Name of the Assignment: Characterization and Applications of Lipases
Course Title: Enzymology Course ID: BTC 517 Date of Submission: 3 August, 2012

Submitted To:
Professor Naiyyum Choudhury
Coordinator,Biotechnology
MNS Department
BRAC University
Mohakhali, Dhaka

Submitted By:

Sultana Rownok Jahan
M.S Biotechnology
Summer 2012
MNS Department
BRAC University
Mohakhali,Dhaka.

Introduction:
Lipases are the special kind of esterases belong to subclass 1 of hydrolytic enzyme class 3 and have been assigned sub-sub class 3.1.1 due to their specificity for carboxylic acid ester bonds. Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are ubiquitous enzymes of considerablephysiological significance and industrial potential. Lipases catalyze the hydrolysis of triacylglycerols to glycerol and free fatty acids. In contrast to esterases, lipases are activated only when adsorbed to an oil–water interface (Martinelle et al., 1995) and do not hydrolyze dissolved substrates in the bulk fluid. A true lipase will split emulsified esters of glycerine and long-chain fatty acids such as triolein and tripalmitin. Lipases are serine hydrolases and contain the consensus sequence G – X1 – S – X2 – G as the catalytic moiety, where G – glycine, S – serine,X1 – histidine and X2 – glutamic or aspartic acid .Lipases display little activity in aqueous solutions containing soluble substrates. In contrast, esterases show normal Michaelis–Menten kinetics in solution. In eukaryotes, lipases areinvolved in various stages of lipid metabolism including fat digestion, absorption, reconstitution, and lipoprotein metabolism. In plants, lipases are found in energy reserve tissues.How lipases and lipids interact at the interface is still not entirely clear and is a subject ofintense investigation (Balashev et al., 2001).

Sources of Lipase:
Lipase occurs widely in nature; however microbial lipases are commercially significant because of low production cost, greater stability and wider availability than other sources.
Fig.1 illustrates the biodiversity of lipases with biological origin.

. Figure 1

Bacterial lipases
Many bacterial lipases are well studied compared to plants and animals. Bacterial lipase is a glycoprotein but some extracellular bacterial lipases are lipoprotein. The organisms are normally grown on nutrient medium containing carbon (oil, sugar and mixed carbon sources), nitrogen, phosphorus sources and mineral salts whereas the production of lipases mostly depends on inducer such as triglycerides, bile salts and glycerol. Lipases from Pseudomonas were probably the first studied and have preponderant role in industries, later on Achromobacter sp., Alcaligones sp.,Pseudomonas sp., Staphylococcus sp., and Chromobacterium sp., have been exploited for production of lipases. The sources and properties of bacterial lipases are given in Table 1(a). These lipases are characterized for pH, temperature, Pi and molecular weight from both gram positive and gram negative bacteria. It is evident from the Table 1(a) that, pH range is between 4.0 to 10.0; temperature range is between 27 to 80oC,whereas, molecular weight varies from 11 to 176 kDa.
Fungal lipases
Fungal lipases have benefits over bacterial lipases due to their low cost of extraction, thermal and pH stability, substrate specificity and activity in organic solvents. Lipase producers are widespread in the fungal kingdom. The chief producers of lipases are Aspergillus sp., Candida sp., Mucor sp., Rhizopus sp., have been studied in great details. The thermophilic Mucor pusillus is well known as a producer of thermostable extracellular lipase and from M. miehei two isoenzymes with slightly different isoelectric points could be isolated. The sources and biochemical properties of fungal lipases are given in Table 1(b). Fungal lipases are characterized for pH,temperature and molecular weight. It is interesting to note that, pH range is between 4.0 to 11.0, temperature range is between 25 to 60oC whereas, molecular weight varies from 27 to 120 kDa.

(Kishore J. Patil et,al,2007)

(Kishore J. Patil et,al,2007)

Plant lipases
In plants mostly lipases are present in the form of food reserve tissues of growing seedlings or especially in those which contains large amount of triacylglycerols. Lipase activity in plant seeds increases during germination because the triacylglycerols are converted to soluble sugars by the action of lipase which is then transported to the growing tissues to supply structural carbon and energy to provide support for the growth of young plants. The sources and properties of plant lipases are given in Table 1(c). Plant lipases are characterized for pH, temperature and molecular weight. It is interesting to note that, pH range is in between 4.0 to 8.0, temperature range is in between 25 to 60oC, whereas, molecular weight varies from 40 to 143 kDa. This data indicates that relatively plant lipases are slightly different from bacterial and fungal lipases.
Animal lipases
Animals are also rich sources of lipases but due to the availability of microbial lipases they are rarely studied, but still they have been isolated from many insects, fishes, mammals. Animal lipase plays an important role in digestion of lipids in biological system (Walton & Cowey1984). Fats required special digestive action before absorption because the end products must be carried in water medium (blood and lymph) in which fats are not soluble. Although little actual fat digestion occurs in the stomach, gastric lipase does digest already emulsified fats such as in egg yolk and cream. The detail biochemical profile of animal lipase is given in Table 1(d).

Properties of lipases
Types of reaction catalyzed by lipase: Lipases are stable and rugged enzyme that act on lipids as well on wide variety of natural and artificial reactant since it has ability to catalyze diversified reaction, few of them are listed below: * Acidolysis * Intertransesterification * Aminolysis * Hydrolysis * Alcoholysis:

Figure2:Reaction mechanism of lipase

Figure3: Non-specificity and 1, 3-Regiospecificity of lipase
Substrates for Lipase
Glycerides are the natural substrate for lipases; they possess a chiral alcohol moiety. It was understood that lipases were particularly useful for the resolution or asymmetrization of esters bearing a chiral alcohol moiety.
General guidelines for the design of substrates for lipase were formulated by the IUPAC7: Figure 3: Types of substrate for lipase

Figure4:Diagrammatic representation of a lipase molecule showing its main features; the substrate can be any triglyceride37

Table 2:List of commonly used substrate for lipase assays (Kishore J. Patil et,al,2007)

i. The center of chirality should be located as close as possible to the site of the reaction (i.e. the ester carboxyl group) to ensure optimal chiral recognition. Thus, esters of secondary alcohols are usually more selectively transformed than those of primary alcohols. ii. There is wide tolerance for the nature of both substituants R1 and R2 but they should differ in size and/or polarity to aid the chiral recognition process. They may also be linked together to form cyclic structures. Polar groups, such as carboxylate, amide or amine—which would be heavily hydrated in an aqueous environment—are not tolerated and, if they are required, they should be protected with a lipophilic unit. iii. The alkyl chain of the acid moiety (R3) should be preferably of straight chain nature, possessing at least three to four carbon atoms. Reaction rates may be improved by using ‘activated’ esters bearing haloalkyl groups, e.g. Cl – CH2 – and Cl – (CH2)2 – for Type I and II, respectively iv. The remaining hydrogen atom in both substrate types must not be replaced by a substituent, since esters of tertiary alcohols and tri substituted carboxylates are usually not accepted by lipases.
v. The stereo-chemical preference of the most commonly used lipases (e.g. from Pseudomonas sp. and Candida sp.) for esters of secondary alcohols follows an empirical model generally referred to as “Kazlauskas rule”. These guidelines may be followed to get fair accuracy to obtain the desired product. pH and temperature kinetics
Lipases are active over broad pH and temperature range and they have molecular weight ranging from 94 to 840 kDa From available literature it can be interpreted that generally lipases have neutral pH optima but the pH and temperature optima of lipases depends on the habitat of its sources. Lipases possess stability over a wide range from pH 4 to 11 and temperature optima in the range from 10 to 960C. (Chandan & Shahani,1964).
Effect of metal ions
The activity of lipase may be inhibited or stimulated by cofactors. Divalent cations such as calcium often stimulated enzyme activity due to the formation of calcium salt of long chain fatty acids (Macrae &Hammond, 1985). Calcium stimulated lipases have been reported in the case of Acinetobacter sp. RAG-1 (Snellman et al., 2002). In contrast, the lipase from P. seruginosa 10145 (Finkelstein et al., 1970) is inhibited by the presence of calcium ions. Further lipases activity is inhibited drastically by heavy metals like Ca+2. Ni+2, Hg+2 and Sn+2 and slightly inhibited by Zn+2 and Mg+2 (Patkar & Bjorkling, 1994). Lipase activity was enhanced in the presence of K+, Ca+2 and Mg+2 ions, but inhibited by Hg+2 ions (Sharma et al, 2009). The addition of Mg+2 did not significantly stimulate lipase production.While many other metal ions including Ca+2, Mn+2, Ba+2, Zn+2, metal ions, including Ca+2, Mn+2, Ba+2, Zn+2, Fe+2, and Cu+2 exerted inhibitory effects. However, lipase production was decreased slightly, to approximately 5%, with the addition of K+ and 30% decrease was observed in lipase production by S5 in an absence of potassium ions. The absence of magnesium ions (Mg+2) in the basal medium was also shown to stimulate lipase production. An alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase (Raja et al. 2006). The lipase activity in presence of a metal ions was compared with control including no metal ion whose activity was taken as 100% and the relative activities at 1mM of Cu+2, Hg+2, Pb+2, Co+2, Cd+2 and Li+ were 0.44, 24.4, 36.2, 49.1, 64.2, 90.0 and 98.2% respectively. Strong inhibition was observed with heavy metals such as Cu+2, Hg+2, Pb+2, Co2+ and Cd+2 in the Todarodes pacificus (Park et al.,2007).
Stability in organic solvents
Stability in organic solvents is desirable in synthesis reaction. From the available information it is concluded that lipases are generally stable in organic solvents with few exceptions of stimulation or inhibition (Gupta & Rathi,2004). Eventually high stimulation is noted in the presence of acetone, isopropanol and ethanol but was unaffected by methanol (Sharma et al., 2009). Stability of lipases in different solvents is described in Table 3. Residual activity ranges from 41% to 100% in Streptomyces ramous, whereas, it was widely ranged from 10% to 100% and enhancement found in Dimethylformamide (120%) and Chloroform (130%) in P. aeruginosa. The effect of various solvents on lipases is given in Table 3. Table 3: Effect of solvents on lipases

(Kishore J. Patil et,al,2007)

Cellular localization of lipase
Mostly the prokaryotes secrete lipase extracellularly,however, eukaryotes synthesis cytosolic origin. The cellular localization lipases indifferent organisms are given in Table 4.

Table 4: Cellular Localisation of Lipase (Kishore J. Patil et,al,2007)

Applications of lipases
Lipases are widely used in the processing of fats and oils, detergents and degreasing formulations, food processing, the synthesis of fine chemicals and pharmaceuticals, paper manufacture, and production of cosmetics, and pharmaceuticals (Rubin and Dennis, 1997a,b;
Kazlauskas and Bornscheuer, 1998). Lipase can be used to accelerate the degradation of fatty waste (Masse et al., 2001) and polyurethane (Takamoto et al., 2001). Major applications of lipases are summarized in Table 5.

Table 5: Industrial Applications of Lipase

Lipases in the detergent industry
Because of their ability to hydrolyzes fats, lipases find a major use as additives in industrial laundry and household detergents.
Detergent lipases are especially selected to meet the following requirements:
(1) a low substrate specificity, i.e., an ability to hydrolyze fats of various compositions
(2) ability to withstand relatively harsh washing conditions (pH 10–11,30–60○C)
(3) ability to withstand damaging surfactants and enzymes [e.g., linear alkyl benzene sulfonates (LAS) and proteases], which are important ingredients of many detergent formulations. Lipases with the desired properties are obtained through a combination of continuous screening (Yeoh et al., 1986; Wang et al., 1995; Cardenas et al., 2001) and protein engineering (Kazlauskas and Bornscheuer, 1998).

Table 6: Commercial Detergent Lipases

Lipases in food industry
Fats and oils are important constituents of foods. The nutritional and sensory value and the physical properties of a triglyceride are greatly influenced by factors such as the position of the fatty acid in the glycerol backbone, the chain length of the fatty acid, and its degree of unsaturation. Lipases allow us to modify the properties of lipids by altering the location of fatty acid chains in the glyceride and replacing one or more of the fatty acids with new ones. This way, a relatively inexpensive and less desirable lipid can be modified to a higher value fat (Colman and Macrae, 1980; Pabai et al., 1995a,b; Undurraga et al., 2001).

Table 7:Lipase applications in the food industry

Table 8:Examples of lipase in cheese production

Lipases in pulp and paper industry
‘Pitch,’ or the hydrophobic components of wood (mainly triglycerides and waxes), causes severe problems in pulp and paper manufacture (Jaeger and Reetz, 1998). Lipases are used to remove the pitch from the pulp produced for paper making. Nippon Paper Industries, Japan, have developed a pitch control method that uses the Candida rugosa fungal lipase to hydrolyze up to 90% of the wood triglycerides. Lipases in organic synthesis
Use of lipases in organic chemical synthesis is becoming increasingly important. Lipases are used to catalyze a wide variety of chemo-, regio-, and stereoselective transformations
(Rubin and Dennis, 1997b; Kazlauskas and Bornscheuer, 1998; Berglund and Hutt, 2000).
Majority of lipases used as catalysts in organic chemistry are of microbial origin. These enzymes work at hydrophilic–lipophilic interface and tolerate organic solvents in the reaction mixtures. The enzymes catalyze the hydrolysis of water-immiscible triglycerides at water–liquid interface. Under given conditions, the amount of water in the reaction mixture will determine the direction of lipase-catalyzed reaction. When there is little or no water, only esterification and transesterification are favored (Klibanov, 1997). Hydrolysis is the favored reaction when there is excess water (Klibanov, 1997). Lipase-catalyzed reactions in supercritical solvents have been described (Rantakyla et al., 1996; Turner et al., 2001; King et al., 2001).

Lipases in bioconversion in aqueous media
Hydrolysis of esters is commonly carried out using lipase in two-phase aqueous media
(Vaysse et al., 1997; Chatterjee et al., 2001). The hydrolysis of p-nitrophenyl palmitate ( pNPP) in n-heptane by a lipase preparation of P. cepacia.Mutagenesis has been used to greatly enhance the enantioselectivity of lipases (Bornscheuer,2000; Gaskin et al., 2001). For example, in one case, the enantioselectivity of lipasecatalyzed hydrolysis of a chiral ester (P. aeruginosa lipase) was increased from e.e. 2% to e.e. 632 R. Sharma et al. / Biotechnology Advances 19 (2001) 627–662 ,81% in just four mutagenesis cycles. The lipase-acyl transferase from C. parapsilosis has been shown to catalyze fatty hydroxamic acid biosynthesis in a biphasic liquid/aqueous medium. The substrates of the reaction were acyl donors (fatty acid or fatty acid methyl ester) and a hydroxylamine. The transfer of acyl group from a donor ester to hydroxylamine (aminolysis) was catalyzed preferentially compared to the reaction of free fatty acids. This feature made the C. parapsilosis enzyme the catalyst of choice for the direct bioconversion of oils in aqueous medium (Vaysse et al., 1997). A novel lipase produced by Burkholderia sp., which could preferentially hydrolyze a bulky ester, t-butyl octanoate (TBO). This lipase was confirmed to be 100-fold superior to commercial lipases in terms of its TBO-hydrolyzing activity.
Lipases in bioconversions in organic media
Enzymes in organic media without a free aqueous phase are known to display useful unusual properties, and this has firmly established nonaqueous enzyme systems for synthesis and biotransformations (Klibanov, 1997). Lipases have been widely investigated for various nonaqueous biotransformations (Therisod and Klibanov, 1987; Klibanov, 1990; Tsai and
Dordick, 1996; Ducret et al., 1998; Dong et al., 1999; Kiran and Divakar, 2001).
Lipases in resolution of racemic acids and alcohols
Stereoselectivity of lipases has been used to resolve various racemic organic acid mixtures in immiscible biphasic systems (Klibanov, 1990). Racemic alcohols can also be resolved into enantiomerically pure forms by lipase-catalyzed transesterification.In one study, a purified lipase preparation from C. rugosa was compared to its crude counterpart in anhydrous and slightly hydrated hydrophobic organic solvents. The purified lipase preparation was less active than the crude enzyme in dry n-heptane, whereas the presence of a small concentration of water dramatically activated the purified enzyme but not the crude enzyme in the esterification of racemic 2-(4-chlorophenoxy) propanoic acid with n-butanol (Tsai and Dordick, 1996).
Lipases in regioselective acylations
Lipases acylate certain steroids, sugars, and sugar derivatives with a high regioselectivity. Monoacylated sugars have been produced in anhydrous pyridine from triethyl carboxylates and various monosaccharides (Therisod and Klibanov, 1987). In contrast ,a lipase from A. niger to catalyze the regioselective deacylation of preacylated methyl b-D-glucopyranoside. Similarly, regioselective deacetylation of preacetylated monosaccharide derivatives in 1,1,1-trichloroethane using a lipase modified with polyethylene glycol.
Lipases in ester synthesis
Lipases have been successfully used as catalyst for synthesis of esters. The esters produced from short-chain fatty acids have applications as flavoring agents in food industry (Vulfson, 1994). Methyl and ethyl esters of long-chain acids have been used to enrich diesel fuels (Vulfson, 1994).
Lipases in oleochemical industry
Use of lipases in oleochemical processing saves energy and minimizes thermal degradation during alcoholysis, acidolysis, hydrolysis, and glycerolysis (Vulfson, 1994; Bornscheuer,
2000). Although lipases are designed by nature for the hydrolytic cleavage of the ester bonds of triacylglycerol, lipases can catalyze the reverse reaction (ester synthesis) in a low water environment. Hydrolysis and esterification can occur simultaneously in a process known as interesterification. Depending on the substrates, lipases can catalyze acidolysis.
Lipases in Medicine
We will now briefly discuss the function of lipases in the digestion of normal fat and mention some applications of these enzymes and their inhibitors in human therapy.
Physiological Function of Lipases in the Digestion of Dietary Fat
Dietary fats are composed of about 95% triacylglycerols (TG).(R. L. Rizek, B. Friend, L. Page, J. Am. ,1974, )Until recently, the hydrolysis of fats was thought to begin in the intestinal lumen and to be catalyzed entirely bypancreatic lipase. In this view, the stomach was a transient storage organ, whose function was limited to mixing and dispersing lipids with the other nutrients. Although many authors have observed the occurrence of preduodenal lipolysis in humans and other species, gastric lipolysis was assumed to be negligible and even attributed to pancreatic contamination after a gastric reflux in the duodenum.Today, the picture has changed, and strong experimental evidence supports the view that the gastric and pancreatic lipases act in synergy. Preduodenal lipases have been purified and biochemically characterized, for example rat lingual, human gastric lipase (HGL), calf a pharyngeal, rabbit gastric (RGL), and dog gastric(DGL). It is now well established that the gastric lipolysis of longchain triacylglycerols is of paramount importance in the physiological absorption of dietary fat, especially in patients suffering from exocrine pancreatic insufficiency, where it could partially compensate for the absence of the pancreatic lipase. (C. K. Abrams, M. Hamosh, V. S. Hubbard, S. K. Datta, P. Hamos, J. 1984)
Furthermore, it was demonstrated that human and rabbit gastric lipases can potentiate in vitro the hydrolysis of triglycerides by human pancreatic lipase.(Y. Gargouri, H. Moreau, R. Verger,1989)
Lipases in Substitution Therapy:
Exocrine pancreatic insufficiency, often found in cystic fibrosis patients, results in two major problems: malnutrition and steatorrhea. These problems can be partly solved by the administration of porcine pancreatic lipase extracts as a replacement therapy for these patients. In the past, such preparations were far from satisfactory, since a large proportion of the enzymes administrated were denaturated in the stomach due to the extreme acidity of the gastric juice. The coadministration of a lipase which could hydrolyze dietary lipids under acidic conditions would probably help in improving some of the problems of malabsorption and steatorrhea. Various clinical studies have been conducted both in animals and humans to assess the efficacy of acid-resistant lipases of fungal origin as enzymatic replacement therapy in exocrine pancreatic insufficiency.(C. Wicker-Planquart, S. Canaan, M. RivieÂre, L. Dupuis, R. Verger,1996).Although some significant effects were observed in weight gain and reduction of steatorrhea, the use of such enzymes is limited as a result of their sensitivity to the proteolytic action of gastric pepsin.
In contrast to the human enzyme, which preferentially hydrolyzes short-chain triacylglycerols, dog gastric lipase has an exceptionally high specific activity on long-chain triacylglycerols, which represent the majority of dietary fat in humans.( F. CarrieÂre, H. Moreau, V. Raphel, R. Laugier, C. Be nicourt, J. L.1991)Dog gastric lipase would probably facilitate the absorption of lipids when given as an enzyme supplement to cystic fibrosis patients together with classical pancreatic extracts.( C. Be nicourt, C. Blanchard, F. CarrieÂre, R. Verger, J. L. ,1993.)

Lipase Inhibitors as Antiobesity Agents:
Conventional treatment for obesity has focused largely on strategies to control energy intake. Under clinical circumstances, the use of an inhibitor of digestive lipases which reduces dietary fat adsorption holds great promise as an antiobesity agent. Tetrahydrolipstatin (THL, 16), derived from lipstatin produced by Streptomyces toxytricini, acts in vitro and in vitro as a potent inhibitor of pancreatic and gastric lipases as well as of cholesterol ester hydrolase.( P. HadvaÂry, H. Lengsfeld, 1988),(B. Borgstrom, Biochim. Biophys.1988)
Lipases in Biofuel industry:
Microbial enzymes are an excellent alternative to produce biofuels. Lipase is an enzyme capable of catalyzing methanolysis reactions.Lipases for biodiesel production from TAG should be nonstereospecific. A wide range of lipases has been used for enzymatic transesterification and esterification. It can be obtained from microorganisms such as bacteria and fungi. They work in mild conditions and have an ability to work with TGs from different origins.They have the ability to catalyze transesterification of both TGs and FFAs to give esters. So all tri-, di and monoglycerides can be converted to FAAE.At the same time, they should also catalyze the esterification of FFA,low product inhibition with high FAAE yield,lipases advantages over acid and base catalysts,low reaction time,possible reuse of the enzyme,temperature and alcohol resistance ,ease of lipase production. These are the key issues to be addressed for industrial use of lipases in biodiesel production to be viable.

Table 9:Biodiesel production with various Lipases

The Future Perspective: Highly Pure and Genetically Engineered Lipases
Highly Pure Lipases
Lipases hold considerable promise in synthetic organic chemistry and have found practical applications already in detergents, oleochemistry, cheese production, medical therapy, and industrial synthesis of specialty chemicals. By now, lipases from over 30 biological sources have been cloned, sequenced, and expressed in host organisms. Pure recombinant lipases from Humicola lanuginosa, Pseudomonas pseudoalcaligenes, Pseudomonas aeruginosa, Mucor miehei, Candida antartica (type B), Bacillus thermocatenulatus, and other sources are now commercially available in free or immobilized form, or as part of a screening set (e.g. Chirazyme from Boehringer Mannheim). Even cross-linked crystals of Candida rugosa and of Pseudomonas cepacia lipase are commercially available from Altus. The tertiary structures of twelve lipases have been resolved, and due to their application potential, this number will grow rapidly. As a result, a more rational approach for how to modify lipases for detergents, oleochemical applications, and organic synthesis is emerging.
Protein Engineering of Lipases
With our increasing knowledge on lipase structure and function, it has become clear that substrate binding domains vary greatly from one lipase to another, providing a more rational explanation for their varying substrate specificities. The different steric and electronic environment prevailing in lipases of different origin. As an example, the distance of the active serine residue from the surface varies between 5 . (ROL) and 16 . (PCL) in the three examples shown. Information on genetically engineered lipases (mostly taken from the patent literature) is summarized in Table 14. As detergents are still the commercially most important field of lipase applications, most pertinent patents deal with enhancing lipase stability and activity in a household detergent matrix.( O. Misset). The scientific literature on modified lipases for

Table 10: Engineered Lipases

oleochemistry and organic synthesis is still scarce. One case deals with the chain-length specificity observed in Rhizopus delemar lipase, which may be related to steric effects involved in the binding of acyl groups. Indeed, site-directed mutagenesis of F95, F112, V206, and V209, sterically demanding points of acyl chain interaction in this lipase, led to a significant shift in the preference of the mutant lipase for the hydrolysis of medium-chain triglycerides.( T. Yoneda et.al,1995) So at the end of the study it can be said that, lipases are versatile enzymes that are used widely. Lipases arebecoming increasingly important in high-value applications in the oleochemical industry and the production of fine chemicals. Lipases are capable of regioselective and stereoselective biotransformations and allow resolution of racemic mixtures. Lipases with improved properties are being produced by natural selection and protein engineering to further enhance usefulness of these enzymes. Lipase-based processing has a promising future

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