Review Article Organic Solvent Tolerant Lipases and ...downloads.hindawi.com/journals/tswj/2014/625258.pdf · bonds. e esteric ation reactions are always carried out in nonaqueous

Review ArticleOrganic Solvent Tolerant Lipases and Applications

Shivika Sharma and Shamsher S Kanwar

Department of Biotechnology Himachal Pradesh University Summer Hill Shimla 171 005 India

Correspondence should be addressed to Shamsher S Kanwar kanwarss2000yahoocom

Received 30 August 2013 Accepted 31 October 2013 Published 2 February 2014

Academic Editors D Fan H Noritomi and B Tian

Copyright copy 2014 S Sharma and S S Kanwar This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Lipases are a group of enzymes naturally endowed with the property of performing reactions in aqueous as well as organic solventsThe esterification reactions using lipase(s) could be performed in water-restricted organic media as organic solvent(s) not onlyimprove(s) the solubility of substrate and reactant in reaction mixture but also permit(s) the reaction in the reverse directionand often it is easy to recover the product in organic phase in two-phase equilibrium systems The use of organic solvent tolerantlipase in organic media has exhibited many advantages increased activity and stability regiospecificity and stereoselectivity highersolubility of substrate ease of products recovery and ability to shift the reaction equilibrium toward synthetic direction Thereforethe search for organic solvent tolerant enzymes has been an extensive area of research A variety of fatty acid esters are now beingproduced commercially using immobilized lipase in nonaqueous solventsThis reviewdescribes the organic tolerance and industrialapplication of lipases The main emphasis is to study the nature of organic solvent tolerant lipases Also the potential industrialapplications that make lipases the biocatalysts of choice for the present and future have been presented

1 Introduction

Lipases have emerged as one of the leading biocatalysts withproven potential for contributing to the multibillion dollarunderexploited lipid bioindustry They have been used inin situ lipid metabolism and ex situ multifaceted industrialapplication(s) [1] and the catalyze the hydrolysis of triacyl-glycerol to glycerol and fatty acids [2] Lipases find potentialapplications in bioprocesses largely due to their availabilityand stability in organic as well as in aqueous media [3ndash5]This enzyme has versatile applications by virtue of its uniqueproperties [6 7] Under natural conditions lipases catalyzethe hydrolysis of ester bonds at the interface between an insol-uble substrate phase and the aqueous phasewhere the enzymeremains dissolved (Figure 1) In nonaqueous conditions theycatalyze the reverse reaction (such as esterification inter-esterification and transesterification) producing glycerides(Figure 2) from glycerol and fatty acids [8 9] In the pastyears a better understanding of enzymes functionalities andcatalytic behaviors together with the progress of molecularengineering has led to new applications for various typesof enzymes as for example proteases acylases oxidases

amylases glycosidases cellulases or lipases Lipases haveimproved substrate specificity and operate in milder reactionconditionsMoreover the fact that they retain their activity inorganic solvents and also their catalytic promiscuity extendtheir range of applications [10 11]

Lipases are ubiquitous in nature and are produced byvarious plants animals and microorganisms However forthe production of industrial enzymes microorganisms arethe most preferred source They have the shortest generationtime high yield of conversion of substrate into productgreat versatility to adapt to environmental conditions andsimplicity in genetic manipulation as well as in cultivationconditions [12] Microbial lipases are currently receivingmuch attention with the rapid development of enzymetechnology [13] and due to their ability to perform catalysisat extremes of temperature pH and organic solvents withchemo- regio- and enantioselectivity Also lipases displayuseful properties related to their stability as organic solvent-tolerant [14] and thermostable [15] enzymes The reason forthe enormous biotechnological potential of microbial lipasesincludes the fact that they are stable in organic solvents donot require cofactors and possess a broad specificity [16]

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 625258 15 pageshttpdxdoiorg1011552014625258

2 The Scientific World Journal

COOH

Serine

Glycosylated area

Hydrophobic batch

Substrate

Water-oil interfaceH2N

H3C

Figure 1 Diagrammatic representation of a lipase molecule show-ing its main features

Fundamental studies on polymerization revealed someremarkable capabilities of lipases for polymerization chem-istry The polymerization and transesterification studies gen-erally demand harsh condition(s) of presence of organicsolvents andor high temperature [17 18] The technolog-ical utility of enzymes can be enhanced greatly by usingthem in organic solvents rather than their natural aqueousreaction media [19] The use of enzyme in organic mediahas exhibited many advantages increased activity and sta-bility regiospecificity and stereoselectivity higher solubilityof substrate ease of products recovery and ability to shiftthe reaction equilibrium toward synthetic direction [20]From the biotechnological point of view there are numerousadvantages of conducting immobilized biocatalyst-promotedenzymatic conversions in organic solvents as opposed toreactions performed in water-based system(s) [21]

2 Sources of Lipases

Lipases are produced by plants animals and microbes butonly microbial lipases are found to be industrially importantsince they are diversified in their enzymatic properties andsubstrate specificity [22] Lipases can be obtained fromanimals mainly from fore-stomach tissue of calves or lambsand pancreatic tissues of pigs The disadvantages of usinganimal lipases include presence of trypsin in pig pancreaticlipases which results in bitter tasting amino acids andpresence of residual animal hormones or viruses as well astheir undesirable effects in the processing of vegetarian orkosher diets [23] Plant lipases are also available but notexploited commercially because of the yield and the processesinvolvedThusmicrobial lipases are currently receivingmoreattention because of their technical and economic advantageswhere the organisms are cultivated in medium containingappropriate nutrient composition under controlled condi-tions Also lipase production by microorganisms variesaccording to the strains the composition of the growthmedium cultivation conditions pH temperature and thekind of carbon and nitrogen sources [24 25] Generallybacteria fungi yeast and actinomycetes are recognized as

Triglyceride

H2O plusmn

H2O plusmn

H2O plusmn

Diglyceride + fatty acid

Monoglyceride + fatty acid

Fatty acid + glycerol

Figure 2 The mediated lipase reaction(s)

preferred sources of extracellular lipases facilitating theenzyme recovery from the culture broth although CandidaPseudomonas Mucor Rhizopus and Geotrichum spp standout as the major commercially viable strains [26] Bacteriallipases are extensively used in food industry for qualityimprovement dairy industry for hydrolysis ofmilk fat cheeseripening beverages to improve aroma and health foodsfor transesterification [6] Many microorganisms have beenreported in the last decade for lipase production in bothsubmerged and solid-state fermentations reported (Table 1)

3 Reactions Catalyzed by Lipases

The lipases catalyze a wide range of reactions includinghydrolysis interesterification alcoholysis acidolysis esterifi-cation and aminolysis They catalyse the hydrolysis of fattyacid ester bond in the triacylglycerol and release free fattyacids The lipase-catalyzed reactions are

(i) Hydrolysis

RCOOR1015840 +H2O 997888rarr RCOOH + R1015840OH (1)

(ii) Synthesis Reactions under this category can be fur-ther separated into the following categories

(a) Esterification

RCOOH + R1015840OH 997888rarr RCOOR1015840 +H2O (2)

(b) Interesterification

RCOOR1015840 + R10158401015840COORlowast 997888rarr RCOORlowast + R10158401015840COOR1015840 (3)

(c) Alcoholysis

RCOOR1015840 + R10158401015840OH 997888rarr RCOOR10158401015840 + R1015840OH (4)

(d) Acidolysis

RCOOR1015840 + R10158401015840COOH 997888rarr R10158401015840COOR1015840 + RCOOH (5)

The Scientific World Journal 3

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

Microorganism Source Submerged fermentation Solid-state fermentationRhizopus arrhizus Fungal 27 28Rhizopus chinensis Fungal 29ndash31 32Aspergillus sp Fungal 33Rhizopus homothallicus Fungal 34 34Penicillium citrinum Fungal 35Penicillium restrictum Fungal 36 37Penicillium simplicissimum Fungal 38Geotrichum sp Fungal 39 40Geotrichum candidum Fungal 40Aspergillus carneus Fungal 41Candida utilis Yeast 42Pichia anomola Yeast 43Yarrowia lipolytica Yeast 44Geobacillus sp Bacterial 45Bacillus tequilensis Bacterial 46Saccharomonospora azurea Bacterial 47

The last three reactions are often grouped together into asingle term namely transesterificationThe term transesteri-fication refers to the exchange of groups between an ester andan acid (acidolysis) between an ester and an alcohol (alcohol-ysis) or between two esters (interesterification) The abilityof lipases to catalyze these reactions with great efficiencystability and versatilitymakes these enzymes highly attractivefrom a commercial point of view The lipase specificities canbe divided [27 28] into three main groups as follows

(1) Substrate Specificity The natural substrates are glycerolesters These enzymes are able to catalyze the hydroly-sis not only of triacylglycerols (TAGs) but also di- andmonoacylglycerols and even phospholipids in the case ofphospholipases

(2) Regioselective Regioselectivity is the preference of onedirection of chemical bondmaking or breaking over all otherpossible directions It is subdivided into the following types

(i) Nonspecific lipases they catalyze the complete hy-drolysis of triacylglycerols into fatty acids and glycerolin a random way monoglycerols and diacylglycerolsare the intermediate products

(ii) Specific 13 lipases they only hydrolyse triacylglyc-erols at the C1 and C3 glycerol bonds producing fattyacids 2-monoacylglycerols and 12-diacylglycerols or23-diacylglycerols the latter two being chemicallyunstable promoting migration of the acyl group pro-ducing 13-diacylglycerol and 1-monoacylglycerols or3-monoacylglycerols

(iii) Specific or selective type fatty acid lipases can bespecific for a particular type of fatty acid or morefrequently for a specific group of fatty acids Theyhydrolyze fatty acid esters located at any triacylglyc-erol position

(3) Enantioselective Lipases have the ability to discriminateenantiomers in a racemic mixture An example of this is theR-isomer of Aspartame which tastes sweet whereas the S-isomer tastes bitter The enantiospecificities of lipases canvary according to the substrate and this variation can beconnected to the chemical nature of the ester [29]

4 Lipase BehaviorProperties inOrganic Solvents

41 Lipase Structure in Organic Solvents Molecular model-ing of R miehei lipase in different environments showed thatthe structure modeled in vacuum was reasonably similar tothe models obtained with hydrophobic solvents as the envi-ronment [30] Generally hydrophilic solvents have relativelymore interactions with the enzyme molecules The structureof an enzyme Subtilisin Carlsberg in a hydrophilic solventacetonitrile which was determined by X-ray crystallographywas shown to be similar to that in water [31]Thewater boundto the enzymemolecules however decreased and acetonitrilemolecules were bound to the enzyme molecules Sequencesfor several lipases and esterases were compared to find theresidues that are either completely or partially conserved[32] Sequence alignment of 16 lipoprotein lipases showeda large area of sequence identity in the proximity of theactive-site serine residue Moreover all the lipases possesseda GlowastSlowastG motif around the active-site serine The sequencesof these lipases showed a close relation with sequences ofhepatic lipases which were less closely related to sequences ofpancreatic lipasesThe parts of lipases that are conservedmaybe important for their structural integrity activity andorspecificity [32]

42 Lipase Stability in OrganicMedia Amajority of enzymesshow good in vitro catalytic rates in aqueous solutionsHowever lipases being activated by interfaces show lower


catalytic rates in homogeneous aqueous solutions than inthe presence of interfaces for example water-organic solventinterface [33] Typically lipases are ubiquitous enzymes thatwere originally characterized by their ability to catalyze thehydrolysis of acylglycerides fatty acid esters and so forthat oil-water interface [34] This is called aqueous-organicsolvent biphasic system Vigorous mixing of the two phasesforms a suspension with a significantly large interfacial area

Enzyme molecules are solubilized in discrete hydratedreverse micelles formed by surfactants within a continuousphase of a nonpolar organic solvent that is in reversemicellar system Under appropriate conditions a reversemicellar solution is homogeneous thermodynamically stableand optically transparent [35] The applications of enzymesin organic media rather than aqueous media have severalimportant advantages such as the shift in thermodynamicequilibrium in favour of the product over the hydrolyticreaction an increased solubility of nonpolar substrates elim-ination of side reactions and an increased thermal stability ofthe enzyme in harsh conditions [21] Most lipases are knownto be active and stable in anhydrous organic solvents Severalcovalent modifications as well as noncovalent modificationtechniques have been developed for solubilization of lipasesin organic solvents [36] The biological origin of lipase thereaction to be carried out (hydrolysis or esterification) thesubstrates used and so forth determine which solvent systemwill be the most suitable one The use of organic solventsin reaction media shifts the thermodynamic equilibriumto favor synthesis over hydrolysis Furthermore in organicsolvent the conformation of the enzyme appears to be morerigid These characteristics enable controlling some of theenzymersquos catalytic properties such as the substrate specificitythe chemo region- and enantioselectivity by variation ofthe solvent [37] Recently lipases from Pseudomonas are themost widely used in biotechnological application becauseof their potential in organic synthesis for highly valuablechemicals [38] Also the cost of downstream processing isa direct function of the solvent system and the impuritiespresent Potential advantages (Table 2) of employing enzymesin nonaqueous media as opposed to aqueous media werepostulated by many authors [39ndash41]

Lipase can undergo deactivation in the synthetic reac-tions due to altered temperature shear stresses exposureto interfaces and chemical denaturants which are gener-ally present in the esterification reaction systems as eithersubstrates or products This enzyme deactivation occurseither due to physical changes in the enzyme structure orchemical changes like deamidation and breakage of disulfidebonds The esterification reactions are always carried out innonaqueous solvents The stability of enzymes in organicsolvents is a strong function of the solvent properties

Deactivation of a lipase from R miehei due to temper-ature and butanol has been studied [42] and a considerabledeactivation of the enzyme by butanol was noticed Thermaldeactivation of enzymes usually occurs due to unfolding ofthe molecule At high temperature various forces maintain-ing the enzyme structure (including hydrogen-bonding ionicand van derWall interactions and hydrophobic interactions)diminish leading to unfolding of the enzyme Thermal

Table 2 Advantages of organic solvents over aqueous media

(i) Better solubility of substrates and product(ii) Shifting of thermodynamic equilibria (synthesis takes placeinstead of hydrolysis)(iii) Simpler removal of solvent (most organic solvents have lowerboiling point than water)(iv) Reduction in water-dependent side reactions such ashydrolysis of acid anhydrides or polymerization of quinines(v) Removal of enzyme after reaction since it is not dissolved(vi) Better thermal stability of enzymes since water is required toinactivate enzymes at high temperatures(vii) Elimination of microbial contamination(viii) Potential of enzymes to be used directly within a chemicalprocess

deactivation of lipase may be reduced considerably by itsimmobilization Thermal deactivation of lipase B from Can-dida antarctica (CALB) and lipase from Candida rugosa(CRL) respectively in their native and immobilized formshas been studied [43 44] Lyophilization of the enzymetogether with certain additives such as carbohydrates [4546] fatty acids [47] or salts [48] has been shown to greatlyimprove the enzyme performance The activating effect ofadditives is more pronounced in dry organic solvents thanin partially hydrated organic solvents [49] The treatmentof Candida rugosa lipase with short-chain polar organicsolvents (methanol ethanol 1- and 2-propanol 1- and 2-butanol orand acetone) enhanced its esterification andtransesterification activity [50] F oxysporum has an increaseof its activity after being incubated in organic solvents whichis an essential feature in many organic syntheses [37]

In organic media the pH dependence of enzymes dis-persed in a solvent has been shown similar to the pHdependence of the enzymes in an aqueous medium [51 52]The dependence of the enzyme activity in organic solventson pH of the aqueous solution in which the enzyme lastexisted is termed pH memory The ionization states oflyophilized compounds are similar to those in solution formin which the compound was lyophilized [53] Although thislatest investigation of lyophilized compounds using Fouriertransform infrared (FTIR) spectroscopy supports the conceptof pH memory there are exceptions to this concept

43 Solubilization of Lipases in Organic Solvents Lipases areinsoluble in organic solvents in their native form Solubilizedlipases are attractive not only because of their higher activities(compared to insoluble lipases) but also because of theiroptical transparency that allows one to perform structuralcharacterization by spectroscopic technique [54] Two typesofmethods namely covalent and noncovalentmodificationsare in common use for lipase solubilization Chemical orcovalent modifications are done by using chemical modifierslike polyethylene glycol (PEG) poly-N-vinylpyrrolidonepolystyrene polymethyl methacrylate [55 56] and nitrocel-lulose membrane [57] Such chemical modifications greatly


affect the activity stability and selectivity of the enzyme[58 59] as well as its reusability [57]

Noncovalent modification of lipases has essentially beenrestricted to coating the lipase molecule with differentsurfactants One of the most widely reported techniquesinvolves dissolution of the lipase and the surfactant inaqueous solution [60 61] When sufficient time is allowedthe hydrophilic tails of the surfactant noncovalently bind topolarionic groups on the surface of lipase thereby creatingan enzyme-surfactant complex whose surface is hydrophobicbecause of the protruding hydrophobic tails of the surfactantThis enzyme-surfactant complex which precipitates fromthe aqueous solution can be dissolved in organic solventsThe solubilized lipase has shown catalytic activities whichare significantly greater than those demonstrated by lipasepowders Another related technique involves preparation ofwater-in-oil emulsions (or reverse micelles) containing lipaseand then drying out the water from the emulsion phasethat yields a lipase-surfactant complex which is soluble andhighly active in organic solvents [62 63]

44 Medium Engineering of Lipases in Organic SolventsOrganic solvent tolerant (OST) lipases are required inbiotechnological applications especially in the production ofbiopolymeric materials biodiesel and the synthesis of finechemicals [38] LipA from Burkholderia cepacia is highlyactive and tolerant to short-chain alcohols [64] Lipasesin nonaqueous systems can be active provided that theessential water layer around them is not stripped offMediumengineering for biocatalysis in nonaqueous media involvesthe modification of the immediate vicinity of the biocatalyst[65 66] Nonpolar solvents are better than polar ones sincethey provide a better microenvironment for the lipase Ifthe enzymersquos microenvironment favors high substrate andlow product solubility the reaction rates would be highThe solvent effects may not be generalized too far Thereare various exceptions of which lipases are a particular caseEnzymes often exhibit diminished activities in nonconven-tional media such as organic solvents than in their naturalmedia that is water-oil interface in the case of lipases [51] Toachieve enhanced rate of enzymatic reactions the operatingconditions are of great importance [67]

5 Immobilization of Lipases

Use of enzymes is still limited due to high cost of enzymeisolation and purification for their single use The enzymesare labile in nature so their isolation from natural envi-ronment can cause denaturation and diminished activityThe low pH temperature and chemical stability in organicsolvents also restrict the use of free enzymes Moreoverthe separation of products in presence of free enzymes istedious These drawbacks of the free enzymes are overcomethrough immobilization technique [68] Immobilizationmayserve two objectives first to improve enzyme stability andsecond to facilitate a decrease in enzyme consumption asthe enzyme can be retrieved and reused for many repeatedcycles of reaction By taking advantage of the ldquointerfacialrdquo

hydrophobicity immobilization of lipases has been per-formed by adsorption on hydrophobic adsorbents includingglass beads coated with hydrophobic materials methylatedsilica phenyl-Sepharose poly-(ethylene glycol)-Sepharosepolypropylene particles polypropylene hollow-fibers andnonwoven fabric and nitrocellulose membranes [57] Immo-bilized enzymes are used in many commercialized productsfor higher yields Lipases are active inorganic solvents andcan catalyze synthesis (esterifications) as well as the reversereaction of synthesis [68] This technique makes use ofenzymes in industriesmore attractive because it offers certainprocessing advantages over free enzyme that include ease ofseparation from the reactant and product improved stabilityand continuous operationThe porous nature of the hydrogeland particulate nature of silica or celite allow the solventand reactants as well as the product(s) to diffuse freely thisenables the substrate to interact with the enzyme easily [69]Immobilized enzymes offer some operational advantagesover soluble enzymes such as choice of batch or continuousprocesses rapid termination of reactions controlled productformation ease of removal from the reaction mixture andadaptability to various engineering designs Immobilizationis therefore often the key to improve the operational per-formance of an enzyme [70] The immobilization of lipasesdepends upon their applications For laundry detergentslipases are not used in immobilized form while synthesis offine chemicals pharmaceuticals and so forth in nonaqueousmedia and sometimes detergent formulations for slow releaseof enzyme needs immobilized lipase

51 Binding to a Carrier The enzyme can be bound to acarrier by covalent ionic or physical interactions Physicalinteractions (adsorption) are weak and enzyme can easilyleach out under the industrial conditions Immobilization oflipases by adsorption occurs through weak forces namelyvan derWaals H-bonds and hydrophobic-hydrophilic orionic interactions It is a simple economical and littletime consuming technique to prepare biocatalytic systems[68] The support used in immobilization of lipases can beinorganic solid or bio- or organic polymer Immobilizationby adsorption is the easiest and least expensive techniqueto prepare solid-support biocatalysts The weak linkagesestablished between enzyme and support (mainly van derWaals hydrogen bonds and hydrophobic interactions) havelittle effect on catalytic activity [71] Regeneration of theimmobilized biocatalyst is often possible However becausethe bonds are so weak the enzyme can easily be desorbedfrom the carrier Adsorption should not be used if enzymecannot be tolerated in the product Immobilization of lipasesby noncovalent adsorption has been shown to be very usefulin nonaqueous systems inwhich desorption can be neglectedowing to the low solubility of lipases in organic solvents[72] For this reason and due to the simplicity of adsorptionprocedure the use of adsorbed lipases is widespread for catal-ysis in water-immiscible solvents on an industrial scale [73]Physical entrapment has been employed inmany commercialcarriers for example controlled pore silica naturalsyntheticpolymers hollow fiber activated charcoal aluminum oxide


SiO2

sphere

SiO2

sphere

SiO2

sphere

SiO2

sphere

(2)

OO

O

O

O

OO

OO

N N

O

NH2

NH2

HO

HO

H

NH2 = Lipase from Mucor javanicus

Glutaraldehyde

Polymerization

H2NCH2CH2NH2

N

HN

7h 80∘C

(1) SurfactantH2O

Figure 3 Lipase immobilization on silica nanoparticle

and celite [4 74ndash76] Because of the current high cost ofsome available commercial support matrixes the possibilityof using inexpensive supports for lipase immobilization suchas rice husk [77] CaCO

3powder [78] grafted hydrogels [79ndash

83] nitrocellulose membrane [57] natural fibers [4] sol-gel matrix [84] chitosan beads [85] butyl Sepabeads [86]and activated silicacelite [83 87] has also been consideredAlthough adsorption seems to be a promising technique forlipase recovery the adsorbents are either expensive or noteasily accessible Also desorption usually involves usage ofa solution containing chaotropic agents or detergents whichleads to complexity in subsequent processing steps and alsoadds to environmental burden

Hydrogels and smart polymers such as polyvinyl alcoholhydrogels and poly-N-isopropylacrylamide (polyNIPAM)are also gaining attention as carriers (Figure 3) for immobi-lization [88]

52 Entrapment It is signified as ldquophysical trappingrdquo of theenzymes into membrane pores It is especially applicable tovery labile biomolecules like enzymes which may degrade orlose activity at extreme conditions (namely temperature pHand harsh reagents) Lipase immobilization by entrapmentis based on porosity of the membrane which retains lipaseswithin the pores and provides substrateproduct diffusion[68]

Incorporation of the enzyme into a polymer networkfor example silica [87] and sol gel during their synthesis isknown to be entrapment Polydimethylsiloxane membranessilicone elastomers microemulsion based organogels andsol-gel matrices [84] are usually employed for entrapmentof enzymes [88] Entrapment of enzymes in silica sol-gelsduring synthesis of polymer was also established [89] Thismethod involved the synthesis of sol-gel by tetraethoxysilanepolymerization (hydrolytic) Sol-gels have been extensivelyused for the lipase immobilization Entrapment can beclassified into lattice and microcapsule types Lattice-type

entrapment involves entrapping enzymes within the intersti-tial spaces of a cross-linked water-insoluble polymer Somesynthetic polymers such as polyacrylamide and polyvinylalcohol and natural polymer (starch) have been used toimmobilize enzymes using this techniqueMicrocapsule-typeentrapping involves enclosing the enzymes within semiper-meable polymer membranes This probably the less devel-oped immobilization technique is very similar to entrap-ment although in this case it is the enzyme and its wholeenvironment that are immobilized Microencapsulation cre-ates artificial cells delimited by a membrane Large moleculessuch as enzymes are not able to diffuse across the syntheticmembrane whereas small molecules for example substratesand products can pass through it [90] The preparationof enzyme microcapsules requires extremely well controlledconditions and the procedures for microencapsulation ofenzymes are liquid drying phase separation and interfacialpolymerization method

53 Cross-Linking (Carrier Free Immobilization) The thirdtype of immobilization that is cross-linking is also knownas carrier free immobilization In the last few years synthesisof carrier-free immobilized biocatalysts by cross-linking ofenzyme aggregates has appeared as a promising technique[91] It involves the use of bifunctional compound such asglutsraldehyde to cross-link the enzyme protein and resultsin the formation of crystal or aggregates There are two typesof carrier free or cross-linked enzymes cross-linked enzymecrystals and cross-linked enzyme aggregates The enzymecan be cross-linked to a polymer for improved mechanicalproperties In such cases the enzyme is first adsorbed on somesupport for example membranes followed by the cross-linking to form support based cross-linked enzyme [92] orthe entrapment of cross-linked enzyme in gel matrix

54 Cross-Linked Enzyme Crystals (CLECs) Cross-linkedenzyme crystals are achieved by the cross-linking of enzyme


crystals by using some bifunctional compound CLECsexhibit superior thermal pH and mechanical stability ascompared to simple amorphous Cross-Linked enzymesCLECs also show better stability against organic solvents[92] In the early 1990s Vertex Pharmaceuticals scientistsestablished the industrial use of CLECs and then AltusBiologics commercialized them for the synthesis of aspar-tame Controlled particle size (1ndash100 120583m) resistance to heatand organic solvents and high activities have made CLECspopular biocatalysts for chromatography or controlled releaseprotein drugs and chiral biocatalysts for asymmetric synthe-ses

55 Cross-Linked Enzyme Aggregates (CLEAs) CLEAs resultfrom the cross-linking of physical aggregates of enzymemolecules The procedure of the synthesis of CLEAs startswith the formation and precipitation of enzymatic proteinaggregates (without perturbation of their tertiary structure)caused by the addition of organic solvents non-ionic poly-mers or salts to an aqueous solutions of proteins Theformation of the physical aggregates [91] that is Cross-linkedenzyme aggregates (CLEAs) [88 93] which are obtainedby precipitation of proteins followed by crosslinking withglutaraldehyde might represent an easy alternative Cross-linked enzyme aggregates (CLEAs) have a prominent advan-tage conferring catalysts with high volume activities [94]Not only did the CLEAs of penicillin acylase have the sameactivity as the CLECs in the synthesis of ampicillin but alsothe cross-linked aggregate catalyzed the reaction in a broadrange of organic solvents However carrier-free immobiliza-tion may not be suitable for the enzyme which catalyzesthe hydrolysis or synthesis of macromolecular substrate orproduct which results from low diffusion in the narrowchannel in enzyme aggregations Moreover something lack-ing in perfection was that CLEAs are too soft and hencemay exhibit poor stability when used in stirred tanks or inpacked-bed reactors [95] If they were encapsulated in largeporous support or a very rigid poly (vinyl alcohol) networkthrough a suitable immobilization technique they would beused widely as a sturdy process biocatalyst

6 Applications

Lipases form an integral part of the industries ranging fromfood dairy pharmaceuticals agrochemical and detergentsto oleo-chemicals tea industries cosmetics leather andin several bioremediation processes [96] Because of thevast applications newer microbes are to be screened forproduction of lipases having desirable properties

61 Lipases in Food Industry Lipases have become an integralpart of the modern food industry [97] They are desirable forthe production of flavors in cheese and for interesterificationof fats and oils The lipase also accelerates the ripeningof cheese and lipolysis of butter fats and cream Lipasesfacilitate the removal of fat from meat and fish products[98] The addition of lipases releases the short-chain (C4and C6) fatty acids which give the sharp tangy flavor while

the release of medium-chain fatty acids (C12 and C14) givesthe soapy taste to the product Cocoa butter is a high valuefat that contains palmitic acid and stearic acid that hasa melting point of 37∘C (Vulfson 1994) Lipases are alsoused for the conversion of triacylglycerols to diacylglycerolsand monoacylglycerols and then these products give rise tononesterified fatty acids and fatty acid propan-2-yl estersLipases are also used as emulsifiers in food pharmaceu-ticals and cosmetics industries [99] Lipases are used forthe production of maltose and lactose like sugar fatty acidesters Some method utilizes the immobilized Rhizomucormiehei lipase for transesterification reaction that replacesthe palmitic acid in palm oil with stearic acid Immobilizedlipases from CALB Candida cylindracea AY30 HumicolalanuginosaPseudomonas sp andGeotrichum candidumwereused for the esterification of functionalized phenols for syn-thesis of lipophilic antioxidants that were used in sunfloweroil [100] Immobilized lipases from Staphylococcus warneriand Staphylococcus xylosus were used for the development offlavor ester [101] Lipases from Mucor miehei and Candidaantarctica were immobilized and used for the synthesis ofshort-chain flavored thioester C rugosa lipases have manyapplications in the food and flavor industry and in theproduction of ice cream [98]

62 Lipases in Resolution of Racemic Acids and AlcoholsLipases catalyze the hydrolysis of ester linkages in lipidswith the release of constituent alcohol and acid moieties Inpharmaceutical industries lipases are used as biocatalysts toresolve racemic alcohols and carboxylic acids through asym-metric hydrolysis and esterification [102] Stereoselectivityof lipases has been used to resolve various racemic organicacid mixtures in immiscible biphasic system [103] Racemicalcohols can also be resolved into enantiomerically pureforms by lipase-catalyzed transesterification Profens (2-arylpropionic acids) an important group of nonsteroidal anti-inflammatory drugs are pharmacologically active mainly inthe (S)-enantiomer form [104] For instance (S)-ibuprofen[(S)-2(4-isobutylphenyl) propionic acid] is 160 times morepotent than its antipode in inhibiting prostaglandin synthesisConsequently considerable effort is being made to obtainoptically pure profens through asymmetric chemical synthe-sis catalytic kinetic resolution [105] resolution of racematevia crystallization and chiral chromatographic separationsMicroorganisms and enzymes have proved particularly use-ful in resolving racemic mixtures

63 Lipases in the Detergent Industry The use of enzymesin detergent formulations is now common in developedcountries with over half of all detergents presently availablecontaining enzymes The detergent industry is the largestindustry for this enzyme [106] The use of enzymes indetergents formulations enhances the detergents ability toremove tough stains and makes the detergent environ-mentally safe Nowadays many laundry detergent productscontain cocktails of enzymes including proteases amylasescellulases and lipases [107] Lipases were developed as deter-gent enzymes after the successful introduction of proteases


in powder and liquid detergents Lipases should meet thefollowing criteria to serve as detergent additives stabilityat alkaline pH solubility in water tolerance to detergentproteases and surfactants and low substrate specificity [108109] Genecor International introduced commercial bacteriallipases namely Lipomax from Pseudomonas alcaligenes andLumafast from Pseudomonas mendocina which could beused as detergent enzymes in the year 1995 [109] Duringlaundering the lipase adsorbs onto the fabric surface toform a stable fabric-lipase complex which then acts on theoil stains and hydrolyses them The complex is resistant tothe harsh wash conditions and is retained on the fabricduring laundering [13] A detergent stable lipase was isolatedfrom Bacillus cepacia by [110] A lipase isolated from Bacilluslicheniformiswas not stable and lost its activity in the presenceof commercial detergents but its activity was restored bythe addition of calcium chloride to the enzyme-detergentcomplex [111] Such lipases lose their activities in the presenceof a chelating agent if any in the detergent

64 Microbial Lipases and Fatty Acid Ester Synthesis Theapplication of lipases in organic media is one of the mostexciting facets of biotechnology industry in recent timesLipases bring about a range of bioconversion reactions suchas hydrolysis interesterification esterification alcoholysisacidolysis and aminolysis [13 112] The concept of lipaseinterfacial activation arises from the fact that their catalyticactivity generally depends on the aggregation state of thesubstrate(s) Lipase-catalyzed condensation in an organicsolvent is useful for the syntheses of esters The fatty acidaliphatic alcohol esters and fatty acid polyol esters have beenused in many chemicals medicines cosmetics or foods bytaking advantage of their particular properties These esterscan be synthesized by condensation reaction of a fatty acidand an alcohol

A variety of fatty acid esters are nowbeing produced com-mercially using immobilized lipase in nonaqueous solvents[21 113ndash115] The esters produced from long-chain fatty acids(12ndash20 carbon atoms) and short-chain alcohols (three to eightcarbon atoms) have been used in food detergent cosmeticand pharmaceutical industries [116] Esters prepared byreaction of long-chain acids with long-chain alcohols haveimportant applications such as plasticizers and lubricants[42] Similarly alcoholic esters of short-chain fatty acids areimportant flavor and aroma compounds whereas esters oflong-chain fatty acids are being explored for their use as fuel(biodiesel) and as waxes in the oleo-chemical industries [13117 118] For these applications natural esters such as thosederived from sperm whale oil carnauba wax and jojoba oilhave been proposed However these oils are expensive andnot readily available in large amountsThus it is economicallyimportant to develop methods for the production of suchesters from cheaper and broadly available raw materials

At present many esters are industrially manufacturedby chemical methods Because chemical methods involvehigh temperature or high pressure it is difficult in manycases to esterify unstable substances such as polyunsaturatedfatty acids (PUFAs) ascorbic acid and polyols Further

the regiospecific acylation of polyols requires protectionand deprotection steps [119] These steps cause a rise inmanufacturing costs The reagents used in synthesis of food-grade esters are limited To overcome these drawbacksenzyme-catalyzed condensations using lipases have beenexploited Lipase-catalyzed condensation has advantages likemild reaction conditions one-step synthesis without protec-tion and deprotection steps and easy application to foodprocessing A lipase catalyzes a reversible reaction and thedirection and equilibrium of the reaction are determined bythe activities of the substrates and products temperature andpressure Although an enzyme-catalyzed reaction is usuallyperformed in an aqueous solution hydrolysis predominatelycauses the production of the desired product to fail when alipase-catalyzed reaction is attempted in an aqueous solutionThus reduction of water in the reaction system would beeffective for improvement in the conversion through thecondensation reaction Some lipases have catalytic activityeven in the presence of little or a small amount of water[120] Fatty acid esters of sugars and sugar alcohols findapplications as surfactantsemulsifiers in food detergentscosmetics and pharmaceutical industries (Table 3) owing totheir biodegradability and low toxicity [83 117]

65 Lipases in Textile Industry Lipases are widely used inthe textile industry to remove size lubricants and to providea fabric with more absorbency for improved levelness indyeing Lipases diminish the frequency of streaks and cracksin the denim abrasion system Lipases together with alphaamylases are used for the desizing of the denim and othercotton fabrics at the commercial scale [140] In the textileindustry polyester has important advantages such as that itincreases strength soft hand stress resistance stain resis-tance wrinkle resistance and abrasion resistance Syntheticfibres have been processed and modified by the action ofenzymes for the production of yarns fabrics textile and rugsIt relates to themodification of the characteristics of polyesterfiber as the result as such polyesters are more susceptible topostmodification treatments The use of polyesterase that isclosely related to lipase can improve the ability of polyesterfabric to uptake chemical compounds dyes antistatic com-pounds antistaining compounds antimicrobial compoundsantiperspirant compounds and deodorant compounds [140]

66 Lipases in Medical and Pharmaceutical ApplicationsLipases are used inmedical and pharmaceutical industry Forinstance enantioselective interesterification and transesteri-fication reactions by the help of lipases have great significancein pharmaceutical industry for selective acylation and dea-cylation reactions [141] The level of lipases in blood serumcan be used as a diagnostic tool for detecting conditionssuch as acute pancreatitis and pancreatic injury [2] Lipasesplay an important role in modification of monoglycerides foruse in emulsifiers in pharmaceutical applications [9] LipasesfromCandida rugosa have been used to synthesize lovastatina drug that lowers serum cholesterol level S marcescenslipase was widely used for the asymmetric hydrolysis of 3-phenylglycidic acid ester which is key intermediate in the


Table 3 Broader applications of fatty acid esters

Ester type Application(s)

Carbohydrates fatty acid esters Antitumorals [121] cosmetics [122] anticaries properties [123]and insecticidals [124]

Fatty acid esters of hydroxyl acids (lactic acid citricacid and alkyl lactates) Surfactants in food industry [125] and cosmetics [126]

Flavonoids a group of polyphenolic compounds foundubiquitously in fruits and vegetables

Broader application like dietetic nutritionalpharmacologicalcosmetic [127 128] and antioxidants [129 130]

Fatty acid esters of sugarssugar alcohol Surfactantemulsifier used in food detergent cosmetics andpharmaceutical industries [117 124]

Esters of long-chain acids with long-chain alcohols(12ndash20 carbon atoms) Plasticizers and lubricants [39]

Aminoacyl esters of carbohydrates

Sweetening agents surfactants microcapsules inpharmaceutical preparations active nucleoside amino acidesters antibiotics and in the delivery of biological active agents[131 132]

Canola phytosterols oleate Cholesterol lowering agents [133]

L-Ascorbyl linoleate Preservative crumb softening agent and inhibition of cancer[134]

FAME Crude palm oil transesterification [135]Cinnamic acid Antioxidant activity [136]Esters of gallic acid Free radical scavenger showing astringent activity [137]

Esters of ferulic acid Flavorfragrance compounds precursors of pharmaceuticalsand as additives in foods cosmetics and sunscreens [114]

Starch esters Used in the food pharmaceutical and biomedical applicationsindustries [138]

Hydroxycinnamic acids and their analogues such as4-hydroxycinnamic (119901-coumaric)34-dihydroxycinnamic (caffeic) and4-hydroxy-35-dimethoxycinnamic (sinapic) acidsincluding their medium- or long-chain alkyl esters

Antioxidant capacity particularly against oxidative attacks bytheir radical-scavenging activity [139]

synthesis of diltiazem hydrochloride [142] The lipase levelin blood serum is a diagnostic indicator for conditionssuch as acute pancreatitis and pancreatic injury [143] Lipaseactivitylevel determination is also important in the diagnosisof heart ailments [144]

67 Lipases as Biosensor A biosensor is a combination ofa biological component with a physicochemical detectorand it assists in analysis of biomolecular interactions [145]The quantitative determination of triacylglycerol is of greatimportance in clinical diagnosis and in food industryThe useof lipid sensing device as a biosensor is rather cheaper andless time consuming as compared to the chemical methodsfor the determination of triacylglycerol Biosensors can be ofthree types (a) chemical (b) biochemical or (c) electronicBiochemical biosensor utilizes the enzymes or other proteins(antibodies) cells or cell extract immobilized on a suitablematrix linked to a transducer An analytical biosensor wasdeveloped for the determination of lipids for the clinicaldiagnosis [146] Here in quantitative determination lipasesare used to generate glycerol from triacylglycerol in theanalytical sample and to quantify the released glycerol byenzymatic or chemical methods This principle enabledthe physician to diagnose the patients with cardiovascular

complaint Candida rugosa lipase biosensor has been devel-oped as a DNA probe [147]

68 Cosmetics and Personal Care Products Lipases havepotential application in cosmetics and perfumeries becausethey show activities in surfactants and in aroma production[148] Monoacyl glycerols and diacylglycerols are producedby esterification of glycerols and are used as surfactants incosmetics and perfume industries Production of flavors bytransesterification and resolution of racemic intermediatesby lipases boost the cosmetic and perfume industry Lipasesproduced by Pseudomonas cepacia have been used to resolvethe racemic rose oxides produced by the bromomethoxy-lation of citronellol [149] Methyl butyrate (MB) or methylester of butyric acid is an ester with a fruity odour ofpineapple apple and strawberry [150] Esters of aliphaticand aromatic acids and alcohols including terpene alcoholsaldehydes and phenols are commonly present in the flavormaterials used in perfumes and other personal care products[151] Retinoids (vitamin A and derivatives) are of greatcommercial potential in cosmetics and pharmaceuticals suchas skin care products Water-soluble retinol derivatives wereprepared by catalytic reaction of immobilized lipase [152]Esters of cinnamic acid ellagic acid ferulic acid and so forth


are organic compounds of biotechnological relevance thatcould be suitably modified as flavorfragrance compoundsprecursors of pharmaceuticals and as additives in foodscosmetics and sunscreens [114] Ferulic acid (4-hydroxy-3-methoxy-cinnamic acid) the most abundant derivative ofcinnamic acid is found in higher plants It is ester linked to cellwall constituents especially to arabinoxylans and lignins Ithas maximum UV absorption at 322 nm which falls betweentheUVBandUVAregion andhence can be used as a potentialUV-absorbing substance for skin protection against sunlight[113]

69 Biodiesel Production Biodiesel is an alternative fuelfor petroleum based diesel and is biodegradable renewablenoninflammable and nontoxic Biodiesel is synthesized bychemocatalytic thermocatalytic and biocatalytic approacheswhere the latter employs lipases as biocatalysts The lipasecatalysed transesterification reaction takes place between alipid and a short-chain alcohol to produce an ester andglycerol [153ndash155] The most commonly employed bacteriallipase for biodiesel synthesis is from Pseudomonas cepacia[156] Natural lipases are often rapidly inactivated by thehigh methanol concentrations used for biodiesel synthesishowever limiting their practical use The lipase from Proteusmirabilis is a particularly promising catalyst for biodieselsynthesis as it produces high yields of methyl esters even inthe presence of large amounts ofwater and expresses verywellin Escherichia coli [64]

610 Lipases in Paper Making Industry Another applicationfield of increasing importance is the use of lipase in removingthe pitch from the pulp produced in the paper industryPitch is the term used to describe collectively the hydrophiliccomponents of wood namely triglycerides and waxes whichcauses severe problems in pulp and paper manufacture [157158] The enzymatic pitch control method using lipase wasput into practice in a large-scale paper making process as aroutine operation in the early 1990s and was the first case inthe world in which an enzyme was successfully applied in theactual paper making process [159]

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] C K Sharma and S S Kanwar ldquoPurification of a novel thermo-philic lipase from B licheniformis MTCC-10498rdquo InternationalResearch Journal of Biological Science vol 1 pp 43ndash48 2012

[2] MNagar S KDwivedi andD Shrivastava ldquoA reviewon indus-trial application in microbial lipasesrdquo International Journal ofPharmaceutical and Research Sciences vol 2 pp 631ndash641 2013

[3] A Kumar and S S Kanwar ldquoLipase production in solid-statefermentation (SSF) recent developments and biotechnologicalapplicationsrdquo Dynamic Biochemistry Process Biotechnology andMolecular Biology vol 6 pp 13ndash27 2012

[4] A Kumar and S S Kanwar ldquoAn innovative approach toimmobilize lipase onto natural fiber and its application for thesynthesis of 2-octyl ferulate in an organic mediumrdquo CurrentBiotechnology vol 1 pp 241ndash248 2012

[5] S S Aulakh and R Prakash ldquoOptimization of medium andprocess parameters for the production of lipase from an oil-tolerantAspergillus sp (RBD-01)rdquo Journal of BasicMicrobiologyvol 50 no 1 pp 37ndash42 2010

[6] B Sumanjelin C S V R Rao and R S Babu ldquoIsolation char-acterization of lipase producing bacteria from crude rice branoil and optimization studies by response surface methodology(RSM)rdquo Journal of Chemical Biological and Physical Sciencesvol 3 pp 289ndash296 2013

[7] V R TembhurkarM B Kulkarni and S A Peshwe ldquoOptimiza-tion of Lipase Production by Pseudomonas spp in submergedbatch process in shake flask culturerdquo Science Research Reportervol 2 pp 46ndash50 2012

[8] R K Saxena A Sheoran B Giri andW S Davidson ldquoPurifica-tion strategies for microbial lipasesrdquo Journal of MicrobiologicalMethods vol 52 no 1 pp 1ndash18 2003

[9] R Sharma Y Chisti and U C Banerjee ldquoProduction purifica-tion characterization and applications of lipasesrdquo Biotechnol-ogy Advances vol 19 no 8 pp 627ndash662 2001

[10] C Carboni-Oerlemans P Domınguez de Marıa B Tuin GBargeman A van der Meer and R van Gemert ldquoHydrolase-catalysed synthesis of peroxycarboxylic acids biocatalyticpromiscuity for practical applicationsrdquo Journal of Biotechnologyvol 126 no 2 pp 140ndash151 2006

[11] K Hult and P Berglund ldquoEnzyme promiscuity mechanism andapplicationsrdquoTrends in Biotechnology vol 25 no 5 pp 231ndash2382007

[12] B D Ribeiro A M de Castro M A Z Coelho and D M GFreire ldquoProduction and use of lipases in bioenergy a reviewfrom the feedstocks to biodiesel productionrdquo Enzyme Researchvol 2011 Article ID 615803 16 pages 2011

[13] F Hasan A A Shah and A Hameed ldquoIndustrial applicationsof microbial lipasesrdquo Enzyme and Microbial Technology vol 39no 2 pp 235ndash251 2006

[14] R N Z R A Rahman S N Baharum M Basri and A BSalleh ldquoHigh-yield purification of an organic solvent-tolerantlipase from Pseudomonas sp strain S5rdquoAnalytical Biochemistryvol 341 no 2 pp 267ndash274 2005

[15] H Li and X Zhang ldquoCharacterization of thermostable lipasefrom thermophilicGeobacillus sp TW1rdquo Protein Expression andPurification vol 42 no 1 pp 153ndash159 2005

[16] S M Chandrasekeran and S Bhartiya ldquoSubstrate specificity oflipase in alkoxycarbonylation reaction QSAR model develop-ment and experimental validationrdquo Biotechnology and Bioengi-neering vol 30 pp 527ndash534 2009

[17] K Tamilarasan and M Dharmendira Kumar ldquoOptimizationof medium components and operating conditions for theproduction of solvent-tolerant lipase by Bacillus sphaericusMTCC 7542rdquo African Journal of Biotechnology vol 10 no 66pp 15051ndash15057 2011

[18] M Khunt and N Pandhi ldquoPurification and characterization oflipase from extreme halophiles isolated from Rann of KutchGujrat Indiardquo International Journal of Life Sciences and PharmaResearch vol 2 pp 55ndash61 2012

[19] W Gang C Guang and W Ming ldquoBiochemical propertiesand potential applications of an organic solvent-tolerant lipaseisolated from Bacillus cereus BF-3rdquo African Journal of Biotech-nology vol 10 no 61 pp 13174ndash13179 2011


[20] R Peng J Lin and D Wei ldquoSynthesis of butyl acetate inn-heptane by the recombinant CS-2 lipase immobilized onkieselguhrrdquoAfrican Journal of Food Science and Technology vol2 pp 59ndash66 2011

[21] A Kumar and S S Kanwar ldquoSynthesis of isopropyl ferulateusing silica-immobilized lipase in an organic mediumrdquo EnzymeResearch vol 2011 Article ID 718949 8 pages 2011

[22] V Ramakrishnan L C Goveas B Narayan and P M HalamildquoComparison of lipase production by Enterococcus faeciumMTCC 5695 and Pediococcus acidilactici MTCC 11361 usingfish waste as substrate optimization of culture conditions byresponse surface methodologyrdquo ISRN Biotechnology vol 2013Article ID 980562 9 pages 2013

[23] J Vakhlu and A Kour ldquoYeast lipases enzyme purificationbiochemical properties and gene cloningrdquo Electronic Journal ofBiotechnology vol 9 no 1 pp 69ndash81 2006

[24] N Souissi A Bougatef Y Triki-ellouz and M Nasri ldquoProduc-tion of lipase and biomass by Staphylococcus simulans grown onsardinella (Sardinella aurita) hydrolysates and peptonerdquoAfricanJournal of Biotechnology vol 8 no 3 pp 451ndash457 2009

[25] H Treichel D de Oliveira M A Mazutti M Di Luccio andJ V Oliveira ldquoA review on microbial lipases productionrdquo Foodand Bioprocess Technology vol 3 no 2 pp 182ndash196 2010

[26] S Ertugrul G Donmez and S Takac ldquoIsolation of lipase pro-ducing Bacillus sp from olivemill wastewater and improving itsenzyme activityrdquo Journal of Hazardous Materials vol 149 no 3pp 720ndash724 2007

[27] P E Sonnet ldquoLipase selectivitiesrdquo Journal of the American OilChemistsrsquo Society vol 65 no 6 pp 900ndash904 1988

[28] P Villeneuve ldquoPlant lipases and their applications in oilsand fats modificationrdquo European Journal of Lipid Science andTechnology vol 105 no 6 pp 308ndash317 2003

[29] H F de Castro and W A Anderson ldquoFine chemicals bybiotransformation using lipasesrdquo Quimica Nova vol 18 pp544ndash554 1995

[30] M Norin F Haeffner K Hult and O Edholm ldquoMoleculardynamics simulations of an enzyme surrounded by vacuumwater or a hydrophobic solventrdquo Biophysical Journal vol 67 no2 pp 548ndash559 1994

[31] P A Fitzpatrick A C U Steinmetz D Ringe and A MKlibanov ldquoEnzyme crystal structure in a neat organic solventrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 90 no 18 pp 8653ndash8657 1993

[32] H W Anthonsen A Baptista F Drabloslashs et al ldquoLipases andesterases a review of their sequences structure and evolutionrdquoBiotechnology Annual Review vol 1 pp 315ndash371 1995

[33] L Sarda and P Desnuelle ldquoInhibition of lipases by proteins akinetic study with dicarpin monolayersrdquo Biochimica et Biophys-ica Acta vol 30 pp 513ndash521 1958

[34] A Bose and H Keharia ldquoProduction characterization andapplications of organic solvent-tolerant lipase by Pseudomonasaeruginosa AAU2rdquo Biocatalysis and Agricultural Biotechnologyvol 2 pp 255ndash266 2013

[35] J W Shield H D Ferguson A S Bommarius and T AHatton ldquoEnzymes in reversed micelles as catalysts for organic-phase synthesis reactionsrdquo Industrial andEngineeringChemistryFundamentals vol 25 no 4 pp 603ndash612 1986

[36] M J Hernaiz J M Sanchez-Montero and J V SinisterraldquoInfluence of the nature of modifier in the enzymatic activityof chemicalmodified semipurified lipase fromCandida rugosardquoBiotechnology and Bioengineering vol 55 pp 252ndash260 1997

[37] P Speranza and G A Macedo ldquoBiochemical characterizationof highly organic solvent-tolerant cutinase from Fusariumoxysporumrdquo Biocatalysis and Agricultural Biotechnology vol 2no 4 pp 372ndash376 2013

[38] B H Cadirci and I Yasa ldquoAn organic solvents tolerant and ther-motolerant lipase from Pseudomonas fluorescens P21rdquo Journal ofMolecular Catalysis B vol 64 no 3-4 pp 155ndash161 2010

[39] N N Gandhi N S Patil S B Sawant J B Joshi P PWangikar and D Mukesh ldquoLipase-catalyzed esterificationrdquoCatalysis Reviews vol 42 no 4 pp 439ndash480 2000

[40] A M Klibanov ldquoImproving enzymes by using them in organicsolventsrdquo Nature vol 409 no 6817 pp 241ndash246 2001

[41] U T Bornscheuer C Bessler R Srinivas and S Hari KrishnaldquoOptimizing lipases and related enzymes for efficient applica-tionrdquo Trends in Biotechnology vol 20 no 10 pp 433ndash437 2002

[42] N N Gandhi S B Sawant and J B Joshi ldquoSpecificity of a lipasein ester synthesis effect of alcoholrdquo Biotechnology Progress vol11 no 3 pp 282ndash287 1995

[43] J-M Moreno M Arroyo M-J Hernaiz and J-V SinisterraldquoCovalent immobilization of pure isoenzymes from lipase ofCandida rugosardquo Enzyme and Microbial Technology vol 21 no8 pp 552ndash558 1997

[44] M Arroyo J M Sanchez-Montero and J V Sinisterra ldquoTher-mal stabilization of immobilized lipase B from Candida antarc-tica on different supports effect of water activity on enzymaticactivity in organic mediardquo Enzyme and Microbial Technologyvol 24 no 1-2 pp 3ndash12 1999

[45] T Yamane T Ichiryu M Nagata A Ueno and S ShimizuldquoIntramolecular esterification by lipase powder in microaque-ous benzene factors affecting activity of pure enzymerdquo Biotech-nology and Bioengineering vol 36 no 10 pp 1063ndash1069 1990

[46] K Dabulis and A M Klibanov ldquoDramatic enhancementof enzymatic activity in organic solvents by lyoprotectantsrdquoBiotechnology and Bioengineering vol 41 no 5 pp 566ndash5711993

[47] A Fishman S Basheer S Shatzmiller and U CoganldquoFatty-acid-modified enzymes as enantioselective catalysts inmicroaqueous organicmediardquoBiotechnology Letters vol 20 no6 pp 535ndash538 1998

[48] Y L Khmelnitsky S H Welch D S Clark and J S DordickldquoSalts dramatically enhance activity of enzymes suspended inorganic solventsrdquo Journal of the American Chemical Society vol116 no 6 pp 2647ndash2648 1994

[49] F Monot F Borzeix M Bardin and J-P VandecasteeleldquoEnzymatic esterification in organic media role of water andorganic solvent in kinetics and yield of butyl butyrate synthesisrdquoApplied Microbiology and Biotechnology vol 35 no 6 pp 759ndash765 1991

[50] S Chamorro J M Sanchez-Montero A R Alcantara andJ V Sinisterra ldquoTreatment of Candida rugosa lipase withshort-chain polar organic solvents enhances its hydrolytic andsynthetic activitiesrdquo Biotechnology Letters vol 20 no 5 pp499ndash505 1998

[51] A Zaks and A M Klibanov ldquoEnzymatic catalysis in nonaque-ous solventsrdquo Journal of Biological Chemistry vol 263 no 7 pp3194ndash3201 1988

[52] F Yang andA J Russell ldquoA comparison of lipase-catalyzed esterhydrolysis in reverse micelles organic solvents and biphasicsystemsrdquoBiotechnology andBioengineering vol 47 no 1 pp 60ndash70 1995


[53] H R Constantino K Griebenow R Langer and A MKlibanov ldquoOn the pH memory of lyophilized compoundscontaining protein functional groupsrdquo Biotechnology and Bio-engineering vol 53 pp 345ndash348 1997

[54] P PWangikar J O Rich D S Clark and J S Dordick ldquoProbingenzymic transition state hydrophobicitiesrdquo Biochemistry vol34 no 38 pp 12302ndash12310 1995

[55] Y Ito H Fujii and Y Imanishi ldquoModification of lipase withvarious synthetic polymers and their catalytic activities inorganic solventrdquo Biotechnology Progress vol 10 no 4 pp 398ndash402 1994

[56] Y Inada M Furukawa H Sasaki et al ldquoBiomedical andbiotechnological applications of PEG- and PM-modified pro-teinsrdquo Trends in Biotechnology vol 13 no 3 pp 86ndash91 1995

[57] A Kumar and S S Kanwar ldquoCatalytic potential of a nitrocel-lulose membrane-immobilized lipase in aqueous and organicmediardquo Journal of Applied Polymer Science vol 124 no 1 ppE37ndashE44 2012

[58] Q M Gu and C J Sih ldquoImproving the enantioselectivity of Ccylindracea lipase by chemicalmodificationrdquo Biocatalysis vol 6pp 115ndash126 1992

[59] M V Calvo F J Plou E Pastor and A Ballesteros ldquoEffect ofchemical modification of isoenzymes A and B from C rugosaon their activity and stabilityrdquo Biotechnology Letters vol 17 no2 pp 171ndash176 1995

[60] K-I Mogi and M Nakajima ldquoSelection of surfactant-modifiedlipases for interesterification of triglyceride and fatty acidrdquoJournal of the American Oil Chemistsrsquo Society vol 73 no 11 pp1505ndash1512 1996

[61] N Kamiya and M Goto ldquoHow is enzymatic selectivity ofmenthol esterification catalyzed by surfactant-coated lipasedetermined in organic mediardquo Biotechnology Progress vol 13no 4 pp 488ndash492 1997

[62] V M Paradkar and J S Dordick ldquoMechanism of extractionof chymotrypsin into isooctane at very low concentrations ofaerosol OT in the absence of reversed micellesrdquo Biotechnologyand Bioengineering vol 43 no 6 pp 529ndash540 1994

[63] S-Y Okazaki N Kamiya M Goto and F Nakashio ldquoEnantios-elective esterification of glycidol by surfactant-lipase complexesin organic mediardquo Biotechnology Letters vol 19 no 6 pp 541ndash543 1997

[64] T P Korman B Sahachartsiri D M Charbonneau G LHuang M Beauregard and J U Bowie1 ldquoDieselzymes devel-opment of a stable and methanol tolerant lipase for biodieselproduction by directed evolutionrdquo Biotechnology for Biofuelsvol 6 p 70 2013

[65] C Torres and C Otero ldquoInfluence of the organic solvents onthe activity in water and the conformation of Candida rugosalipase description of a lipase-activating pretreatmentrdquo Enzymeand Microbial Technology vol 19 no 8 pp 594ndash600 1996

[66] S W Tsai and J S Dordick ldquoExtraordinary enantiospecificityof lipase catalysis in organic media induced by purification andcatalyst engineeringrdquo Biotechnology and Bioengineering vol 52pp 296ndash300 1996

[67] P P Wangikar P C Michels D S Clark and J S DordickldquoStructure and function of subtilisin BPNrsquo solubilized in organicsolventsrdquo Journal of the American Chemical Society vol 119 no1 pp 70ndash76 1997

[68] S Gupta A Bhattacharya and C N Murthy ldquoTune to immo-bilize lipases on polymer membranes techniques factors andprospectsrdquo Biocatalysis and Agricultural Biotechnology vol 2pp 171ndash190 2013

[69] A Harun M Basri M B Ahmad and A B Salleh ldquoEnan-tioselective esterification reaction using immobilized Can-dida rugosa lipase on poly(N-vinyl-2-pyrrolidone-co-styrene)hydrogelrdquo Journal of Applied Polymer Science vol 92 no 5 pp3381ndash3386 2004

[70] A Idris andA Bukhari ldquoImmobilizedCandida antarctica lipaseB hydration stripping off and application in ring openingpolyester synthesisrdquo Biotechnology Advances vol 30 no 3 pp550ndash563 2012

[71] J D Andrade and V Hlady ldquoProtein adsorption and materialsbiocompatibility a tutorial review and suggested hypotehesesrdquoAdvances in Polymer Science vol 79 pp 1ndash63 1986

[72] Z Knezevic LMojovic andBAdnadjevic ldquoPalmoil hydrolysisby lipase from Candida cylindracea immobilized on zeolite typeYrdquoEnzyme andMicrobial Technology vol 22 no 4 pp 275ndash2801998

[73] F Xavier Malcata H R Reyes H S Garcia C G Hill Jr andCHAmundson ldquoImmobilized lipase reactors formodificationof fats and oilsmdasha reviewrdquo Journal of the American Oil ChemistsrsquoSociety vol 67 no 12 pp 890ndash910 1990

[74] S S Kanwar S Gehlot M L Verma R Gupta Y Kumarand G S Chauhan ldquoSynthesis of geranyl butyrate with thepoly(acrylic acid-co-hydroxy propyl methacrylate-cl-ethyleneglycol dimethacrylate) hydrogel immobilized lipase of Pseu-domonas aeruginosa MTCC-4713rdquo Journal of Applied PolymerScience vol 110 no 5 pp 2681ndash2692 2008

[75] S S Kanwar C Sharma M L Verma S Chauhan S SChimni and G S Chauhan ldquoShort-chain ester synthesis bytransesterification employing poly (MAc-co-DMA-cl-MBAm)hydrogel-bound lipase of Bacillus coagulons MTCC-6375rdquoJournal of Applied Polymer Science vol 109 no 2 pp 1063ndash10712008

[76] S Sharma and S S Kanwar ldquoProperties and stability ofcelite immobilized lipase of a thermophilic Bacillus sp STL-7rdquoInternational Journal of Biochemistry vol 108 pp 173ndash182 2013

[77] J Tantrakulsiri N Jeyashoke and K Krisanangkura ldquoUtiliza-tion of rice hull ash as a support material for immobilizationof Candida cylindracea lipaserdquo Journal of the American OilChemistsrsquo Society vol 74 no 2 pp 173ndash175 1997

[78] R Rosu Y Iwasaki N Shimizu N Doisaki and T YamaneldquoIntensification of lipase performance in a transesterificationreaction by immobilization on CaCO

3powderrdquo Journal of

Biotechnology vol 66 no 1 pp 51ndash59 1998[79] M Basri A Harun M B Ahmad C N A Razak and

A B Salleh ldquoImmobilization of lipase on poly(N-vinyl-2-pyrrolidone-co-styrene) hydrogelrdquo Journal of Applied PolymerScience vol 82 no 6 pp 1404ndash1409 2001

[80] S S Kanwar R K Kaushal A Jawed and S S ChimnildquoEvaluation of methods to inhibit lipase in colorimetric assayemploying p-nitrophenyl palmitaterdquo Indian Journal of Biochem-istry and Biophysics vol 42 pp 233ndash237 2005

[81] S S Kanwar R K Kaushal M L Verma et al ldquoSynthesisof ethyl laurate by hydrogel immobilized lipase of BacilluscoagulansMTCC-6375rdquo Indian Journal of Microbiology vol 45no 3 pp 187ndash193 2005

[82] S S KanwarHKVerma R KKaushal et al ldquoEffect of solventsand kinetic parameters on synthesis of ethyl propionate catal-ysed by poly (AAc-co-HPMA-cl-MBAm)-matrix-immobilizedlipase of Pseudomonas aeruginosa BTS-2rdquo World Journal ofMicrobiology and Biotechnology vol 21 no 6-7 pp 1037ndash10442005


[83] M L Verma W Azmi and S S Kanwar ldquoSynthesis of ethylacetate employing celite-immobilized lipase of Bacillus cereusMTCC 8372rdquo Acta Microbiologica et Immunologica Hungaricavol 56 no 3 pp 229ndash242 2009

[84] N B Carvalho J M P Barbosa M V S Oliveira A T Fricksand A S L C M F Soares ldquoBiochemical properties of Bacillussp itp-001 lipase immobilized with a sol gel processrdquo QuimicaNova vol 36 pp 52ndash58 2013

[85] E C Egwim A A Adesina O A Oyewole and I N OkoliegbeldquoOptimization of lipase immobilized on chitosan beads forbiodiesel productionrdquo Global Research Journal of Microbiologyvol 2 pp 103ndash112 2012

[86] R V Branco M L E Gutarra D M G Freire and R VAlmeida ldquoImmobilization and characterization of a recom-binant thermostable lipase (Pf2001) from Pyrococcus furiosuson supports with different degrees of hydrophobicityrdquo EnzymeResearch vol 2010 Article ID 180418 8 pages 2010

[87] A Kumar V Sharma P Sharma and S S Kanwar ldquoEffectiveimmobilisation of lipase to enhance esterification potential andreusabilityrdquo Chemical Papers vol 67 pp 696ndash702 2013

[88] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis and Catalysis vol 349 no 8-9 pp 1289ndash1307 2007

[89] D Avnir S Braun O Lev and M Ottolenghi ldquoEnzymes andother proteins entrapped in sol-gel materialsrdquo Chemistry ofMaterials vol 6 no 10 pp 1605ndash1614 1994

[90] P Villeneuve J M Muderhwa J Graille and M J Haas ldquoCus-tomizing lipases for biocatalysis a survey of chemical physicaland molecular biological approachesrdquo Journal of MolecularCatalysis B vol 9 no 4ndash6 pp 113ndash148 2000

[91] M P G Torres M L Foresti and M L Ferreira ldquoCross linkedenzyme aggregates (CLEAs) of selected lipases a procedure forthe proper calculation of their recovered activityrdquoAMBExpressvol 3 p 25 2013

[92] L Cao L van Langen and R A Sheldon ldquoImmobilisedenzymes carrier-bound or carrier-freerdquo Current Opinion inBiotechnology vol 14 no 4 pp 387ndash394 2003

[93] L Cao F Van Rantwijk and R A Sheldon ldquoCross-linkedenzyme aggregates a simple and effective method for theimmobilization of penicillin acylaserdquoOrganic Letters vol 2 no10 pp 1361ndash1364 2000

[94] L Chen Y D Hu N Li and M H Zong ldquoCross-linkedenzyme aggregates of b-glucosidase from Prunus domesticaseedsrdquo Biotechnol Letters vol 34 pp 1673ndash1678 2012

[95] J J Roy and T E Abraham ldquoStrategies in making cross-linkedenzyme crystalsrdquo Chemical Reviews vol 104 no 9 pp 3705ndash3721 2004

[96] K J Patil M Z Chopda and R T Mahajan ldquoLipase biodiver-sityrdquo Indian Journal of Science and Technology vol 4 pp 971ndash982 2011

[97] F Theil ldquoLipase-supported synthesis of biologically activecompoundsrdquo Chemical Reviews vol 95 no 6 pp 2203ndash22271995

[98] A Ray ldquoApplication of lipase in industryrdquo Asian Journal ofPharmacy and Technology vol 2 pp 33ndash37 2012

[99] H P Fleming ldquoMixed cultures in vegetable fermentationsrdquo inMixed Cultures in Biotechnology J G Zeikus and E A JohnsonEds pp 69ndash103 McGrawHill New York NY USA 1991

[100] HXuM Li andBHe ldquoImmobilization ofCandida cylindracealipase on methyl acrylate-divinyl benzene copolymer and itsderivativesrdquo Enzyme andMicrobial Technology vol 17 no 3 pp194ndash199 1995

[101] R Talon N Dublet M-C Montel and M Cantonnet ldquoPurifi-cation and characterization of extracellular Staphylococcuswarneri lipaserdquo Current Microbiology vol 30 no 1 pp 11ndash161995

[102] S Abraham N R Kamini and M K Gowthaman ldquoProcessstrategies for alkaline lipase production using Aspergillus NigerMTCC 2594rdquo Journal of Applied Pharmacy vol 1 pp 34ndash1152011

[103] A M Klibanov ldquoAsymmetric transformations catalyzed byenzymes in organic solventsrdquo Accounts of Chemical Researchvol 23 no 4 pp 114ndash120 1990

[104] A J Hutt and J Caldwell ldquoThe importance of stereochemistryin the clinical pharmacokinetics of the 2-arylpropionic acidnon-steroidal anti-inflammatory drugsrdquo Clinical Pharmacoki-netics vol 9 no 4 pp 371ndash373 1984

[105] S M O Van Dyck G L F Lemiere T H M Jonckers RDommisse L Pieters and V Buss ldquoKinetic resolution of adihydrobenzofuran-type neolignan by lipase-catalysed acetyla-tionrdquo Tetrahedron Asymmetry vol 12 no 5 pp 785ndash789 2001

[106] D H Tambekar S P Mundekar and V B Bombode ldquoPartialcharacterization and optimization of lipase production fromBacillus cereus isolated from haloalkaliphilic lonar lakerdquo Inter-national Journal of Life Sciences Biotechnology and PharmaResearch vol 2 pp 249ndash257 2013

[107] J H Jeon J-T Kim Y J Kim et al ldquoCloning and character-ization of a new cold-active lipase from a deep-sea sedimentmetagenomerdquo Applied Microbiology and Biotechnology vol 81no 5 pp 865ndash874 2009

[108] W J Quax WJ ldquoBacterial enzymesrdquo in The Prokaryotic Sym-biotic Associations Biotechnology Applied Microbiology MDworkin and S Falkow Eds pp 777ndash796 Springer New YorkNY USA 2006

[109] R N Z R A Rahman A B Salleh and M Basri ldquoLipasesintroductionrdquo in New Lipases and Proteases A B Salleh R NZ R A Rahman and M Basri Eds pp 1ndash22 Nova ScienceNew York NY USA 2006

[110] P Rathi R K Saxena and R Gupta ldquoA novel alkaline lipasefrom Burkholderia cepacia for detergent formulationrdquo ProcessBiochemistry vol 37 no 2 pp 187ndash192 2001

[111] R A Beyoumi S S El-louboudeyNM Sidkey andMAAbd-El-Rahman ldquoProduction purification and characterizationof thermoalkalophilic liapse for application in bio-detergentindustryrdquo Journal of Applied Sciences Research vol 3 pp 1752ndash1765 2007

[112] B Rajesh and I Bhaskar Reddy ldquoLipase from organic solventtolerant Bacillus strain C5 isolation and identificationrdquo Inter-national Journal of Scientific Research vol 2 pp 26ndash28 2013

[113] A Kumar and S S Kanwar ldquoSynthesis of ethyl ferulate inorganic medium using celite-immobilized lipaserdquo BioresourceTechnology vol 102 no 3 pp 2162ndash2167 2011

[114] C Chandel A Kumar and S S Kanwar ldquoEnzymatic synthesisof butyl ferulate by silica-immobilized lipase in a non-aqueousmediumrdquo Journal of Biomaterials andNanobiotechnology vol 2pp 400ndash408 2011

[115] H S Ruela F K Sutili I C R Leal N M F Carvalho L S MMiranda and R O M A de Souza ldquoLipase-catalyzed synthesisof secondary glucose esters under continuous flow conditionsrdquoEuropean Journal of Lipid Science and Technology vol 115 pp464ndash467 2013

[116] A R Bauer D Garbe and H Surgurg In Common Fragranceand Flavour Material VCH New York NY USA 2nd edition1990


[117] R K Saxena P K Ghosh R Gupta W S Davidson S Bradooand R Gulati ldquoMicrobial lipases potential biocatalysts for thefuture industryrdquo Current Science vol 77 no 1 pp 101ndash115 1999

[118] A Pandey S Benjamin C R Soccol P Nigam N Krieger andV T Soccol ldquoThe realm of microbial lipases in biotechnologyrdquoBiotechnology and Applied Biochemistry vol 29 no 2 pp 119ndash131 1999

[119] J A Arcos M Bernabe and C Otero ldquoQuantitative enzymaticproduction of 6-O-acylglucose estersrdquo Biotechnology and Bio-engineering vol 57 pp 505ndash509 1998

[120] G Kirchner M P Scollar and A M Klibanov ldquoResolutionof racemic mixtures via lipase catalysis in organic solventsrdquoJournal of the American Chemical Society vol 107 no 24 pp7072ndash7076 1985

[121] S Okabe M Suganuma Y Tada et al ldquoDisaccharide estersscreened for inhibition of tumor necrosis factor-120572 release arenew anti-cancer agentsrdquo Japanese Journal of Cancer Researchvol 90 no 6 pp 669ndash676 1999

[122] Y Watanabe S Adachi K Nakanishi and R Matsuno ldquoCon-densation of L-ascorbic acid and medium-chain fatty acids byimmobilized lipase in acetonitrile with lowwater contentrdquo FoodScience and Technology Research vol 5 no 2 pp 188ndash192 1999

[123] K S Devulapalle A Gomez De Segura M Ferrer M AlcaldeGMooser and F J Plou ldquoEffect of carbohydrate fatty acid esterson Streptococcus sobrinus and glucosyltransferase activityrdquo Car-bohydrate Research vol 339 no 6 pp 1029ndash1034 2004

[124] Z Song S Li X Chen L Liu and Z Song ldquoSynthesis ofinsecticidal sucrose estersrdquo Forestry Studies in China vol 8 pp26ndash29 2006

[125] A J Angello and J R Vercellotti ldquoPhospholipids and fatty acidesters of alcoholsrdquo in Food Emulsifier Chemistry TechnologyFunctional Properties and Applications G Charalambous andG Doxastakis Eds Elsevier Amsterdam The Netherlands1989

[126] S-W Chang J-F Shaw K-H Yang I-L Shih C-H Hsiehand C-J Shieh ldquoOptimal lipase-catalyzed formation of hexyllauraterdquo Green Chemistry vol 7 no 7 pp 547ndash551 2005

[127] J B Harborne and C A Williams ldquoAdvances in flavonoidresearch since 1992rdquo Phytochemistry vol 55 no 6 pp 481ndash5042000

[128] T Guardia A E Rotelli A O Juarez and L E Pelzer ldquoAnti-inflammatory properties of plant flavonoids Effects of rutinquercetin and hesperidin on adjuvant arthritis in ratrdquo Farmacovol 56 no 9 pp 683ndash687 2001

[129] G Cao E Sofic and R L Prior ldquoAntioxidant and prooxidantbehavior of flavonoids structure-activity relationshipsrdquo FreeRadical Biology and Medicine vol 22 no 5 pp 749ndash760 1997

[130] B H Havsteen ldquoThe biochemistry and medical significance ofthe flavonoidsrdquo Pharmacology andTherapeutics vol 96 no 2-3pp 67ndash202 2002

[131] M Tamura M Shoji T Nakatsuka K Kinomura H Okaiand S Fukui ldquoMethyl 2 3-di- (Lmdash120572-amimobutyryl)-120572-D-glucopyranoside a sweet substance and tastes of related com-pounds of neutral amino acids and d-glucose derivativesrdquoAgricultural and Biological Chemistry vol 49 pp 2579ndash25861985

[132] O Kirk F Bjorkling S E Godfredsen and T S Larsen ldquoFattyacid specificity in lipase catalyzed synthesis of glucoside estersrdquoBiocatalysis vol 6 pp 127ndash134 1992

[133] P Villeneuve F Turon Y Caro et al ldquoLipase-catalyzed synthe-sis of canola phytosterols oleate esters as cholesterol lowering

agentsrdquoEnzyme andMicrobial Technology vol 37 no 1 pp 150ndash155 2005

[134] T Luhong Z Hao M M Shehate and S Yunfei ldquoA kineticstudy of the synthesis of ascorbate fatty acid esters catalysedby immobilized lipase in organic mediardquo Biotechnology andApplied Biochemistry vol 32 no 1 pp 35ndash39 2000

[135] J H Sim G K Khor A H Kamaruddin and S BhatialdquoThermodynamic studies on activity and stability of immobi-lized Thermomyces lanuginosus in producing fatty acid methylester (FAME)rdquo International Journal of Scientific and ResearchPublications vol 3 pp 2250ndash3153 2013

[136] C K Sharma G S Chauhan and S S Kanwar ldquoSynthesis ofmedically important ethyl cinnamate ester by porcine pancre-atic lipase immobilized on poly(AAc-co-HPMA-cl-EGDMA)hydrogelrdquo Journal of Applied Polymer Science vol 121 no 5 pp2674ndash2679 2011

[137] V Leela and A Saraswathy ldquoQuantification of pharmacologi-cally activemarkers gallic acid quercetin and lupeol fromacacialeucophloea wild flowers by HPTLC Methodrdquo Analytical andBioanalytical Techniques vol 4 160 2013

[138] J Y Xin Y Wang T Liu K Lin L Chang and C G XialdquoBiosysthesis of corn starch palmitate by lipase novozym 435rdquoInternational Journal of Molecular Sciences vol 13 pp 7226ndash7236 2012

[139] N N L Castillo A D R Rodrıguez B M Porta and MJ Cruz-Gomez ldquoProcess for the obtention of coumaric acidfrom coumarin analysis of the reaction conditionsrdquo Advancesin Chemical Engineering and Science vol 3 pp 195ndash201 2013

[140] H D Rowe ldquoBiotechnology in the textileclothing industry areviewrdquo Journal of Consumer Studies and Home Economics vol23 pp 53ndash61 2001

[141] S C Stinson ldquoFine and intermediate chemicals makers empha-size new products and processesrdquo Chemical and EngineeringNews vol 73 no 29 pp 10ndash26 1995

[142] H Matsumae M Furui and T Shibatani ldquoLipase-catalyzedasymmetric hydrolysis of 3-phenylglycidic acid ester the keyintermediate in the synthesis of diltiazem hydrochloriderdquo Jour-nal of Fermentation and Bioengineering vol 75 no 2 pp 93ndash981993

[143] A L Gutman K Zuobi and T Bravdo ldquoLipase-catalyzedpreparation of optically active 120574-butyrolactones in organicsolventsrdquo Journal of Organic Chemistry vol 55 no 11 pp 3546ndash3552 1990

[144] J D Schnatz J W Ormsby and R H Williams ldquoLipoproteinlipase activity in human heartrdquo The American Journal of Physi-ology vol 205 pp 401ndash404 1963

[145] S C B Gopinath P Anbu T Lakshmipriya and A HildaldquoStrategies to characterize fungal lipases for applications inmedicine and dairy industryrdquo BioMed Research Internationalvol 2013 Article ID 154549 10 pages 2013

[146] A Masahiko K Masahiro K Takasi M Kenji and M AyarildquoProcess for preparation of polyol fatty acid ester and glyceridemixture obtainedrdquo European Patent EP-658629 1995

[147] S Benjamin and A Pandey ldquoIsolation and characterization ofthree distinct forms of lipases from Candida rugosa producedin solid state fermentationrdquo Brazilian Archives of Biology andTechnology vol 44 no 2 pp 213ndash221 2001

[148] J O Metzger and U Bornscheuer ldquoLipids as renewableresources current state of chemical and biotechnological con-version and diversificationrdquo Applied Microbiology and Biotech-nology vol 71 no 1 pp 13ndash22 2006


[149] S C Taneja V K Sethi S S Andotra S Koul and G N QazildquoRose oxides a facile chemo and chemo-enzymatic approachrdquoSynthetic Communications vol 35 no 17 pp 2297ndash2303 2005

[150] V K Garlapati and R Banerjee ldquoSolvent-free synthesis offlavour esters through immobilized lipasemediated transesteri-ficationrdquo Enzyme Research vol 2013 Article ID 367410 6 pages2013

[151] M C R Franssen L Alessandrini and G Terraneo ldquoBiocat-alytic production of flavors and fragrancesrdquo Pure and AppliedChemistry vol 77 no 1 pp 273ndash279 2005

[152] T Maugard B Rejasse and M D Legoy ldquoSynthesis of water-soluble retinol derivatives by enzymatic methodrdquo BiotechnologyProgress vol 18 no 3 pp 424ndash428 2002

[153] Y Chen B Xiao J Chang Y Fu P Lv and X Wang ldquoSynthesisof biodiesel from waste cooking oil using immobilized lipase infixed bed reactorrdquo Energy Conversion and Management vol 50no 3 pp 668ndash673 2009

[154] N Dizge C Aydiner D Y ImerM Bayramoglu A Tanrisevenand B Keskinler ldquoBiodiesel production from sunflower soy-bean and waste cooking oils by transesterification using lipaseimmobilized onto a novel microporous polymerrdquo BioresourceTechnology vol 100 no 6 pp 1983ndash1991 2009

[155] M Raita V Champreda and N Laosiripojana ldquoBiocatalyticethanolysis of palm oil for biodiesel production using micro-crystalline lipase in tert-butanol systemrdquo Process Biochemistryvol 45 no 6 pp 829ndash834 2010

[156] Q Li and Y Yan ldquoProduction of biodiesel catalyzed by immobi-lized Pseudomonas cepacia lipase from Sapium sebiferum oil inmicro-aqueous phaserdquo Applied Energy vol 87 no 10 pp 3148ndash3154 2010

[157] R L Farell K Hata and M B Wall ldquoSolving pich problemsin pulp and paper processes by the use of enzymes or fungirdquoAdvances in Biochemical EngineeringBiotechnology vol 57 pp197ndash212 1997

[158] A Gutierrez J C Del Rıo and A T Martınez ldquoMicrobial andenzymatic control of pitch in the pulp and paper industryrdquoAppliedMicrobiology and Biotechnology vol 82 no 6 pp 1005ndash1018 2009

[159] P Bajpai ldquoApplication of enzymes in the pulp and paperindustryrdquo Biotechnology Progress vol 15 no 2 pp 147ndash157 1999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


COOH

Serine

Glycosylated area

Hydrophobic batch

Substrate

Water-oil interfaceH2N

H3C

Figure 1 Diagrammatic representation of a lipase molecule show-ing its main features

Fundamental studies on polymerization revealed someremarkable capabilities of lipases for polymerization chem-istry The polymerization and transesterification studies gen-erally demand harsh condition(s) of presence of organicsolvents andor high temperature [17 18] The technolog-ical utility of enzymes can be enhanced greatly by usingthem in organic solvents rather than their natural aqueousreaction media [19] The use of enzyme in organic mediahas exhibited many advantages increased activity and sta-bility regiospecificity and stereoselectivity higher solubilityof substrate ease of products recovery and ability to shiftthe reaction equilibrium toward synthetic direction [20]From the biotechnological point of view there are numerousadvantages of conducting immobilized biocatalyst-promotedenzymatic conversions in organic solvents as opposed toreactions performed in water-based system(s) [21]

2 Sources of Lipases

Lipases are produced by plants animals and microbes butonly microbial lipases are found to be industrially importantsince they are diversified in their enzymatic properties andsubstrate specificity [22] Lipases can be obtained fromanimals mainly from fore-stomach tissue of calves or lambsand pancreatic tissues of pigs The disadvantages of usinganimal lipases include presence of trypsin in pig pancreaticlipases which results in bitter tasting amino acids andpresence of residual animal hormones or viruses as well astheir undesirable effects in the processing of vegetarian orkosher diets [23] Plant lipases are also available but notexploited commercially because of the yield and the processesinvolvedThusmicrobial lipases are currently receivingmoreattention because of their technical and economic advantageswhere the organisms are cultivated in medium containingappropriate nutrient composition under controlled condi-tions Also lipase production by microorganisms variesaccording to the strains the composition of the growthmedium cultivation conditions pH temperature and thekind of carbon and nitrogen sources [24 25] Generallybacteria fungi yeast and actinomycetes are recognized as

Triglyceride

H2O plusmn

H2O plusmn

H2O plusmn

Diglyceride + fatty acid

Monoglyceride + fatty acid

Fatty acid + glycerol

Figure 2 The mediated lipase reaction(s)

preferred sources of extracellular lipases facilitating theenzyme recovery from the culture broth although CandidaPseudomonas Mucor Rhizopus and Geotrichum spp standout as the major commercially viable strains [26] Bacteriallipases are extensively used in food industry for qualityimprovement dairy industry for hydrolysis ofmilk fat cheeseripening beverages to improve aroma and health foodsfor transesterification [6] Many microorganisms have beenreported in the last decade for lipase production in bothsubmerged and solid-state fermentations reported (Table 1)

3 Reactions Catalyzed by Lipases

The lipases catalyze a wide range of reactions includinghydrolysis interesterification alcoholysis acidolysis esterifi-cation and aminolysis They catalyse the hydrolysis of fattyacid ester bond in the triacylglycerol and release free fattyacids The lipase-catalyzed reactions are

(i) Hydrolysis

RCOOR1015840 +H2O 997888rarr RCOOH + R1015840OH (1)

(ii) Synthesis Reactions under this category can be fur-ther separated into the following categories

(a) Esterification

RCOOH + R1015840OH 997888rarr RCOOR1015840 +H2O (2)

(b) Interesterification

RCOOR1015840 + R10158401015840COORlowast 997888rarr RCOORlowast + R10158401015840COOR1015840 (3)

(c) Alcoholysis

RCOOR1015840 + R10158401015840OH 997888rarr RCOOR10158401015840 + R1015840OH (4)

(d) Acidolysis

RCOOR1015840 + R10158401015840COOH 997888rarr R10158401015840COOR1015840 + RCOOH (5)

































SiO2

sphere

SiO2

sphere

SiO2

sphere

SiO2

sphere

(2)

OO

O

O

O

OO

OO

N N

O

NH2

NH2

HO

HO

H


Glutaraldehyde

Polymerization

H2NCH2CH2NH2

N

HN

7h 80∘C

(1) SurfactantH2O














6 Applications










































References















































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































SiO2

sphere

SiO2

sphere

SiO2

sphere

SiO2

sphere

(2)

OO

O

O

O

OO

OO

N N

O

NH2

NH2

HO

HO

H


Glutaraldehyde

Polymerization

H2NCH2CH2NH2

N

HN

7h 80∘C

(1) SurfactantH2O














6 Applications










































References















































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















SiO2

sphere

SiO2

sphere

SiO2

sphere

SiO2

sphere

(2)

OO

O

O

O

OO

OO

N N

O

NH2

NH2

HO

HO

H


Glutaraldehyde

Polymerization

H2NCH2CH2NH2

N

HN

7h 80∘C

(1) SurfactantH2O














6 Applications










































References















































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










SiO2

sphere

SiO2

sphere

SiO2

sphere

SiO2

sphere

(2)

OO

O

O

O

OO

OO

N N

O

NH2

NH2

HO

HO

H


Glutaraldehyde

Polymerization

H2NCH2CH2NH2

N

HN

7h 80∘C

(1) SurfactantH2O














6 Applications










































References















































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


SiO2

sphere

SiO2

sphere

SiO2

sphere

SiO2

sphere

(2)

OO

O

O

O

OO

OO

N N

O

NH2

NH2

HO

HO

H


Glutaraldehyde

Polymerization

H2NCH2CH2NH2

N

HN

7h 80∘C

(1) SurfactantH2O














6 Applications










































References















































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




6 Applications










































References















































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































References















































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























References















































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







References















































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Review Article Organic Solvent Tolerant Lipases and ...downloads.hindawi.com/journals/tswj/2014/625258.pdf · bonds. e esteric ation reactions are always carried out in nonaqueous