailable to monogastrics because of the presence of nonstarch polysaccharides (NSP) which interfere with digestion. It prevents access of the animal’s own digestive enzymes to the nutrients contained in the cereals and also NSP can become solubilized in the gut and cause problems of high gut viscosity, which further interferes with digestion. The addition of selected carbohydrases will break down NSP, releasing nutrients (energy and protein), and reducing the viscosity of the gut contents (Ao et al. 2010; Montoya et al., 2011; Novozymes, 2011). The carbohydrase class of enzymes includes xylanases, glucanases, and amylases. They act in the stomach to break down and degrade carbohydrates such as fiber, starch and non-starch polysaccharides into simple sugars that provide energy for use by the animal. The overall effect is improved feed utilization and a more “healthy” digestive system for monogastric animals. One of the most common carbohydrases is xylanase. Xylanase attacks the arabinoxylan structure of corn or wheat, allowing the animal to absorb its components as an energy source. This limits the requirement for supplemental fat or energy in the final diet.

4.7.3 PROTEASES

Protease enzymes are an important factor in protein digestion as they hydrolyze the less digestible proteins in animal feeds and break them down into more usable peptides. Improving the digestibility of dietary protein with a quality protease can reduce feed cost by allowing the use of lower crude protein feedstuffs with lesser quality amino acids, effectively lowering protein and digestible amino acids levels required from the feedstuffs up to 10%.

Protease breaks down anti-nutritional factors associated with various proteins. Proteases improve the digestion of proteins and increase amino acid availability, which helps release valuable nutrients. The result is improved animal growth and performance and minimal negative effects of undigested protein in the hindgut. Raw ingredients with low digestibility of amino acid respond greatest to an exogenous protease. This is why its greatest value is

Download free eBooks at bookboon.com 72 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes when alternative ingredients are used in the diet. Proteases help producers manage the nutritional risks associated with feedstuff quality and allow them to best utilize all available feed ingredients. Proteases are not limited to diets with alternative ingredients. Animals consuming a traditional corn-soybean meal diet cannot utilize 100 percent of the protein fraction. Therefore, adding a protease enzyme to a corn-soybean meal diet will enhance amino acid digestibility and animal performance.

The benefits of enzymes are becoming better realized as more research is done. For the animal, enzymes optimize gut health, produce uniform growth and enhance overall health. For the producer, they decrease feed costs and improve profitability. Each type of enzyme has its own specific function and therefore do not interfere with one another.

Mode(s) of action of enzymes Different feed enzymes will have different modes of action (Table 4.7.3). Despite their increasing acceptance as feed additives, the exact mode(s) of action of feed enzymes remains to be elucidated. The general consensus is that one or more of the mechanisms are responsible for the observed benefits.

Enzyme Target substrate

Phytases Phytic acid

β-Glucanases β-Glucan

Xylanases Arabinoxylans

α-Galactosidases Oligosaccharides

Proteases Proteins

Amylase Starch

Lipases Lipids

Mannanases, cellulases, Cell wall matrix (fiber components) hemicellulases pectinases

Table 4.7.1: Type of feed enzymes Based on Ravindran (2013)

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Must act under acidic pH condition of stomach

Resist low pH

Resist pepsin’s proteolytic action

It should act other parts of digestive tract

Table 4.7.2: Mode of action of different feed enzymes

Degradation of specific bonds in ingredients that are not usually hydrolyzed by endogenous digestive enzymes.

Degradation of antinutritional factors that limit nutrient digestion directly, increase intestinal digest viscosity indirectly, or both.

Disruption of endosperm integrity and the release of nutrients that are bound to or entrapped by the cell wall.

Shift of digestion to more efficient digestion sites.

Reductions in endogenous secretions and protein losses from the gut resulting in reduced maintenance requirements.

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Reduction in the weight of the intestinal tract and changes in the intestinal morphology.

Changes in the microflora profile in the small intestine. As enzymes influence the amounts and form of substrate present within the gut, their use has a direct effect on the bacteria that make up the microfloral populations.

Augmentation of endogenous digestive enzymes, which are either insufficient or absent in the bird, resulting in improved digestion. This will be especially true for newly hatched chicks with immature digestive systems.

Table 4.7.3: Pre-requisite of enzymes used in animal nutrition Based on Ravindran (2013)

4.8 ORGANIC SYNTHESIS Biocatalysis has been the focus of intense scientific research and is now a well-established technology within the chemical industry. Biocatalysis is the general term for the transformation of non-natural compounds by enzymes. The accelerated reaction rates, together with the unique stereo-, regio-, and chemoselectivity (highly specific action) and mild reaction conditions offered by enzymes, make them highly attractive as catalysts for organic synthesis. Additionally, improved production techniques are making enzymes inexpensive and more widely available. Enzymes work across a broad pH and temperature range, and often also in organic solvents. Many enzymes have been found to catalyze a variety of reactions that can be dramatically different from the reaction and substrate with which the enzyme is associated in nature.

Enzymes are preferred in industrial chemical synthesis over conventional methods for their high selectivity, i.e., chiral, positional and functional group specific. Such high selectivity is extremely advantageous in chemical synthesis as it may offer several benefits such as minimal or no byproduct formation, easier separation, and less environmental problems. Besides, mild operational conditions and high catalytic efficiency are advantages of enzyme mediated commercial applications.

Chemical synthesis is an area where the use of enzyme catalysis has long been seen as having great promise. In spite of that, the chemical industry has been slow to implement enzyme- based processes and the use of enzymes in the chemical industry is still low in comparison with other industries. At present, however, very significant growth in this area is being seen and enzyme based processes are now, finally, being widely introduced for the production of a diversity of different chemicals; one major example is in the production of single-enantiomer intermediates used in the manufacture of drugs and agrochemicals (Schmidt, 2001). This market is characterized by a very high degree of fragmentation, as very few enzymes have

Download free eBooks at bookboon.com 75 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes applicability in a broad range of different processes. The class of enzymes most widely applied to organic synthesis is the hydrolases. Members of the hydrolase family that have been used extensively include lipases, esterases, and proteases. Now days, lipases are widely used for organic reactions. Enzymatic processes recently introduced include the usage of lipases for the production of enantiopure alcohols and amides, nitrilases for the production of enantiopure carboxylic acids, and acylases for the production of new semisynthetic penicillins (Schmidt, 2001). As many companies are currently at an early stage in the use of enzyme- based catalysis, many new developments are expected in this area over the next few years. Lipases are used to catalyze a wide variety of regioselective and stereoselective transformations (Kazlauskas, 1994; Berglund and Hunt,2000; Rajendra et al., 2016). Applications for lipases include kinetic resolution of racemic alcohols, acids, esters or amines (Ghanem and Aboul- Enein, 2004), and also the desymmetrization of prochiral compounds (Garca-Urdiales et al., 2005). They are also successfully used in regioselective esterification or transesterification of polyfunctional compounds, for example in the chemoenzymatic synthesis of nucleoside derivatives (Ferrero and Gotor, 2000). Non-conventional processes, such as aldol reactions or Michael addition have been achieved using lipases (Bornscheuer and Kazlauskas, 2004).

Most of lipases used currently as catalysts in organic chemistry are derived from microorganisms. These enzymes work at hydrophilic-lipophilic interface and tolerate organic solvents in the reaction mixtures. Use of lipases in the synthesis of enantiopure compounds has been reported by Berglund and Hutt (2000). For example, Pseudomonas lipases are extensively used in industry, especially for the production of chiral chemicals which serve as basic building blocks in the synthesis of pharmaceuticals, pesticides and insecticides. These enzymes show distinct differences in regioselectivity and enantioselectivity, despite a high amino acid sequence homology.

Lipases are used as biocatalyst in the production of significant biodegradable compounds. Trimethylolpropane esters were synthesized as lubricants. Lipases are able to catalyze ester syntheses and transesterification reactions in organic solvent systems. This has opened up the possibility of enzyme catalyzed production of biodegradable polyesters. Aromatic polyesters can also be synthesized by lipase biocatalysis (Bailey and Ollis, 1986).

Lipases are the most frequently used, particularly, in the formation of a wide range of optically active alcohols, acids, esters, and lactones (Jaegera and Reetz, 1998; Hasan et al., 2006). Lipases are used for the production of (S, R)-2, 3-p-ethoxyphenylglycyclic acid, an intermediate for diltiazem (Gentile et al., 1992). Oxidoreductases, such as polyphenol oxidase is involved in the synthesis of 3,4-dihydroxylphenyl alanine (DOPA), a chemical used in the treatment of Parkinson’s disease (Faber, 1997). Oligosaccharides and polysaccharides, play vital roles in cellular recognition and communication processes, are synthesized industrially using high regio- and stereoselectivity of glycosyltransferases (Ginsburg and Robbins,

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1984). Lyases are used in organic synthesis of cyanohydrins from ketones, acrylamide from acrylonitrile, malic acid from fumaric acid (Faber, 1997; Zaks, 2001). The nitrile hydratase mediated process for the production of acrylamide is carried out by the Nitto Chemical Company of Japan at a scale of more than 40,000 tons per year (Zaks, 2001).

4.9 PHARMACEUTICALS Enzymes are being explored for pharmaceutical applications (Choi et al., 2015; Anbu et al., 2015). Bornscheuer et al. (2012) reviewed the biocatalytic routes scaled up for pharmaceutical manufacturing showing the competitiveness of enzymes versus traditional chemical processes. 360° One of the most successful examples in the practical application of enzymes in the pharmaceutical industry is the anti-diabetic compound, sitagliptin (Desai, 2011; Savile et al., 2010). Sitagliptin is a drug for type II diabetes that has been marketed under the thinking trade name Januvia by Merck (Desai, 360°2011). . Enzymes have been found useful for preparingthinking beta-lactam antibiotics such as semisynthetic penicillins and cephalosporins (Volpato et al., 2010). The semisynthetic. penicillins have

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© Deloitte & Touche LLP and affiliated entities. INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes largely replaced natural penicillins and about 85% of penicillins marketed for medicinal use are semisynthetic. 6-Aminopenicillanic acid is obtained by the hydrolysis of the amide bond of the naturally occurring penicillin with the enzyme penicillin amidase, which unlike chemical hydrolysis does not open the β-lactam ring.

The most important applications in biocatalysis are the synthesis of complex chiral pharmaceutical intermediates efficiently and economically. Esterases, lipases, proteases, and ketoreductases are widely applied in the preparation of chiral alcohols, carboxylic acids, amines, or epoxides (Zheng and Xu, 2011). Kinetic resolution of racemic amines is a common method used in the synthesis of chiral amines. Acylation of a primary amine moiety by a lipase is used by BASF for the resolution of chiral primary amines in multi-thousand ton scale (Sheldon, 2008).

Atorvastatin, the active ingredient of Lipitor, a cholesterol-lowering drug can be produced enzymatically. The process is based on three enzymatic activities, such as a ketone reductase, a glucose dehydrogenase, and a halohydrin dehalogenase. Several iterative rounds of DNA shuffling for these three enzymes led to a 14-fold reduction in reaction time, a 25-fold reduction in enzyme use, a sevenfold increase in substrate loading and a 50% improvement in isolated yield (Ma et al., 2010).

Therapeutic enzymes have a wide variety of specific uses such as oncolytics, thrombolytics, or anticoagulants and as replacements for metabolic deficiencies. Enzymes are being used to treat many diseases like cancer, cardiac problems, cystic fibrosis, dermal ulcers, inflammation, digestive disorders etc. Proteolytic enzymes serve as good anti-inflammatory agents. Collagenase enzyme, which hydrolyzes native collagen and spares hydrolysis of other proteins, has been used in dermal ulcers and burns. Papain has been shown to produce marked reduction of obstetrical inflammation and edema in dental surgery. Deoxyribonuclease is used as a mucolytic agent in patients with chronic bronchitis. Trypsin and chymotrypsin have been successfully used in the treatment of athletic injuries and postoperative hand trauma. Hyaluronidase has hydrolytic activity on chondroitin sulphate and may help in the regeneration of damaged nerve tissue (Moon et al., 2003). Lysozyme hydrolyzes the chitins and mucopeptides of bacterial cell walls. Hence, it is used as antibacterial agent usually in combination with standard antibiotics. Lysozyme has also been found to have activity against HIV, as the RNase A and urinary RNase U present selectively degrades viral RNA (Lee-Huang et al., 1999) showing possibilities for the treatment of HIV infection.

Cancer research has some good examples of the use of enzyme therapeutics (Gurung et al., 2013). Studies have proved that arginine-degrading enzyme (PEGylated arginine deaminase) can inhibit human melanoma and hepatocellularcarcinomas (Ensor et al., 2002). Another PEGylated enzyme, Oncaspar1 (pegaspargase), is showing good results for the treatment

Download free eBooks at bookboon.com 78 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes of children newly diagnosed with acute lymphoblastic leukemia. The further application of enzymes as therapeutic agents in cancer is described by antibody-directed enzyme prodrug therapy (ADEPT). A monoclonal antibody carries an enzyme specific to cancer cells where the enzyme activates a prodrug and destroys cancer cells but not normal cells. This approach is being used for the discovery and development of cancer therapeutics based on tumor-targeted enzymes that activate prodrugs. Certain enzymes such as l-asparaginase have been found to be useful in treating cancer. l-asparaginase, by reducing the concentration of asparagine, retards the growth of cancer cells. It has proven particularly useful in treating lymphoblastic leukemia and certain forms of lymphomas. Genetic engineering basically involves taking the relevant gene from the microorganism that naturally produces a particular enzyme (donor) and inserting it into another microorganism that will produce the enzyme more efficiently (host). The first step is to cleave the DNA of the donor cell into fragments using restriction enzymes. The DNA fragments with the code for the desired enzyme are then placed, with the help of ligases, in a natural vector called a plasmid that can be transferred to the host bacterium or fungus. In recombinant DNA technology, restriction enzymes recognize specific base sequences in double helical DNA and bring out cleavage of both strands of the duplex in regions of defined sequence. Restriction enzymes cleave foreign DNA molecules. The term restriction endonuclease comes from the observation that certain bacteria can block virus infections by specifically destroying the incoming viral DNA (Adrio and Demain, 2014). Such bacteria are known as restricting hosts, since they restrict the expression of foreign DNA. Certain nicks in duplex DNA can be sealed by an enzyme-DNA ligase which generates a phosphodiester bond between a 5′-phosphoryl group and a directly adjacent 3′-hydroxyl, using either ATP or NAD+ as an external energy source.

4.10 PERSONAL CARE The global beauty market (cosmetics and toiletries or personal care products (PCPs)) in the last 20 years, has grown by 4.5% a year on average (CAGR), with annual growth rates ranging from around 3–5.5% (Barbalova, 2011; Sunar et al., 2016).

The world’s cosmetic industry is worth tens of billions of US dollars and the industry is continuously seeking new products with ingredients having specific actions for which enzymes have been the most preferred choice for enhancement of personal care products.

Enzymes have recently been started to be used for cosmetic application in developing PCPs for wide acceptability as they have good consumer appeal and improved performance (Sunar et al., 2016). But these have always been poorly evaluated for their functionality in cosmetic science. Proteolytic enzymes like bromelain, papain, etc. have been used in PCPs for skin

Download free eBooks at bookboon.com 79 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes peeling and smoothing for several years, but, the general problem associated with such use is the irritation caused by some enzymes on the skin surfaces due to their proteolytic activities. The area where the topical applications of enzymes are widely explored and have shown substantial benefits is in skin protection, with enzymes having excellent stability. The enzymes used for skin protection can capture free radicals caused by environmental pollution, microorganisms, sunlight, radiations etc. The trend on use of enzymes in PCPs shows ample variability in terms of enzymes used from different types of classes for their specific function and roles. Studies of enzyme formulations suitable for topical use have also shown that such dosage forms are relatively easy to handle. However, the choice of base, surface active agent, etc., is important to provide for a stable formulation, and proper vehicle selection is also crucial for the proper activity. Another approach to cosmetics and skin care product development is to increase the effectiveness of existing ingredients that might improve skin functioning. Many new topical ingredients (from mushrooms to salmon caviar to sea urchin spines to green algae to knotweed) have been placed in complex anti-aging formulations (Draelos, 2012). Nanoparticles are revolutionizing many areas of chemistry, physics, and possibly cosmetic formulation. The long term effects of nanoparticles are not known currently. Yet, nanoparticles could be the next frontier in cosmetic dermatology (Sonneville-Aubrun et al., 2004). Nanoparticles have great potential to create topical cosmeceutical formulations that behave in ways that enable better penetration of active

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Download free eBooks at bookboon.com Click on the ad to read more 80 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes skin ingredients. In the future nanoparticle therapy, nanoemulsions, polymeric nanoparticle spheres, and nanoliposomes may be used for improving the appearance of the skin (Tadros et al., 2004). Nanotechnology may allow ingredients to show new skin effects, improving effectiveness of cosmetics and skin care product.

Table 4.10.1 shows the use of enzymes in cosmetics.

Superoxide dismutase Superoxide dismutase (SOD) is an antioxidant naturally found in the body, and catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. This enzyme has been frequently used in various fields for its effective role in catalyzing superoxide free radicals. SOD is one of the most effective and popular tropical enzymes so far being used in skin care products. It is also found in barley grass, brussels sprouts, broccoli, wheatgrass, cabbage, and most green plants. As such, they are an important antioxidant defense in nearly all cells exposed to oxygen, like skin cells. Superoxide dismutase overcomes the harmful effect of superoxide, and protects the cell from superoxide toxicity; it is the most common free radical in the body. It has the fastest turnover number of any known enzyme. It is used in cosmetics and personal care products as an anti-aging ingredient and antioxidant because of its ability to reduce free radical damage in the skin, thereby preventing wrinkles, fine lines, age spots, help with wound healing, soften scar tissue, protect against UV rays, and reduce other signs of aging (Sunar et al., 2016). It was discovered as a blue/green protein in 1938 by Mann and Leilin and subsequently characterized as an enzyme and named as superoxide dismutase by McCord and Fridovitch in 1969.

Peroxidase There are two different types of hydroxyl free radical-scavenging enzymes, belonging to the oxidoreductase class of enzymes. These are known as peroxidase and catalase. Plants are known to have heme-containing peroxidases, which are nonspecific peroxidases and are capable of acting on a variety of substrates including hydrogen peroxide. Similar nonspecific enzymes in animals are lactoperoxidase (thiocyanate ion oxidation), myeloperoxidase (phagocytosis), and thyroid peroxidase (iodine ion oxidation). However, the most studied one is the horseradish peroxidase obtained from the roots of horseradish. These free radical-scavenging enzymes have been extensively used in PCPs. For example, fennel seed extracts containing peroxidase are being used in cosmetics because of their high-lipid peroxidation activities and low odor. The pale yellow/green liquid extract is also shown to have nonirritating and

Download free eBooks at bookboon.com 81 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes nonsensitizing activity and has shown much better protection activity than tocopherol. Lignin peroxidase, a novel skin-lightening active agent derived from a fungus is being studied with some interest for developing as an ingredient in products to treat pigmentation disorders. From these discoveries, the development of lignin peroxidase as a skin-lightening agent resulted (US Patent and Trademark Office Patent Application 20060051305). This novel skin-lightening active ingredient is produced extracellularly during submerged fermentation of the fungus Phanerochaete chrysosporium (Woo et al., 2004). It is then purified from the fermented liquid broth. The lignin peroxidase enzyme (trademarked as Melanozyme) identifies eumelanin in the epidermis and specifically breaks down the pigment without affecting melanin biosynthesis or blocking tyrosinase. Melanozyme is a glycoprotein active at pH 2–4.5 and is currently proprietary and is available only in a new skin-lightening product known as ‘Elure’. The safety of lignin peroxidase as a skin-lightening active ingredient has been shown in preclinical studies. Lignin peroxidase is nonmutagenic and nonirritating to eyes. The potential for skin irritation is very low.

Tyrosinase Tyrosinase is an oxidase and is the rate-limiting enzyme for controlling the production of melanins. This enzyme is mainly involved in two distinct reactions of melanin synthesis (Hideya et al., 2007; Kumar et al., 2011):

-- the hydroxylation of a monophenol -- the conversion of an o-diphenol to the corresponding o-quinone. o-Quinone undergoes several reactions to form melanin. The melanin synthesis in melanocytic cells is regulated by tyrosinase enzyme. This is a membrane-bound copper containing glycoprotein and is the critical rate-limiting enzyme. Tyrosinase is produced by melanocytic cells, and following its synthesis and subsequent processing in the endoplasmic reticulum and Golgi, it is sent to specialized organelles. These are termed melanosomes, wherein the pigment is synthesized and deposited. In the hair and skin, the melanosomes are transferred from melanocytes to neighboring keratinocytes and are distributed in those tissues for producing visible color (Hideya et al., 2007). The cosmetic industry worked with substances involved in natural melanin formation during the past years. Not similar to the melanoidin process, a natural tan is induced and protection against UV radiation also is provided. The tyrosinase enzyme converts the tyrosine (amino acid) into dihydroxyphenylalanine (DOPA) and into its quinoid form, the DOPA quinone, which is the base for the formation of both types of melanin – eumelanin (dark brown) and pheomelanin (reddish yellow). The combination of both types is responsible for the skin tone, which is found to vary from skin to skin. The tyrosinase is induced by the α-melanocytes stimulating hormone and controlled

Download free eBooks at bookboon.com 82 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes by UV radiation. Other tyrosinase stimulators are the β-endorphins. Endorphin-related substances are found in vegetable extracts and together with synthetic acetyl tyrosine they are able to induce the UV independent formation of melanin. Additional UV radiation will accelerate and stimulate the melanin formation process after the product has been used. New developments focus on additional tyrosinase activators and adequate transport systems for integrating the substances into the skin (Lautenschltens, 2007). Zymotan complex, which is a tanning activator, consists of tyrosine amino acids (precursors of melanine) and tyrosinase. Tyrosinase enzyme catalyzes the reaction forming the melanin in the presence of solar radiation. This enzyme is present in several plants and has been also isolated from yeast, milk and leucocytes.

Proteases Proteases are group of enzymes which hydrolyze the protein bonds of amino acids. Proteases play an important role in industrial biotechnology, especially in detergents, foods, pharmaceuticals, and in PCPs (Gupta and Khare, 2007; Kalpana Devi et al., 2008). Proteolytic enzyme is essential for several physiological processes like digestion of food proteins, protein turnover, cell division, blood clotting cascade, signal transduction, processing of polypeptide

Download free eBooks at bookboon.com Click on the ad to read more 83 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes hormones, etc. Proteases are used extensively in the pharmaceutical industry for preparation of medicines, such as ointments for debridement of wounds. They are also used in denture cleaners and as contact lens enzyme cleaners (Ogunbiyi et al., 1986). Proteases used in the detergent and food industries are produced in bulk quantities and are used in crude form; whereas those used in medicine are produced in small amounts but need extensive purification before use (Bholay and Patil, 2012).

Lipases Lipases are ubiquitous enzymes present in all types of living organisms. Lipases exert their activity on the carboxyl ester bonds of triacylglycerols and other substrates. Their natural substrates are insoluble lipid compounds prone to aggregation in aqueous solution. Among the lipases from higher eukaryotes, porcine pancreatic lipase has been used for many years as a technical enzyme (Lotti and Alberghina, 2007). Active lipases can mostly be found in cosmetics for cleansing (anticellulite treatment) or overall body slimming, where they are responsible for the mild loosening and removal of dirt and/or small flakes of dead corneous skin and/or assist in breaking down fat deposits, often in combination with further enzymes, such as proteases. Further applications have been mentioned for nose cleansing, makeup beauty masks, and hair care. Based on the broad variety of compounds derived from fats and carboxylic acids in cosmetic products, lipases and their hydrolytic, esterifying, and acylating activities show enormous potential for implementation in the production of cosmetic ingredients.

Immobilized lipases are used for the preparation of water-soluble retinol derivatives and are commercially very important in cosmetics and pharmaceuticals such as skin care products. Lipases are used in hair waving preparation and have also been used as an ingredient of topical antiobese creams or as oral administration (Gurung et al., 2013).

Hyaluronidase Hyaluronidase (HA) enzymes catalyze the hydrolysis of certain complex carbohydrates such as hyaluronic acid and chondroitin sulfates. The enzymes have been found in mammalian tissues (testis being the richest mammalian source), insects, leeches, snake venom, and in bacteria. HA has gained much importance in cosmetics for its popularity in cosmetic facial augmentation. HA is a naturally occurring glycosaminoglycan disaccharide. It is available in almost all body fluids and tissues, such as the synovial fluid, the vitreous humor of the eye, and hyaline cartilage. These varying properties may inform clinicians as to which HA

Download free eBooks at bookboon.com 84 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes filler would be most suitable for a specific clinical use. For example, a more highly cross- linked HA filler would likely be resilient in its ability to hold its form, making it suitable for the correction of deep wrinkles. In addition, a more monophasic filler might cleanly retain its form and clinically have a smoother appearance. Hyaluronidase is US Food and Drug Administration (FDA) approved as a temporary dispersion agent for injectable fluids, typically local anesthetics during retrobulbar blocks. It has been used clinically for over 60 years (Silverstein et al., 2012).

Proteases Peeling/antiaging/antiwrinkle

Lipases Anticellulitis

Hyaluronidase Moisturising agent

Tyrosinase Tanning agent

Superoxide dismutase Antifree radicals

Peroxidase Antifree radicals

Alkaline Phosphatase Antiwrinkle

Table 4.10.1: Use of enzymes in cosmetics

4.11 BIOFUEL

4.11.1 BIOETHANOL

Over the last decade, there has been a lot of interest in fuel ethanol as a result of increased environmental concern, higher crude oil prices and, by the ban in certain regions of the gasoline additive methyl tert-butyl ether (MTBE), which can be interchanged directly with ethanol (Kirk et al., 2002). Therefore, extensive efforts are being made to develop improved enzymes that can enable the use of inexpensive and partially utilized substrates such as lignocellulose, to make bioethanol more competitive with fossil fuels. The cost of enzymes required to convert lignocellulose into a suitable fermentation feed-stock is a main issue, and the recent work focuses both on the development of enzymes with high activity and stability and also on their efficient production. Governmental programs have been launched in USA by the Department of Energy to support these developments, spurred by the general emphasis on reducing pollution and the need to work towards fulfilling the Kyoto protocol.

Bioethanol production processes vary significantly depending on the raw material involved, but some of the main stages in the process remain the same, even though they take place

Download free eBooks at bookboon.com 85 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes in different conditions of pressure and temperature, and they sometimes involve different microorganisms. These stages include hydrolysis fermentation and distillation (Olsson et al., 2005). Hydrolysis is achieved chemically and enzymatically. Currently, there are mainly two types of process technologies called first and second generation technology.

First generation process technology produces ethanol from sugars (a dimer of the monosaccharides glucose and fructose) and starch-rich (polysaccharides of glucose) crops such as grain and corn. Sugars can be directly converted to ethanol but starches must first be converted to fermentable sugars by enzymes from malt or molds. The technology is well-known but high prices of the raw material and the ethics about using food products for fuel are the main problems.

The raw material in second generation is lignocellulosic materials such as wood, straw, and agricultural residues, which are often available as wastes. These kinds of materials are inexpensive but the process technology is more advanced than converting sugar and starch (Fan et al. 1987; Badger, 2002). Basically, the lignocellulosic biomass comprises of lignin, cellulose and hemicelluloses.

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Table 4.11.1 shows first generation and second generation feed stocks for bioethanol production (Bajpai, 2013). Table 4.11.2 shows enzymes involved in biofuel production.

Ethanol is “a wonderfully clean burning fuel that can be produced from farm crops, agricultural wastes, even garbage.”

– Alexander Graham Bell, 1917

Enzymes have a significant role in the production of biofuels – the fuels of future. Cellulases, xylanases and amylases act on cellulosics and starchy substrates to yield a cocktail of carbohydrates that can be converted into motor fuel (ethanol) after fermentation with appropriate microorganisms.

Burning ethanol obtained from cellulose produces 87% reduced emissions than burning petrol, whereas for the ethanol from cereals the figure is no more than 28%. Ethanol obtained from cellulose contains 16 times the energy required to produce it, petrol only 5 times and ethanol from maize only 1.3 times. The problem is a matter of how to break the bonds of this molecule in order to convert it into fermentable sugars. In fact, this is undoubtedly the type of raw material that is the most complicated to process. Lignin binds together pectin, protein and the two types of polysaccharides, cellulose and hemicellulose, in lignocellulosic biomass. Lignin resists attack by microorganisms and adds strength to the plant. Pretreatment is therefore required to open the biomass by degrading the lignocellulosic structure and releasing the polysaccharides. Pretreatment is followed by treatment with enzymes which hydrolyze cellulose and hemicellulose. The cellulose fraction releases glucose (C6 monosaccharide – sugar with six carbon atoms) and the hemicellulose fraction releases pentoses (C5 monosaccharide – sugar with five carbon atoms) such as xylose. Out of carbohydrate monomers in lignocellulosic materials, xylose is second most abundant after glucose. Glucose is easily fermented into ethanol, but another fermentation process is required for xylose – for example using special microorganisms. The second generation holds great advantages with the fermentation of biomass in the form of agricultural waste materials but there are some challenges such as efficient pretreatment and fermentation technologies together with environmentally friendly process technology.

Ethanol-from-cellulose (EFC) holds great promise due to, abundance, the widespread availability and relatively low cost of cellulosic materials. Significant investment into research, pilot and demonstration plants is going on to develop commercial processes using the biochemical and thermochemical conversion technologies for ethanol. Johnson et al. (2010) have reviewed the current status of commercial lignocellulosic ethanol production.

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In US, in 2016 the production of a record 15.25 billion gallons of ethanol supported 74,420 direct jobs in renewable fuel production and agriculture as well as 264,756 indirect and induced jobs across all sectors of the economy.

Currently the US has a target of 136,260 million liters per year (ML/yr) of renewable fuels production by 2022. This target is only achievable with a majority of this renewable fuel coming from lignocellulosic material, such as wood, corn stover, switch grass, wheat straw and purpose grown energy crops. Demonstration-scale cellulosic ethanol plants are under construction as part of the government’s objective to make cellulosic ethanol cost competitive. The plants cover a wide variety of feed stocks, conversion technologies and plant configurations to help identify viable technologies and processes for full-scale commercialization. All demonstration plants, which are sized at 10% of a commercial- scale biorefinery, are expected to be operational soon. Commercial-scale plants are in the planning stages. Demonstration and commercial plants include – Abengoa – Alico, Alltech, American Energy Enterprises (AEE), Bluefire Ethanol, Coskata, Flambeau River Papers, Park Falls, Wisconsin, Fulcrum-Bioenergy, Sierra Biofuels Plant, ICM,Mascoma, The Wisconsin Rapids, Pacific Ethanol, Red Shield Environmental (RSE), The BioGasol process, Poet, Pure Energy & Raven BioFuels, Range Fuels, Verenium, Virent. Several efforts are underway in North America to commercially produce ethanol from wood and other cellulosic materials as a primary product.

NREL and its partners say that the research conducted in this area is an important step toward realizing the potential of biorefineries (www.ethanol.org/documents/6-05_Cellulosic_Ethanol. pdf). Biorefineries, analogous to today’s oil refineries, will use plant and waste materials to produce an array of fuels and chemicals – not just ethanol. Biorefineries will extend the value-added chain beyond the production of renewable fuel only. Progress towards a commercially viable biorefinery depends on the development of real-world processes for biomass conversion. With these new technologies for the production of cellulosic ethanol, its promise becomes closer to reality with each passing day.

Cellulosic ethanol is on track be cost competitive with corn-based ethanol by 2016, a development that could drive the fuel’s production, according to an industry survey conducted by Bloomberg New Energy Finance (BNEF). The survey focused on 11 major producers in the cellulosic ethanol industry, all of which use a technique known as enzymatic hydrolysis to break down and convert the complex sugars in non-food crop matter, and a fermentation stage to convert the material into ethanol, BNEF said. Cellulosic ethanol cost 94 cents a liter to produce in 2012, about 40 percent more than ethanol made from corn, BNEF said. That price gap will close by 2016, surveyed cellulosic ethanol producers predicted. Project capital expenditures, feed stock and enzymes used in the production process are still

Download free eBooks at bookboon.com 88 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes the largest costs of running a cellulosic ethanol plant, the respondents said in the survey. But technology has pushed operating costs lower. For example, enzyme costs for a liter of cellulosic ethanol dropped 72 percent between 2008 and 2012 due to technological improvements, BNEF said.

4.11.2 OTHER BIOFUELS MADE BY ASSISTANCE FROM ENZYMES

Biofuels include products made via sustainable processing; substantiated by reducing the need for energy from fossil fuel, obtaining better production efficiencies and reducing environmental impact. Biodiesel is an example of such a product having combustion properties like petro- diesel. Biogas is a renewable energy source resulting from biomass – mainly waste products from industrial or agricultural production. A biorefinery is a facility that integrates biomass conversion processes and equipment to produce fuels, power, and value-added chemicals from biomass. Enzymatic catalysis is needed as the way to a sustainable, selective and mild production technique.

Biodiesel is methyl or ethyl esters of fatty acids made from renewable biological resources: vegetable oils or animal fat. The esters are typically made by catalytic reactions of free

Download free eBooks at bookboon.com Click on the ad to read more 89 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes fatty acids (FFA) or triglycerides with alcohols, preferably methanol or ethanol. The overall reaction is a sequence of consecutive and reversible reactions, in which diglycerides and monoglycerides are formed as intermediate compounds. The complete stoichiometric reaction requires 1 mol of triglycerides and 3 mol of alcohol. The reaction is reversible and therefore excess alcohol is used to shift the equilibrium to the products’ side. Methanol and ethanol are frequently used in the process. Transesterification as an industrial process is generally carried out by heating an excess of the alcohol under different reaction conditions in the presence of an acid or a base, or by heterogeneous catalysts such as metal oxides or carbonates, or by a lipase enzyme. The biodiesel yield in the transesterification process is affected by process parameters like moisture, content of free fatty acids (FFAs), reaction time, reaction temperature, catalyst type and molar ratio of alcohol to oil.

First generation feed stocks

Sugar beet Sweet sorghum Sugar cane Maize Wheat Barley Rye Grain Sorghum Triticale Cassava Potato

Second generation feed stocks

Corn stover Wheat straw Sugar cane bagasse Municipal solid waste

Table 4.11.1: First generation and second generation feed stocks for bioethanolBased on Walker (2010)

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Bioethanol

First generation ethanol Alpha amylase, Beta amylase, Glucoamylase

Second generation ethanol Endoglucanase, Cellobiohydrolase, Beta-glucosidase

Biodiesel

Lipase

Table 4.11.2: Enzymes involved in biofuel production

4.12 PROCESSING OF OIL AND FATS The use of enzymes in the oils and fats industry is new, providing several solutions to both the industry problems and the key to produce novel oils and fats.

Processing enzymes can have many benefits in the oils and fats industry such as increasing the yield improving the extraction of oil and lowering the energy required in the process.

Table 4.12.1 shows the enzymes used in processing of fats and oils.

Lipases catalyze reactions under mild reaction conditions (i.e., the industrial hydrolysis of fats and oils or the manufacture of fatty acid amides), allowing high specificity; they can therefore be used to obtain high-value chemicals for food and industrial uses at competitive production costs. For example, cocoa butter fat required for chocolate production generally is in short supply and the price can fluctuate widely. However, lipase catalyzed transesterification of inexpensive oils can be used, for example to produce cocoa butter from palm mid-fraction. The transesterification in organic solvents catalyzed by lipase is a developing industrial application such as production of cocoa butter equivalent, human milk fat substitute, pharmaceutically important polyunsaturated fatty acids (PUFA) and production of biodiesel from vegetable oils (Nakajima et al., 2000). So, lipase enzyme-based technology involving mixed hydrolysis and synthesis reactions are mostly used in commercial activity to upgrade some of the less desirable fats to cocoa butter substitutes (Undurraga et al., 2001). One of the applications of lipase-based technology used the immobilized Rhizomucor miehei lipase for the transesterification reaction which replaces the palmitic acid in palm oil with stearic acid. Similarly, a lipase-catalyzed interesterification of butter fat was used to reduce the long-chain saturated fatty acids and a corresponding increase in C18:0 and C18:1 acid at position 2 of the selected triacylglycerol (Pabai et al., 1995). Another example is the use of

Download free eBooks at bookboon.com 91 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes lipase enzymes to enrich polyunsatured fatty acids (PUFAs) from animal and plant lipids. Free PUFAs and their mono-and diglycerides are subsequently used to produce a variety of pharmaceuticals (anti-inflammatories, thrombolytics, etc.) (Jaeger and Reetz, 1998; Belarbi et al., 2000). Because of their metabolic effects, PUFAs are increasingly used as pharmaceuticals, nutraceuticals and food additives (Belarbi et al., 2000). Many of the PUFAs are required for normal synthesis of lipid membranes and prostaglandins. Microbial lipase enzymes are used to obtain PUFAs from animal and plant lipids such as menhaden oil, tuna oil and borage oil. In addition, the flavor development for dairy products (cheese, butter, margarine, bakery products, alcoholic beverages, milk chocolate and sweets) is obtained by selective hydrolysis of fat triglycerides to release free fatty acids which act as flavor precursors (Jaeger and Reetz, 1998). Immobilized M. miehei lipase in organic solvent catalyzed the reactions of enzymatic interesterification for production of vegetable oils such as sunflower oil, corn oil, peanut oil, olive oil and soybean oil containing omega-3 polyunsaturated fatty acids. Lipase enzymes are important to hydrolyze lipids so as to obtain fatty acids and glycerol, both of which have important industrial applications. For example, fatty acids are used in soap production (Hoq, 1985) and glycerol is used as raw material for pharmaceutical industries.

Enzymatic interesterification is an effective way of controlling the melting characteristics of edible fats and oils (Christensen et al., 2001). No chemicals are used in the process and

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Download free eBooks at bookboon.com Click on the ad to read more 92 INDUSTRIAL ENZYMES: AN UPDATE Industrial application of enzymes no trans-fatty acids are produced. The technology was not widely used until recently due to the high cost of the enzyme, but now enzymatic interesterification is a cost-effective technique to both chemical interesterification and hydrogenation as neither washing nor bleaching of the inter- esterified fat is required, and the low-temperature enzymatic process does not produce any side products. The capital investment costs are low because the enzymatic process requires only one simple column/tank as special equipment. A specific melting profile of the fat is obtained by passing the oil once through the enzyme column. Unlike both hydrogenation and chemical interesterification, the enzymatic process does not require any chemicals. The enzyme is fixed in the column during the production process; therefore the only handling of the enzyme is when it is changed after the production of several hundreds of tons of fat.

Another process is the removal of phospholipids in vegetable oils (‘de-gumming’), using a highly selective microbial phospholipase (Clauson, 2001). This is another example where the introduction of an enzyme step has resulted in both energy and water savings for the benefit of the industry and the environment. Enzymatic degumming is a physical refining process in which one group of phospholipases converts nonhydratable phosphatides into fully hydratable lysolecithin. In industrial degumming, this facilitates gum removal. In most physical refining methods, a fundamental criterion should be that the crude oil is degummed as effectively as possible. Using different phospolipases a variety of products, for example lyso-phospholipids, free fatty acids, diacylglycerols, choline phosphate, and phosphatidates are produced. Traditionally, chemical refining uses large amounts of caustic soda as a main refining agent. The enzymatic degumming process has several advantages. An overall higher yield is obtained because the gums contain up to 25% less residual oil, and because no soap stock is produced, no oil is lost. Furthermore, enzymatic degumming works with crude oil and also water-degummed.

Lipase

Transesterification

Phospholipase

De-gumming, lyso-lecithin production

Table 4.12.1: Enzymes used in processing of fats and oils Based on Kirk et al. (2002)

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Sunar K, Kumar U and Deshmukh S (2016). Recent applications of nzymes in personal care products. In: Dhillon, G. Singh, Kaur, S. (Eds.), Agro-Industrial Wastes as Feedstock for Enzyme Production: Apply and Exploit the Emerging and Valuable Use Options of Waste Biomass. Academic Press, 279–298.

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5 ENZYME MARKET

Major enzyme producers are located in USA, Europe and Japan. The important players in global enzyme market are: Novozymes A/S, Associated British Foods plc (AB Enzymes), Danisco/DuPont (Genencor Industrial Biosciences), BASF Corp., Specialty Enzymes and Biotechnology Ltd., DSM, Amano Enzymes among several others (Sarrouh et al., 2012).

Demand for industrial enzymes in developed countries such as the USA, Western Europe, Japan and Canada has been relatively stable during the recent times, while developing countries of Asia-Pacific, Eastern Europe and Africa, and Middle East regions emerged as the fastest growing markets for industrial enzymes (Global Industry Analysts, Inc., 2011). Increase in demand for nucleases, polymerases and many specialty enzymes, coupled with the strong growth in markets for animal feed are expected to steer growth in industrial enzymes market. USA and Europe collectively command a major share of the world industrial enzymes market.

Carbohydrases market is projected to be the fastest growing product segment and Protease enzymes constitute the largest product segment in the global industrial enzymes market. Lipase enzymes represent the other major product segment showing high growth potential (Global Industry Analysts, Inc., 2011). Food and feed represents the largest segment for industrial enzymes in terms of end-use. Developing countries are expected to emerge as the fastest growing consumers of industrial enzymes for food and feed applications, as increase in per capita income in these regions would continue to drive the demand for meat. Detergents constitute the other major end-use segment for industrial enzymes. However, demand for detergent enzymes, is likely to be affected by the fluctuating prices of raw materials and the continuous efforts by the manufacturers to reduce the costs. Nevertheless, a large percentage of mid-tier and low tier-detergent manufacturers are increasing the use of enzymes in their products for offering better performance.

Pharmaceuticals and bioethanol sectors have succeeded in attracting significant attention of the investors and are self-sufficient in undertaking new product development activities and in launching unique and novel products in the market, thus offering new opportunities to the industrial enzyme producers. But, segments such as wastewater treatment, chemicals and pulp & paper lack sufficient funding for carrying out new product developments (Sarrouh et al., 2012). Market research show that industrial demands for enzymes is being driven by new enzyme technologies and increase use of organic compounds in place of petrochemical- based ingredients.

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By 2024, the global industrial enzymes market is expected to reach USD 9.63 billion, (Grand View Research, Inc., 2016). The market is expected to see significant growth because of increasing substitution of chemicals with industrial enzymes especially in food and beverage and nutraceutical applications.

Enzyme market is dominated worldwide by the food and beverage products, and drug industry that go directly or indirectly for human consumption. Growing applications of industrial enzymes in food processing industry and detergents is expected to increase the demand. Protease enzymes are widely used in the detergent industry. These enzymes have superior stain removal properties. But, the demand in the detergent application is expected to see sluggish growth because of market saturation. Growing use of protease in bakery products is expected to drive market growth. Furthermore, increasing application scope of the product in nutraceutical industry as a digestive enzyme is expected to increase demand. Technological developments in the field of industrial enzymes have led to the use of the product as cleaning agents. The increasing use of enzymes in treatment of waste water is also expected to increase the demand. Novozymes, Danisco and DSM dominated the global industrial enzymes market in 2015 with the industry being characterized by forward integration by manufacturers to distribution and end-use. Manufacturers such as DuPont and DSM produce industrial enzymes for specialized applications. The global industrial enzymes market is dominated by North America due to the presence of a large number of manufacturers in the USA and Canada.

Industrial enzymes demand for lipase enzymes is expected to see significant growth, growing at over 8.0% from 2016 to 2024. Increasing demand for the product in food & beverage and textile industry is expected to augment growth during 2016 to 2024.

The feed enzymes are expected to grow at over CAGR 9.0% from 2016 to 2024. The increasing use of feed enzymes as a protein source in animal feed is to improve performance in livestock which in turn is expected to increase demand during 2016 to 2024.

Asia Pacific is expected to witness significant growth, growing at a CAGR of over 10.0% from 2016 to 2024. Vigorous expansion in food processing industries particularly in Asia- Pacific is expected to augment growth. Moreover, the supportive regulations promoting the expansion of manufacturing industries are expected to have a positive effect on market growth.

Europe accounted for over 29.0% of the market share in 2015 and is expected to see significant growth because of rising demand in pharmaceutical and textile industry. Strict regulations, prohibiting the use of toxic chemicals and catalysts in various applications, are expected to positively affect the market over the next eight years.

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Carbohydrases dominated the global market in 2017. Food processing dominated the global market and accounted for more than 30% of the global market in 2017. North America dominated the global market in 2017. The United States market was estimated at USD 1,388.27 million in 2017 and is estimated to register a CAGR of 5.6% through 2023 (Industrial Enzymes Market, 2017).

The top three producers – Novozymes, DowDuPont, and DSM together – account for almost 74% of the global market. Other important producers in the market are Cargill Incorporated, Dyadic International, Inc., Maps Enzymes Ltd., and Advanced Enzyme (Industrial Enzymes Market, 2017).

In July 2017, Swissaustral, a leader in the development of novel enzymatic products and processes from extremophiles and Ginkgo Bioworks, the organism company, announced a new partnership to develop microorganisms that will produce industrial enzymes at scale. These enzymes will be used as a safe and low-energy replacement for conventional chemical reaction processes in many industries such as pharmaceuticals, textiles, foods and household goods. In July 2017, Indian enzyme manufacturer Advanced Enzyme Technologies Ltd. announced to take over German industrial biotech company Evoxx Technologies GmbH for EUR 7.65 million in cash (Industrial Enzymes Market, 2017).

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References Global Industry Analysts, Inc. (2011). In: Report – Global Strategic Business

Grand View Research (2016). https://www.grandviewresearch.com/press-release/global- industrial-enzymes-market

Industrial Enzymes Market – Growth, Share, Trends and Forecasts (2018–2023) https://www.mordorintelligence.com/industry-reports/global-industrial-enzymes-market-industry

Sarrouh B, Santos TM, Miyoshi A, Dias R, Azevedo V (2012). Up-to-date insight on industrial enzymes applications and global market. J Bioprocess Biotechniq S4:002 doi:10.4172/2155- 9821.S4-002.

2000

1500

1000 $ Millions

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0 2008 2009 2010 2015

Technical enzymes Food and beverage enzymes Others

Figure 5.1: Global industrial enzyme market 2008–2015 (Sarrouh et al., 2012)

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6 FUTURE PERSPECTIVES

The prospects of industrial uses of microbial enzymes have increased greatly in 21st century and continuously increasing. Enzymes have significant potential for many industries to meet demand of rapidly growing population and cope exhaustion of natural resources. Enzymes are currently being used in many different industrial products and processes and new areas of application are continuously being added up (Gurung et al., 2013; Kirk et al., 2002; Chandel et al., 2007; Singh et al., 2016). Enzymes can be developed today for processes where no one would have expected an enzyme to be applicable just a decade ago. Thanks to advances in modern biotechnology! Common to most applications, the introduction of enzymes as effective catalysts working under mild reaction conditions results in substantial savings in resources such as energy and water for the benefit of both the environment and the industry in question. In a world approaching exhaustion of several natural resources, and a rapidly increasing population, enzyme technology offers a great potential for many industries to help meet the challenges they are expected to face in the coming years.

The enzyme market and number of competitive enzyme based processes is growing rapidly, because of new enzymes, cheaper production methods and new application areas (Novozymes, 2011; Schafer et al., 2002; Schmid et al. 2001). The possibility to dramatically change enzyme properties by directed evolution and gene shuffling, and effective methods to screen for new enzymes in the environment, makes it possible to use enzymes which are specifically tailored to their application and process conditions. Enzyme technology is close to a major breakthrough, because of several factors ranging from simple cost savings, the increasing demand for chiral chemicals, the opportunities created by emerging technologies and the trend towards sustainable industrial development.

Enzymes have tremendous potential in various industrial sectors that may be pharmaceuticals, food, feed, beverages, detergents, leather processing and pulp and paper industry (Kirk et al., 2002; Leisola et al., 2002; Li et al., 2012). Alternatively, these biomolecules may be used consistently to meet continuously rising demand of food supply. Enzymes of microbial origin have significant potential in waste management, and consequently in the development of green environment. The enzymes are efficiently used in many industries for higher quality productions at higher reaction rates with innocuous pollution and cost effectiveness.

Around the globe, enzyme market is dominated by the food and beverage products, and drug industry that go directly or indirectly for human consumption. One of the biggest challenges in front of fast growing economies such as India is to provide food and healthcare to even their larger population. India, an agriculture-based economy, is predicted to grow at 7.9% by 2018 (http://data.worldbank.org/country/india) and an attractive market that is

Download free eBooks at bookboon.com 115 INDUSTRIAL ENZYMES: AN UPDATE Future perspectives opening her doors for industrial enzyme based manufacturing sector. Indian biotech sector accounts 2% of the global biotech market, but it is gaining worldwide visibility due to the investment opportunities as well as its research output (Binod et al., 2013). Bharat Biotech, a Hyderabad-based vaccines manufacturer has announced a breakthrough in developing the world’s first Zika vaccine. This vaccine is ready for pre-clinical trials demonstrating the “Make in India” efforts. Almost 50% of total enzyme demand covers pharma sector. The detergent manufacturing and textile processing sector cover almost 20% each. Food and feed industries, leather and paper industries demand 5% each (Binod et al., 2013). Industrial biotechnology has an important role to play in the way modern foods are processed. New ingredients and alternative solutions to current chemical processes will be the challenge for the enzyme industry. Compared with traditional chemical reactions, the more specific and cleaner technologies made possible by enzyme-catalyzed processes will promote the continued trend towards natural processes in the production of food.

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References Binod P, Palkhiwala P, Gaikaiwari R, Namppthiri, KM, Duggal, A, Dey K, Pandey A (2013). Industrial enzymes – present status and future perspectives for India. J. Sci. Ind. Res. 72: 271–286

Chandel A, Rudravaram R, Rao L, Ravindra P, Lakshmi NM (2007). J Commer Biotechnol 13: 283. https://doi.org/10.1057/palgrave.jcb.3050065

Gurung N, Ray S, Bose S, Rai V (2013). A broader view: microbial enzymes and their relevance in industries, medicine, and beyond. Biomed Res. Int. 2013: 329121.10.1155/2013/329121 http://data.worldbank.org/country/india

Kirk O, Borchert TV, Fuglsang CC (2002). Industrial enzyme applications. Curr. Opin. Biotechnol. 13: 345.

Leisola M, Jokela J, Pastinen O, Turunen O, and Schoemaker H (2002). Industrial use of enzymes, In: Encyclopedia of Life Support Systems (EOLSS), OOP H¨anninen and M Atalay, Eds., pp. 1–25, EOLSS, Oxford, UK, 2002.

Li S, Yang X, Yang S, Zhu M, Wang X (2012). Technology prospecting on enzymes: Application, marketing and engineering, Comput. Struct. Biotechnol. J., 2: p. e201209017.

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Schafer T, Kirk O, Borchert TV, Fuglsang, CC, Pedersen S, Salmon S, Olsen HS, Deinhammer R, Lund H (2002). Enzymes for technical applications. In Biopolymers; Fahnestock SR, Steinbü chel SR, Eds.; Wiley-VCH: Weinheim, Germany, 2002; pp. 377−437.

Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, et al. (2001). Industrial biocatalysis today and tomorrow. Nature 409: 258–268.

Singh R, Kumar M, Mittal A, Mehta PK (2016). Microbial enzymes: industrial progress in 21st century. 3 Biotech 6:174–175. doi:10.1007/s13205-016-0485-8.

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