May-July.2012, Vol.2.No.3, 1379-1389. e- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences

An International Peer Review E-3 Journal of Sciences Available online at www.jcbsc.org Section B: Biological Sciences CODEN (USA): JCBPAT

Review Article

An Overview of Microbial Lipases

K.S.S.*, M.V.V.Chandana , V. and M.Manasa 1*,2,3,4 Centre for Biotechnology, Department of Chemical Engineering, A.U College of Engineering, Andhra University, Visakhapatnam -530003, , . Received: 8 May 2012; Revised: 31 May 2012; Accepted: 8 June 2012

ABSTRACT Lipases are the most pliable biocatalyst and bring about a wide range of bioconversion reactions, such as hydrolysis, interesterification, esterification, alcoholysis, acidolysis and aminolysis that play a key role in fat digestion by cleaving long-chain triglycerides into polar lipids and these Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and the synthesis of esters formed from glycerol and long-chain fatty acid and they are ubiquitous enzymes which catalyze the hydrolysis of triacylglycerols to glycerols and free fatty acid. Lipases have been utilized for fat modification, fragrance development in dairy product and for the synthesis of personal care product, oil industry, dairy industry, pharmaceuticals and bakery industry and cosmetics. The most important microbial lipase-producing strains for submerged and solid-state fermentations are reviewed as well as the main substrates, including the use of agro- industrial residues. Current process techniques (batch, repeated-batch, fed-batch, and continuous mode) are discussed and the importance of lipase catalyzed fat splitting process, kinetics, deactivation kinetics, pre-purification and novel purification technologies. Keywords: Lipases, Current process techniques, Kinetics, Deactivation kinetics, Pre-purification and Novel purification technologies.

INTRODUCTION Lipases are ubiquitous enzymes of considerable physiological significance industrial potential. Lipases (Triacylglycerol acylhydrolases EC 3.1.1.3) are a class of hydrolase which catalyze the hydrolysis of triglycerides to glycerol and free fatty acids over an oil-water interface and reversing the reaction in non- aqueous media. This phenomenon of interfacial activation with a lid covering the active site is today an area of extensive research involving X-ray crystallographers, biochemists, molecular biologists, chemists and biochemical engineers. Lipases catalyze the hydrolysis and transesterification of other esters, synthesis of esters and exhibit enantioselective properties specific chemical transformation of lipases has make them increasingly popular in the food, detergent, cosmetic, organic synthesis and pharmaceutical industries1-4 .Commercially useful lipases are usually obtained from microorganisms that produce a wide variety of extra cellular lipases. 1379 J. Chem. Bio. Phy. Sci. Sec. B. 2012, Vol.2, No.3, 1379-1389.

An Overview ... K.S.S.Rekha et al.

Lipase have emerged as one of the leading biocatalysts contributing to the multibillion dollar underexploited lipid technology bio-industry and have been used in situ lipid metabolism and ex situ multifaceted industrial applications 5 .This is mainly result of the huge achievements made in the cloning and expression of enzymes from microorganisms, as well as of an increasing demand for these biocatalyst with novel and specific properties such as specificity, stability, pH and temperature 6,7 . Lipases are produced by animals, plants and microorganisms. Many microorganisms are known as potential producers of extracellular lipases, including bacteria, yeast and fungi 8. Fungal species are preferably cultivated in Solid-State Fermentation (SSF), while bacteria and yeast are cultivated in submerged fermentation (SmF)9. SOURCES OF MICROBIAL LIPASES

Lipases are ubiquitous in nature and are produced by several plants, animals, and microorganisms. A review of the most recent potential microorganisms for lipase production in both submerged and solid-state fermentations are reported in Table 1. The main microorganisms used before 2004 have already been well described in the reports of Gupta et al 10 and Sharma et al 11 .

Table-1: Microorganisms cited in the recent literature as potential lipase producers

Microorganism Source Submerged fermentation Solid-state fermentation Rhizopus arrhizus FungalTan Fungal Tan and Yin 12 , Yang et al.13 and Yin , Yang et al 12 .

Rhizopus chinensis Fungal Teng et al 14. , Wang et al.15 Teng Sun and Xu 17 and Xu, 16 Aspergillus sp. Fungal Cihangir and Sarikaya 18 Rhizopus homothallicus Fungal Diaz et al.19 Diaz et al.19. Penicillium citrinum Fungal D’Annibale et al.20 Penicillium restrictum Fungal Azeredo et al.21 Palma et al.22 Penicillium simplicissimum Fungal Vargas et al.23

Geotrichum sp. Fungal Yan and Yan 24, Burkert et al.25 Geotrichum candidum Fungal Burkert et al.26 Aspergillus carneus Fungal Kaushik et al.27 Rhizopus sp. Fungal Bapiraju et al.28 Martinez-Ruiz et al.29

Candida utilis Yeast Grbavcic et al 4. Trichosporon asahii Yeast Kumar and Gupta 30 Pseudomonas sp. Bacterial Kiran et al.31.

Bacillus stearothermophilus Bacterial Abada 8 Bacillus coagulans Bacterial Alkan et al.32 Burkholderia multivorans Bacterial Gupta et al.2

1. Filamentous Fungi: Some of the most commercially important lipase-producing fungi are recognized as belonging to the genera Rhizopus sp., Aspergillus sp., Penicillium sp., Geotrichum sp., Mucor sp., and Rhizomucor sp. Lipase production by filamentous fungi varies according to the strain, the composition of the growth medium, cultivation conditions, pH, temperature, and the kind of carbon and nitrogen sources 18 . 2. Yeast: According to Vakhlu and Kour 33 , the main terrestrial species of yeasts that were found to produce lipases are: Candida rugosa, Candida tropicalis, Candida Antarctica. 3. Bacteria: Among bacterial lipases being exploited, those from Bacillus exhibit interesting properties that make them potential candidates for biotechnological applications. Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus coagulans, Bacillus stearothermophilus, and Bacillus alcalophilus are the most 1380 J. Chem. Bio. Phy. Sci. Sec. B, 2012, Vol.2, N o.3, 1379 -1389. An Overview ... K.S.S.Rekha et al. common bacterial lipases. In addition, Pseudomonas sp., Pseudomonas aeruginosa, Burkholderia multivorans, Burkholderia cepacia, and Staphylococcus caseolyticus are also reported as bacterial lipase producers. PRODUCTION PROCESSES Fermentative processes have been conducted in batch, repeated-batch, fed-batch, and continuous mode. The mode of operation is, to a large extent, dictated by the characteristics of the product of interest. This section will consider recent applications of batch, repeated-batch, fed-batch, and continuous processes to lipase production in SmF and SSF. Table 2 summarizes the different forms of production, processes, and bioreactor configurations employed in the last 10 years for lipase production both in SmF and SSF.

Table: 2- Process modes and main bioreactor configuration employed for lipase production in the last 10 years

Process conduction Bioreactor References Submerged fermentation Batch STR Gupta et al.2 , Burkert et al.26 , Montesinos et al.34 , Batch Airlift Burkert et al.26 Repeated-batch STR Benjamin and Pandey 35 (1997), Li et al.36 Yang et al.13 Fed-batch STR Boareto et al.37 , Montesinos et al.38 Continuos STR Montesinos et al.34 , 38

Batch Packed-bed Benjamin and Pandey 35

1. Batch Processes: Most papers reporting lipase production use batch mode in shaken flasks. However, there are a considerable number of studies focusing on the use of bubble, airlift, and stir tank bioreactors 2. Repeated-Batch Processes: The repeated-batch processes combines the advantages of fed-batch and batch processes, mainly making possible to conduct the process by long periods and improving the productivity compared to the batch process. The work of Yang et al.13 investigated the lipase production by immobilized mycelium from R. arrhizus in submerged fermentation using repeated-batch fermentations. Immobilized cells showed high stability for repeated use. Li et al. 36 used repeated fed-batch strategy to produce lipase from Acinetobacter radioresistens. 3. Fed-Batch Processes: The fed-batch processes are characterized by the addition of one or more nutrients to the bioreactor during the process, maintaining the products inside the bioreactor until the final of fermentation. The fed-batch processes are amply employed to minimize the effects of the cell metabolism control and, mainly, prevent the inhibition by substrate or metabolic products. 4. Continuous Processes: Montesinos et al.38 investigated the production of extra- and intracellular lipases in continuous cultures of C. rugosa using pure or carbon source mixtures. Lipase productivity in continuous cultures increased by 50% compared to data obtained from batch fermentation and was dependent on the dilution rate applied. Maximum yields relative to consumed substrate were obtained with oleic acid at low dilution rates. The authors found that during nitrogen limitation, lipase activity was suppressed. PRODUCTION AND MEDIA DEVELOPMENT FOR LIPASE Microbial lipases are produced mostly by submerged culture, but solid state fermentation methods can be used also. Immobilized cell culture has been used in a few cases. Many studies have been undertaken to define the optimal culture and nutritional requirements for lipase production by submerged culture. Lipase production is influenced by the type and concentration of carbon and nitrogen sources, the culture pH, the growth 1381 J. Chem. Bio. Phy. Sci. Sec. B, 2012, Vol.2, N o.3, 1379 -1389. An Overview ... K.S.S.Rekha et al. temperature, and the dissolved oxygen concentration 39 . Lipidic carbon sources seem to be generally essential for obtaining a high lipase yield; however, a few authors have produced good yields in the absence of fats and oils. 1. Effect of carbon sources: Fructose and palm oil were considered as the best carbohydrate and lipid sources, respectively, for the production of an extracellular lipase by Rhodotorula glutinis. 2. Effect of nitrogen sources:For an extracellular lipase of Pe. citrinum , obtained maximal production in a medium that contained 5% (wt/vol) peptone (pH 7.2). Nitrogen sources such as corn steep liquor and soybean meal stimulated lipase production but to a lesser extent than peptone. APPLICATION OF LIPASES Major applications of lipases are summarized in Table 3. Most of the industrial microbial lipases are derived from fungi and bacteria.

Table-3: Industrial applications of microbial lipases

Industry Action Product or application Detergents Hydrolysis of fats Removal of oil strains from fabrics

Dairy foods Hydrolysis of milk fat, Development of flavoring agents in cheese ripening, milk, cheese and butter modification of butter fat Bakery foods Flavor improvement Shelf-life prolongation Beverages Improved aroma Beverages Food dressings Quality improvement Mayonnaise, dressings and whippings Health foods Transesterification Health foods Meat and fish Flavor development Meat and fish products; fat removal Fats and oils Transesterification, Cocoa butter, margarine, fatty acids, hydrolysis glycerol, mono and diglycerides Chemicals Enantioselectivity, Chiral building blocks, chemicals synthesis Pharmaceuticals Transesterification, Specialty lipids, digestive aids hydrolysis Cosmetics Synthesis Emulsifiers, moisturizers Leather Hydrolysis Leather products paper Hydrolysis Paper with improved quality Cleaning Hydrolysis Removal of fats

KINETICS OF ENZYME REACTION Several mechanisms have been proposed for lipase catalysis reactions 40 . The vast majorities of these mechanisms were developed for the case of soluble lipases acting on insoluble substrates ( e.g. oil droplets dispersed in water). However, in the absence of diffusional limitations the validity of the aforementioned mechanism easily extended to include the most complex case of having the lipases present in immobilized states. The simplest kinetic model applied to describe lipase-catalyzed reaction is based on the classic Michaelis-Menten mechanism as applied to emulsified oil/water systems 41 . The kinetic steps can be represented schematically as:

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here E denotes the immobilized enzyme, S the substrate (glycerides), ES the enzyme-substrate complex and P and Q the products (hydrolyzed glycerides and free fatty acids). The rate of formation of free fatty acids per unit volume of reacting fluid ( rv) can be represented in terms of this mechanism as:

v = Vmax[S] /( Km+ S) (2)

Vmax = kcat [E] tot (3)

−1 1 Km = kcat +k k (4)

Where Vmax is the rate observed when the lipase is saturated with substrate, Km is the Michaelis-Menten constant, the bracket denote molar concentrations of the various species, and the subscript tot denotes the total amount. In the case of feed stocks from natural origin which contain more one chemical species susceptible to lipase action ( e.g. , butter fat), the Michaelis-Menten mechanism denoted as equation (2) may be extended in order to include competitive inhibition by every substrate, Si with respect to each other. For extent of hydrolysis below 70%, a pseudo-zero order rate expression arises which takes the form

The above equation is based on the following assumptions: (1) the Michaelis-Menten constants, Kmi , associated with every substrate Si (out of N possible substrates) are approximately equal; and (2) all [ Si]/ Kmi are very large compared to unity. Three rate expressions were utilized based on Michaelis-Menten kinetics and a ping-pong bi-bi mechanism to fit the uni-response experimental data for the total rate of release of fatty acids.

where rV,hyd,A , rV,hyd,B and rV,hyd,C correspond to the effective rate of reaction per unit volume (M/h) for the various models, [G] is the concentration (M) of glyceride moieties (accessible ester bonds, and i represent lumped parameter related to the rate parameter appearing in the various Michaelis-Menten models ( Vmax parameter, and Michaelis and inhibition constant). These rate expressions are identical in mathematical form to those employed by Malcata et al 42,43 ,. If the feedstock does not contain, initially, significant amount of free fatty acids, then [G] can be determined by the following equation.

[G] = [ G0] - [FA ] (10)

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Where [ G0] represents the initial concentration of glyceride bonds accessible to hydrolysis and [ FA ] is the concentration of free fatty acids resulting from hydrolysis. 1. Deactivation Kinetics: One of disadvantages, and probably the most serious disadvantage, of biocatalyst is that they lose their catalytic activities during the reaction. This phenomenon is well recognized in biotechnology and is called decay, inactivation, or denaturation of the biocatalyst. Even if the life of a biocatalyst can be prolonged by its immobilization, it still loses its activity sooner or later. The catalytic durability of the biocatalyst during continuous operation is called operation stability. The operational stability of a biocatalyst is estimated by its half-life, which is the elapsed time at which the catalytic activity is reduced to half. The half-life is a very important parameter that governs the economical feasibility of the bioprocess concerned 44 . Two methods are known to evaluate the deactivating catalyst during continuous operation. For the most common case continuous operation is performed so as to keep the conversion fixed (constant conversion). This requires decreasing flow rate as the enzyme activity drops gradually. In other method the flow rates (or the space velocity) is kept constant throughout the continuous operation (constant feed rate or constant space velocity) to observe the decrease in the outlet conversion. Generally, deactivation rates are determined in the absence of substrate, but enzyme deactivation rates can be considerably modified by the presence of substrate and other materials. Biocatalyst thermal stability is a fundamental aspect in the reactor performance. Despite this, most information on biocatalyst stability, being gathered under nonreactive conditions, is of limited use, leaving aside modulation effects by substrate and products, which certainly play a role during catalysis. Different mechanisms have been proposed to describe enzyme thermal inactivation. The simplest and most used is one stage first-order kinetics, which proposes the transition of a fully active native enzyme to a fully inactivated species in a single step. Such mechanism leads to a model of exponential decay:

(11)

Thermal inactivation is certainly more complex and series and parallel mechanisms have been proposed to describe it 45 . Models derived from such mechanisms contain a high number of parameters, which are difficult to determine experimentally. However, a two phase series mechanism usually represents well the phenomenon in terms of a limited number of parameters susceptible to reliable experimental determination. A model based on such mechanism is represented by equation (12):

Where e stands for enzyme activity, eo is its initial value, t stands for time, k1 and k2 are the transition rate constants in each inactivation stage and A is the specific activity ratio between the intermediate and initial enzyme stage. These models have been traditionally used to evaluate enzyme stability under non-reactive conditions 46 , so that parameters obtained do not reflect the behavior in the presence of substrate and products as it occurs in the reactor. It has been postulated that any substrate that interacts with the enzyme during catalysis is a potential modulator of enzyme stability 47 Therefore -d[Ei J ]/ dt = kiJ [ Ei J ] (13)

kiJ = ki(1-niJ) where niJ represents the modulation factor -of modulator J in inactivation stage i. For instance, in the case of an enzyme subjected to product competitive inhibition, three enzyme species will exist : the free enzyme (E) and the secondary enzyme-substrate (ES) and enzyme-product (EP) complexes, among which the enzyme will be distributed during the course of catalysis.

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PRE-PURIFICATION STEPS Most of the microbial lipases are extracellular and the fermentation process is usually followed by the removal of cells from the culture broth, either by centrifugation or by filtration. The cell-free culture broth is then concentrated by ultrafiltration, ammonium sulphate precipitation or extraction with organic solvents. About 80% of the purification schemes attempted thus far have used a precipitation step, with 60% of these using ammonium sulphate and 35% using ethanol, acetone or an acid (usually hydrochloric) followed by a combination of several chromatographic methods such as gel filtration and affinity chromatography. Precipitation is usually used as a fairly crude separation step, often during the early stages of a purification procedure, and is followed by chromatographic separation. 1. Chromatographic steps: Most of the time, a single chromatographic step is not sufficient to get the required level of purity. Hence, a combination of chromatographic steps is required. Ion exchange chromatography is the most common chromatographic method; used in 67% of the purification schemes analysed and in 29% of these procedures, it is used more than once. The most frequently employed ion- exchangers are the diethylaminoethyl (DEAE) group in anion exchange (58%) and the carboxymethyl (CM) in cation exchange (20%). Strong ion exchangers based on triethylaminoethyl groups, and Q-Sepharose are becoming more popular in lipase purification. Gel filtration is the second most frequently employed purification method, used in 60% of the purification schemes and more than once in 22% of them. Affinity chromatography has been used as a purification step in 27% of the schemes. Hydrophobic interaction chromatography has been used in 18% of the cases with the most popular hydrophobic adsorbents being octyl or phenyl functional groups. Concanavalin A (Con A) and heparin are employed for purification of fungal and mammalian lipases on account of the glycoprotein nature of these lipases. Adsorption chromatography is applied in 16% of the purification schemes and the adsorbent hydroxyapatite is used frequently. Affinity methods can be applied at an early stage, but as the materials are expensive, the less costly ion exchange and gel filtration are usually preferred after the precipitation step. Although gel filtration has the lower capacity for loaded protein, it can be used at an early stage in the purification or as one of the last steps for fine polishing of the protocol. NOVEL PURIFICATION TECHNOLOGIES Some novel purification technologies have recently been applied to the purification of lipases. These include membrane processes, immunopurification, hydrophobic interaction chromatography employing epoxy- activated spacer arm as a ligand and polyethylene glycol–Sepharose gel, poly(vinyl alcohol) polymers as column chromatography stationary phases and aqueous two-phase systems. Membrane processes Cross-flow membrane filtration has been used in the downstream processing of lipases, namely, for microbial cell removal and concentration of the supernatant of the spent media containing lipases. CONCLUSION Critical analysis of current literature is focused on lipases, discussing the new lipase producing microorganisms, optimization of media composition, purification strategies, applications used in this specific field. Lipases are becoming increasingly important in high-value applications in the oleo-chemical industry and the production of fine chemicals. Simultaneously, advances are being made in bioreactor and reaction technologies for effectively using the lipases. REFERENCES 1. H.Park, K. Lee, Y. Chi and & S.Jeong; Effects of methanol on the catalytic properties of porcine pancreatic lipase. Journal of Microbiology and Biotechnology; 2005, 15 (2), 296. 2. N.Gupta, V. Shai & R.Gupta; Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor. Process Biochemistry , 2007, 42 (2), 518.

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*Correspondence Author: K.S.S.Rekha ,Centre for Biotechnology, Department of Chemical Engineering, A.U College of Engineering, Andhra University, Visakhapatnam -530003, Andhra Pradesh, India.

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