cold active microbial lipases: some hot issues and recent developments

Biotechnology Advances 26 (2008) 457–470

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Research review paper

Cold active microbial lipases: Some hot issues and recent developments

Babu Joseph a,⁎, Pramod W. Ramteke a, George Thomas b

a Department of Microbiology and Microbial Technology, College of Biotechnology and Allied Sciences, Allahabad Agricultural Institute-Deemed University,Allahabad 211 007, Uttar Pradesh, Indiab Department of Molecular Biology and Genetic Engineering, College of Biotechnology and Allied Sciences, Allahabad Agricultural Institute-Deemed University,Allahabad 211 007, Uttar Pradesh, India

⁎ Corresponding author. Tel.: +91 532 2684296; fax: +E-mail address: [email protected] (B. Joseph).

0734-9750/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.biotechadv.2008.05.003

A B S T R A C T
A R T I C L E I N F O
Article history:
Lipases are glycerol ester h Received 6 November 2007Accepted 9 May 2008Available online 18 May 2008
Keywords:BiocatalystsCold active lipaseEnzymesIndustrial applicationLipolyticPsychrophiles

ydrolases that catalyze the hydrolysis of triglycerides to free fatty acids andglycerol. Lipases catalyze esterification, interesterification, acidolysis, alcoholysis and aminolysis in additionto the hydrolytic activity on triglycerides. The temperature stability of lipases has regarded as the mostimportant characteristic for use in industry. Psychrophilic lipases have lately attracted attention because oftheir increasing use in the organic synthesis of chiral intermediates due to their low optimum temperatureand high activity at very low temperatures, which are favorable properties for the production of relativelyfrail compounds. In addition, these enzymes have an advantage under low water conditions due to theirinherent greater flexibility, wherein the activity of mesophilic and thermophilic enzymes are severelyimpaired by an excess of rigidity. Cold-adapted microorganisms are potential source of cold-active lipasesand they have been isolated from cold regions and studied. Compared to other lipases, relatively smallernumbers of cold active bacterial lipases were well studied. Lipases isolated from different sources have a widerange of properties depending on their sources with respect to positional specificity, fatty acid specificity,thermostability, pH optimum, etc. Use of industrial enzymes allows the technologist to develop processesthat closely approach the gentle, efficient processes in nature. Some of these processes using cold activelipase from C. antarctica have been patented by pharmaceutical, chemical and food industries. Cold activelipases cover a broad spectrum of biotechnological applications like additives in detergents, additives in foodindustries, environmental bioremediations, biotransformation, molecular biology applications and hetero-logous gene expression in psychrophilic hosts to prevent formation of inclusion bodies. Cold active enzymesfrom psychrotrophic microorganisms showing high catalytic activity at low temperatures can be highlyexpressed in such recombinant strains. Thus, cold active lipases are today the enzymes of choice for organicchemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, microbiologistsand biochemists.

© 2008 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4582. Cold active lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4583. Structural features of lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458

3.1. General lipase structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4583.2. Modifications of lipase structure for cold adaptation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4593.3. Structure of Candida antarctica lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

4. Production of cold active lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4594.1. Psychrophiles as sources of cold active lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4594.2. Fermentation conditions for cold active lipase production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4604.3. Purification and characterization of cold active lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

5. Biotechnological approaches in cold active lipase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4625.1. Gene cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4635.2. Protein engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464

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458 B. Joseph et al. / Biotechnology Advances 26 (2008) 457–470

6. Industrial applications of cold active lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4646.1. Medical and pharmaceutical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4656.2. Synthesis of fine chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4656.3. Applications in food industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4666.4. Domestic applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4666.5. Environmental application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4666.6. Patents in cold active lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

7. Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

1. Introduction

Lipasebelongs to theenzymeclass ofhydrolases (E.C.3). It acts onesterbonds (E.C.3.1) of carboxylic esters (E.C.3.1.1). They hydrolyze triacylgly-cerols to fatty acids, diacylglycerol, monoacylglycerol, and glycerol(Carriere et al., 1994) and known as triacylglycerol acyl hydrolases(E.C.3.1.1.3). Lipids constitute a large part of earth's biomass, and lipolyticenzymes play an important role in the turnover of these water insolublecompounds. Lipases break and or modify the carboxyl ester bonds oflipids and its derivatives. Hydrolysis of fat is the primary reaction oflipases (Khare et al., 2000). Lipolytic enzymes are involved in breakdownand thus in the mobilization of lipids within cells of individual organismas well as in the transfer of lipids from one organism to another (Beissonet al., 2000). Lipases catalyze esterification, interesterification, acidolysis,alcoholysis and aminolysis in addition to the hydrolytic activity on trigly-cerides. As hydrolases, they do not require cofactors. They usually exhibitgood chemioselectivity, regioselectivity and enantioselectivity. Finally,lipases possess broad substrate specificity and found with optimumactivities over awide range of temperatures. These interesting propertiesmake lipases the most versatile biocatalyst (Kademi et al., 2005).

Lipases have emerged as one of the leading bio-catalyst/bio-ac-celerators with proven potential for contributing to the multibilliondollar under exploited lip-tech bio-industry and are used both in situlipid metabolism and ex situ multi-faceted industrial application(Benjamin and Pandey, 1998; Pandey et al., 1999). Few reports revealthat lipases have emerged as key enzymes in swiftly growing bio-technology, owing to their multi-faceted properties, which find usage ina wide array of industrial applications, such as food technology, deter-gent, chemical industry and biomedical sciences (Jaeger et al., 1999;Pandey et al., 1999). The commercial use of lipases is a billion dollarbusiness that comprises awidevarietyof differentapplications includingsynthesis of biopolymer and biodiesel, production of pharmaceuticals,agrochemicals, cosmetics and flavors (Haki and Rakshit, 2003). Theglobalmarket for industrial enzymeswas estimated nearly US $ 2 billionin 2004. As a result, the market is expected to rise at an average annualgrowth rate (AAGR) of a little over 3% over the next four years, and thetotal industrial enzymemarket in 2009 is expected to reach nearly US$ 2.4 billion (Rajan, 2004; Hasan et al., 2006). Through the number ofreview articles (Sharma et al., 2001; Gupta et al., 2004; Akai and Kita,2007), importance of lipases can be easily envisioned. Over the last fewyears, there has been a dramatic increase in the number of publicationsin the field of lipase-catalyzed reactions performed in common organicsolvents, ionic liquids or even non-conventional solvents. Considerableresearch has shown that reactions catalyzed by enzymes are moreselective and efficiently performed than many of their analogues in theorganic chemistry laboratory (Ghanem, 2007). The present reviewadopts a brief consideration on the structural modification, source,production and industrial applications of cold active lipases.

2. Cold active lipases

Cold-adapted lipolytic microorganisms produce lipases, whichfunction effectively at cold temperatures with high rates of catalysis in

comparison to the lipases from mesophiles or thermopiles, whichshows little or no activity at low temperature. These lipases haveevolved a range of structural features that confer a high level offlexibility, particularly around the active site are translated into lowactivation enthalpy, low-substrate affinity, and high specific activity atlow temperatures. Moreover, the maximum level of activity of theselipases is shifted towards lower temperatures with a concomitantdecrease in thermal stability. The knowledge of cold active lipolyticenzymes is increasing at a rapid and exciting rate. Unfortunately, thestudies on cold active lipases are incomplete and scattered. Till date,no attempts have been undertaken to organize this information.Hence, an overview of this biotechnologically and industrially im-portant enzyme and its characteristics has been collected and com-piled from the information available in the literature. From the limitednumber of available reports on cold active lipases, it is clear that mostof the studies were focused on isolation, purification and character-ization of these enzymes followed by gene cloning, expression andsequencing. The genes encoding for cold active lipases were isolatedand cloned into mesophilic bacteria (E. coli) as host organism and usedfor their expression. However, the review of Gerday et al. (1997)revealed an extremely unstable condition for the expression of cold-adapted lipases within their host (Feller et al., 1990, 1991a). In otherreports related to expression studies, the stability of gene encodinglipase production in the host is not clear. A worldwide initiative hastaken up for exploring cold active lipase producing microorganismsand their industrial applications.

3. Structural features of lipase

3.1. General lipase structure

The lipase consists of a single domain molecule and all lipasesconform to a common structural organization, viz., the alpha/betahydrolase fold (Ollis et al., 1992; Nardini and Dijkstra, 1999). The activesite of lipase contains the catalytic triad, Ser105-His224-Asp187,common to all serine hydrolases where Ser as the nucleophile, His asthe basic residue, and Asp or Glu as the acidic residue (Uppenberget al., 1994a,b). Such a catalytic triad exists in enzymes with differentfolding, including trypsin and subtilisin, and is an example of con-vergent evolution (Holmquist, 2000). Access to the active site, con-sisting a serine, histidine, carboxylic acid triad may be shielded by amobile lid, whose position closed or open determines the enzyme inan inactive or active conformation. The activation can be explained bythe opening of a lid (flap) structure of the enzyme at an interface. Thelipase with open lid is the active form of the enzyme and gives thesubstrate access to the active site. The substrate-binding site is locatedinside a pocket on top of the central β-sheet that is typical of this fold.Size and geometry of the substrate-binding cleft have been related tosubstrate specificity (Pleiss et al., 1998) and residues that contact thesubstrate have been identified by crystallography and docking.However, it is recognized that other protein regions, such as the liditself (Brocca et al., 2003) and the reaction conditions (Verger, 1980)may play a role in lipases specificity.

459B. Joseph et al. / Biotechnology Advances 26 (2008) 457–470

3.2. Modifications of lipase structure for cold adaptation

Cold active lipases are structurally modified by an increasingflexibility of the polypeptide chain enabling an easier accommodationof substrates at low temperature. The fundamental issues concerningmolecular basis of cold activity and the interplay between flexibilityand catalytic efficiency are important in the study of structure–function relationships in enzymes. Such issues are often approachedthrough comparison with the mesophilic or thermophilic counter-parts, by site-directed mutagenesis and 3D crystal structures (Narinxet al., 1997; Wintrode et al., 2000). The molecular modelling of Pseu-domonas immobilis lipase revealed several features of cold-adaptedlipases (Arpigny et al., 1997). A very low proportion of arginineresidues as compared to lysines, a low content in proline residues, asmall hydrophobic core, a very small number of salt bridges and ofaromatic–aromatic interactions are the possible features of lipase forcold adaptation. Similarly the weakening of hydrophobic clusters, thedramatic decrease (40%) of the Proline content and of the ratio Arg/Arg+Lys makes lipases active at low temperature (Gerday et al., 1997).Moreover when compared to the dehalogenase from Xanthobacterautotrophicus, the cold active lipase displays a very small number ofaromatic–aromatic interactions and of salt bridges. The location ofsome salt bridges which are absent in the cold lipase seems to becrucial for the adaptation to cold. A large amount of charged residuesexposed at the protein surface, have been detected in the cold activelipase from Pseudomonas fragi (Alquati et al., 2002). They alsoobserved a reduced number of disulphide bridges and of Prolines inloop structures. Arginine residues were distributed differently than inmesophilic enzymes, with only a few residues involved in stabilizingintramolecular salt bridges and a large proportion of them exposed atthe protein surface that may contribute to increased conformationalflexibility of the cold-active lipase. In addition to this, the structuralfactors possibly involved in cold adaption are increased number andclustering of glycine residues (providing local mobility), lower numberof ion pairs and weakening of charge-dipole interactions in α helices(Georlette et al., 2004; Gomes and Steiner, 2004). The substitution ofGlycine with Proline by mutation caused a shift of the acyl chainlength specificity of the enzyme towards short-chain fatty acid estersand enhanced themostability of the enzyme (Kulakovaa et al., 2004). Amutation in the lid region of catalytic triad of cold active lipases fromP. fragi improved substrate selectivity and thermostability (Santarossaet al., 2005). Introduction of polar residues in the surface of exposedlid might be involved in improved substrate specificity and proteinflexibility. The sequence alignment study of cold active lipase fromPhotobacterium lipolyticum showed three aminoacid residues(Ser174, Asp236 and His312) constitute the active site and RG residues(Arg236 and Gly91) making an oxyanion sequence (Ryu et al., 2006). Itis understood that the catalytic cavity of the psychrophillic lipase ischaracterized by high plasticity. These structural adaptations mayconfer on the enzyme a more flexible structure, in accordance with itslow activation energy and its low thermal stability. The above dis-cussions may help to obtain information for insights into the molec-ular mechanisms of cold adaption and thermolability of cold activelipases.

3.3. Structure of Candida antarctica lipase

Literature reviews reveal that the Antarctic yeast (C. antarctica) isthe most extensively studied microorganismwith respect to its lipasesecretion. The psychrophilic yeast, C. antarctica, originally isolatedfrom Antarctic habitat expresses two lipase variants viz., C. antarcticalipase A (CAL A) and C. antarctica lipase B (CAL B) with differentphysiochemical properties (Kirk and Christensen, 2002). Many of thelipases show interfacial activation; their activity is much higher whenacting on substrates at a water–micelle interface compared to thedissolved substrates. CAL A shows interfacial activation, while CAL B

does not show such behavior and is therefore not considered to be atrue lipase (Martinelle and Hult,1995). CAL Bwas found to be selectivefor position sn-3 and CAL A could preferentially cleave sn-2 ester bond(Rogalska et al., 1993). The interfacial activation can be explained bythe opening of a lid (flap) structure of the enzyme at an interface. Thislid structure covering the active site of true lipases is absent or verysmall in CAL B. The structure of CAL B was solved in 1994 (Uppenberget al., 1994a,b,1995). The CAL B contains the sequence, Thr-x-Ser-x-Glyaround the active-site Ser, whereas in all other microbial andmammalian lipases, Thr is replaced by Gly. When Thr was replacedby a Gly in a CAL B site-directed mutant (expected to create localflexibility), thermostability increased (rather than decreased) with aconcomitant decrease in specific activity. The unexpected increase inthermostability and the decrease in specific activity were thought toarise from the replacement of Thr side-chains with Gly enhancingcontacts between secondary structural elements around the activesite (Patkar et al., 1997). The active site of CAL B possesses an oxyanionhole that stabilizes the transition state and the oxyanion in thereaction intermediate. This oxyanion hole is a spatial arrangement ofthree hydrogen-bond donors, one from the side chain of Thr40and two from the back-bone amides of Thr40 and Gln106. The activesite also contains a small cavity called the stereospecificity pocket(Uppenberg et al., 1995), in which secondary alcohols have to orientone substituent during catalysis. This gives CAL B a high enantios-electivity towards chiral secondary alcohols. The enantioselectivity ofCAL B towards secondary alcohols is determined by the steric require-ments of the stereospecificity pocket. The fast-reacting enantiomer ofthe secondary alcohols orients its large substituent towards the active-site entrance and its medium-sized substituent in the stereospecificitypocket. To react, the slow-reacting enantiomer has to have the op-posite orientation for its substituents compared to the fast-reactingenantiomer. The large substituent is not easily accommodated in thestereospecificity pocket, which explains the low reaction rate of thisenantiomer (Rotticci et al.,1998). Enzyme variantswere createdwhereamino acids predicted to play key roles for the lipase activity in thedifferent models were replaced by an inert amino acid (alanine).Kasrayan et al. (2007) studied activity measurements of the over-produced and purified mutant CAL A. Moreover, found that the activesite consists of amino acid residues Ser184, His366, and Asp334 and inwhich there is no lid. They suggested that this model could be used forfuture targeted modifications of the enzyme to obtain new substrateacceptance, better thermostability, and higher enantioselectivity.

4. Production of cold active lipases

4.1. Psychrophiles as sources of cold active lipases

Cold active lipases are largely distributed in microorganisms sur-viving at low temperatures near 5 °C. Although a number of lipaseproducing sources are available, only a few bacteria and yeast wereexploited for the production of cold active lipases. Attempts have beenmade from time to time to isolate cold active lipases from thesemicroorganisms having high activity at low temperatures. A list ofvarious cold active lipase producing psychrophillic and psychro-trophic bacteria is presented in Table 1. These bacterial strains wereisolated mostly from Antarctic and Polar regions which represents apermanently cold (0±2 °C) and constant temperature habitat. Variousstudies shows that a high bacterial count has been recorded as high as105 ml−1 and 106 ml−1 in water column and in the sea ice respectively(Sullivan and Palmisano, 1984; Delille, 1993). Another potential sourceof cold active lipases is deep-sea bacteria. A marine bacterium Aero-monas hydrophila growing at a temperature range between 4 and37 °C produced cold active lipolytic enzyme (Pemberton et al., 1997).Few bacterial genera have been isolated and characterized from deep-sea sediments where temperature is below 3 °C. They include Aero-monas sp. (Lee et al., 2003); Pseudoalteromonas sp. and Psychrobacter

Table 2Fungi producing cold active lipases

Microorganism Sources References

Aspergillus nidulans Ns Mayordomo et al. (2000)Candida antarctica Antarctic habitat Patkar et al. (1993)

Uppenberg et al. (1994a)Uppenberg et al. (1994b)Patkar et al. (1997)Koops et al. (1999)Zhang et al. (2003)Siddiqui and Cavicchioli (2005)

C. lipolytica Frozen food Alford and Pierce (1961)Geotrichum candidumPencillium roqueforti

Ns: Not specified.

Table 1Bacteria producing cold active lipases

Microorganism Sources References

Acinetobacter sp. strain no. 6 Siberian tundra soil Suzuki et al. (2001)Acinetobacter sp. strain no. O16 Ns Breuil and Kushner

(1975)Achromobacter lipolyticum Ns Khan et al. (1967)Aeromonas sp. strain no. LPB 4 Sea sediments Lee et al. (2003)

Aeromonas hydrophila Marine habitat Pemberton et al. (1997)Bacillus sphaericus MTCC 7526 Gangothri Glacier

(Western Himalaya)Joseph (2006)

Microbacterium phyllosphaeraeMTCC 7530Corynebacteriumpaurametabolum MTCC 6841,

Naukuchiatal lakeUttaranchal(Western Himalaya)

Joshi et al. (2006)

Moraxella sp. Antarctic habitat Feller et al. (1990)Morexella sp TA144 Antarctic habitat Feller et al. (1991a)Photobacteriumlipolyticum M37

Marine habitat Ryu et al. (2006)

Pseudoalteromonas sp. Deep-sea sediments Zeng et al. (2004)wp27Pseudoalteromonas sp. Antarctic marine Giudice et al. (2006)Psychrobacter sp.Vibrio sp.Pseudomonas sp.strain KB700A

Subterraneanenvironment

Rashid et al. (2001)

Pseudomonas sp. B11-1: Alaskan soil Choo et al. (1998)Pseudomonas P38 Ns Tan et al. (1996)Pseudomonas fluorescens Refrigerated milk samples Dieckelmann et al. (1998)Pseudomonas fluorescens Refrigerated food Andersson (1980)Pseudomonas fluorescens Refrigerated human

placental extractsPreuss et al. (2001)

Pseudomonas fragistrain no. IFO3458

BCCM™/LMG2191T Alquati et al. (2002)

BCUG, BelgiumPseudomonas fragistrain no.IFO 12049

Ns Aoyama et al. (1988)

strain no. IFO 12049Psychrobacter okhotskensis sp. Sea coast Yumoto et al. (2003)Psychrobacter sp. wp37 Deep-sea sediments Zeng et al. (2004)Psychrobacter sp. Ant300 Antarctic habitat Kulakovaa et al. (2004)Psychrobacter immobilisstrain B 10

Antarctic habitat Arpigny et al. (1997)

Psychrobacter sp. 7195 Antarctic habitat Zhang et al. (2007)Psychrobacter sp. Antarctic habitat Parra et al. (2007)Serratia marcescens Raw milk Abdou (2003)Staphylococcus aureus Ns Alford and Pierce (1961)Staphylococcus epidermidis Frozen fish samples Joseph et al. (2006)

Ns: Not specified.


sp. (Zeng et al., 2004) P. lipolyticum (Ryu et al., 2006). Bacterial generaincluding P. fragi (Aoyama et al., 1988; Alquati et al., 2002), Pseudo-monas fluorescens (Dieckelmann et al., 1998) and S. marcescens(Abdou, 2003) which produces cold active lipases were isolatedfrom refrigerated milk and food samples. Permanently cold regionssuch as glaciers and mountain regions are another habitat for coldactive lipase producing microorganisms. The soil and ice in Alpineregion also harbor psychrophillic microorganisms, which produceslipases. In addition to all these permanently cold regions, there aremany other accessible and visible soil and water which become coldboth diurnally and seasonally fromwhich cold active lipase producingmicrobes can be isolated using appropriate low temperature techni-ques. The widespread use of refrigeration to store fresh and preservedfoodstuffs provides a great diversity of nutrient rich habitat for somewellknownpsychrotolerant food spoilagemicroorganisms. Cold activelipases were also reported in psychrophilic fungi and yeast. Theyinclude Candida lipolytica, Geotrichum candidum and Pencilliumroqueforti isolated from frozen food samples for the cold active lipasesproduction (Alford and Pierce, 1961). Aspergillus nidulans (Mayordomo

et al., 2000) and C. antarctica have been reported to produce coldactive lipolytic enzymes. However, a deep research has been done onthe C. antarctica (Table 2).

4.2. Fermentation conditions for cold active lipase production

Cold active lipases are mostly extra cellular and are highly in-fluenced by nutritional and physicochemical factors such as tempera-ture, agitation, pH, nitrogen source, carbon source, inducers, inorganicsources and dissolved oxygen. Submerged fermentation is the mostcommonmethod used for cold active lipase production (Dieckelmannet al., 1998; Lee et al., 2003). A list of various production parametersfor different cold active lipase producing microorganisms is given inTable 3. Cold-adapted microorganisms tend to have good growth rateat low temperature. The production of cold active lipase is consideredtemperature dependent and thermolabile (Rashid et al., 2001). Mor-axella sp. isolated from Antarctican habitat grows well at 25 °C andproduced cold active lipolytic enzyme (Feller et al., 1990). Pseudomo-nas sp. strain B11-1 utilized yeast extract and tryptone as best carbonand nitrogen sources for growth and production of lipases. Tween 80and Tributyrin induced production of cold active lipases at 4 °C withan optimum pH 7.6 (Choo et al., 1998). A. nidulans WG312 producedcold active lipase by utilizing olive oil as an inducer at 30 °C(Mayordomo et al., 2000). Soybean oil induced the production of coldactive lipases from Acinetobacter sp. strain no. 6 at 4 °C within fourdays (Suzuki et al., 2001). Aeromonas sp. LPB 4 produced lipase at 10 °Cin eight days time duration by using tryptone and yeast extract ascarbon and nitrogen source and trybutylin as an inducer (Lee et al.,2003). Serratia marcescens produced cold active lipase in presence ofskim milk as energy source at 6 °C in 6 days of incubation. Tween 80and Tween 20 were the best inducers for cold-adapted lipase pro-duction with yeast extract as carbon source in 14 days at 25 °C forPsychrobacter sp. wp37. Another isolate of Pseudoalteromonas sp.wp27 produced lipases at 25 °C in 14 days with yeast extract as carbonsource and olive oil and Tween 80 as inducers (Zeng et al., 2004).

4.3. Purification and characterization of cold active lipases

Most purification schemes for lipases are based on multistep stra-tegies. However, in these years new techniques have been developedthat may yield high recovery. Based on the nature of lipase producedby the organism one has to design the protocol for purification(Saxena et al., 2003). The significance of cold active lipases is exten-sively recognized in a number of applications (Houde et al., 2004). Thepurified lipase is needed for the synthesis of fine chemicals, cosmeticsand in pharmaceutical industries. However, homogenous preparationof cold-adapted lipases is not required for all industrial applications. Alist of purified cold active lipases and results obtained duringpurification studies are given in Table 4. Lipases from Antarctic cold-adapted bacteria have been the subject of several studies, all of themfailing to obtain purified forms due to the difficulty of eliminating

Table 3Overview of production parameters for cold active lipase

Microorganism pH Temp. Incubation period C source N source References

(°C)

BacteriaAeromonas sp. LPB 4 Ns 10 8 days Trybutylin Tryptone, Yeast extract Lee et al. (2003)Acinetobacter sp. strain no. 6 Ns 4 7 days Soybean oil Ns Suzuki et al. (2001)Bacillus sphaericus MTCC 7526 8 15 48 h Lactose/Tributyrin Peptone Joseph (2006)Corynebacterium paurometabolum MTCC 6841 8.5 25 Ns Soybean oil/olive oil NaNO3 and KNO3 Joshi et al. (2006)Microbacterium phyllosphaerae MTCC 7530 8 15 36 h Tributyrin/Lactose Peptone Joseph (2006)Moraxella sp. Ns 25 Ns Ns Ns Feller et al. (1990)Pseudoalteromonas sp. wp27 Ns 25 14 days Olive oil, Tween80 Yeast extract Zeng et al. (2004)Pseudomonas sp. strain KB700A 7.2 −5 Ns Tributyrin Tryptone, Yeast extract Rashid et al. (2001)Pseudomonas sp. strain B11-1 7.6 4 Ns Tween80, Tributyrin Yeast extract, Tryptone Choo et al. (1998)Psychrobacter sp. wp37 Ns 25 14 days Tween80, Tween20 Yeast extract Zeng et al. (2004)Serratia marcescens Ns 6 3 days Ns Skim milk Abdou (2003)

FungusAspergillus nidulans WG312 Ns 30 Ns Ns Olive oil Mayordomo et al. (2000)

C: Carbon; N: Nitrogen; Ns: Not specified.


lipopolysaccharides produced by Antarctic microorganisms, andfound strongly associated with the lipid hydrolases (Gerday et al.,1997).

The investigations on cold active lipases from Psychrobacterimmoblis B10 were perused on semi-purified preparations, thenucleotide sequence of which is also available (Arpigny et al., 1995).Lipase purity is evaluated after each purification step by measuringthe overall activity and specific activity. The purification efficiency isdetermined by total yield and purification factor (Kademi et al., 2005).For industrial applications the purification step should be economical,rapid, high yielding and easy to produce in large scale operations(Gupta et al., 2004). Prepurifaction step involves concentration of theprotein containing lipases by ammonium sulphate precipitation,ultrafiltration or extraction with organic solvents. Since lipases areknown to be hydrophobic in nature having large hydrophobic surfacesaround the active site, the purification may be achieved by optingfor affinity chromatographic techniques. The widely used chromato-graphic technique involves columns packed with QAE sephadex, CMcellulose, DEAE cellulose, phenyl-sepharose etc. However in certainapplications, further purification can be achieved by gel filtrationchromatography. The usual procedures for purification of lipases aretroublesome, time consuming and results in low yield. Low thermo-stability of these cold active lipases is also a major problem in puri-fication. Novel purification steps are therefore needed to increase theoverall enzyme yield and purification fold.

The effective catalytic properties of enzymes have led to introduc-tion into several industrial products and processes (Dordick, 1991;Koeller and Wong, 2001; Park et al., 2001a; Schmid et al., 2001).Recent developments in biotechnology, particularly in areas such asprotein engineering (Kim and Choi, 1984; Joo et al., 1998; Eijsink et al.,2004) and directed evolution, have provided important tools for theefficient development of new enzymes. The characterization and

Table 4Purification of cold active lipases

Organism Purification technique

Aeromonas sp. LPB 4 QAE Sephadex columnBacillus sphaericus MTCC 7526 DEAE celluloseMicrobacterium phyllosphaerae MTCC 7530 DEAE celluloseMoraxella sp. AcA 34 column, UltrafiltrationPseudomonas sp. Strain B11-1: —[gene cloned into E. coli and purified]

DEAE Cellulofine

Psychrobacter sp. 7195 DEAE Sepharose CL-4B, and SephadexSerratia marcescens CM Cellulose, DEAE cellulose

Sephadex G-150Aspergillus nidulans WG312 phenyl-sepharose chromatography, lin

Ns: Not specified.

kinetic study of cold active lipase were studied in terms of optimumpH and stability, optimum temperature, thermo-stability and effect ofchelating agents, inhibitors, solvents and metal ions. The cold activelipases from microorganisms have an optimum activity at 20 °C andare stable at a wide range of temperatures. However, these cold activeenzymes are unstable above 65 °C (Table 5). These cold active lipasespossessing stability at various physical and chemical conditions mayhave potentials in biotechnological and industrial applications at lowtemperatures. The molecular weight of these purified proteins variedfrom 50–85 kDa and they have broad substrate specificity. Lipases arewidely used as industrial catalyst; there are several advantages anddisadvantages for industrial application of cold active lipases. They areeasily deactivated when subjected to heat, extreme pH range or inorganic solvents (Jensen, 1983; Longo and Combes, 1999; Matsumotoet al., 2001; Noel and Combes, 2003).

Numbers of strategies have been proposed to overcome such alimitation including the use of soluble additives, immobilization, pro-tein engineering, and chemical modification (Kwon and Rhee, 1984;Chae et al.,1998; Park et al., 2001b; Lee et al., 2002). Themodification ofprotein surface with modifiers by chemical binding appears to be agood strategy to improve biocatalyst performance. Modified enzymeswere typically macroscopic catalysts that retained in the reactor;therefore, continuous replacement of the enzyme is not necessary. Theactivity of the cold enzyme presents an apparent optional activityaround 35 °C and retains about 20% of its activity at 0 °C. The activity ofmesophilic lipase is close to zero below 20 °C and still increases attemperatures above 60 °C. Properties of cold lipase from P. immobilisstrain B10 was compared with lipase from mesophillic bacterium,Pseudomonas aeruginosa (Arpigny et al., 1997). The activation energiesevaluated from the Arrhenius plots are 63 and 110 kJ/mol for the coldand mesophilic enzymes, respectively underlining the cold characterof lipase produced by the Antarctic bacterium. This characteristics also

Fold increase/yield (%) References

53.5/7.5 Lee et al. (2003)17.74/4.70 Joseph (2006)22.03/7.50 Joseph (2006)Ns Feller et al. (1990)38/17 Choo et al. (1998)

G-75 Ns Zhang et al. (2007)20/45 Abdou (2003)

olenic acid-agarose Ns Mayordomo et al. (2000)

Table 5Characterization of cold active lipases

Organism Optimum Stability MW(kDa)

Comments References

Temp./pH Temp./pH

Acinetobacter sp. strain no. 6 20 °C/Ns Ns/Ns Ns Broad specificity towards the acyl group (C8−C16)of ethyl esters

Suzuki et al. (2001)

Aeromonas sp. LPB 4 35 °C/Ns 50 °C/Ns 50 Medium chain acyl group p-nitrophenyl esters seemedto be good substrate; Increased activity with detergents

Lee et al. (2003)

Bacillus sphaericus MTCC 7526 15 °C/8.0 30 °C/8 40 Stable in presence of organic solvents and compatiblewith detergents

Joseph (2006)

Microbacterium phyllosphaeraeMTCC 7530

20 °C/8.0 35 °C/8 42 Presence of organic solvents activity compatible withdetergents

Joseph (2006)

Pseudo-alteromonas sp. wp27 20–30 °C/7.0–8.0 Ns/Ns 85 Enzymes were 60% active at 4 °C Zeng et al. (2004)Pseudomonas sp. strain KB700A 35 °C/8.0–8.5 Ns/Ns 49.9 Highest activity with p-nitrophenyl caprate Rashid et al. (2001)Pseudomonas sp. strain B11-1:[recombinant]

45 °C/8.0 5–35 °C/6.0–9.0 33.7 Strongly inhibited by Zn2+, Cu2+, Fe3+, Hg2+ Choo et al. (1998)

Psychrobacter sp. wp37 20–30 °C/7.0–8.0 Ns/Ns 85 Enzymes were 60% active at 4 °C Zeng et al. (2004)Psychrobacter sp 7195 [recombinant] 30 °C/9.0 Ns/7.0–10.0 Ns Ca2+ and Mg2+ enhanced activity Zhang et al. (2007)Serratia marcescens 37 °C/8.0 65 °C/6.6 52 Observed 90% activity at 5 °C Abdou (2003)Aspergillus nidulans WG312 40 °C/6.5 Low thermal stability/Ns 29 preference toward esters of short- and middle-chain

fatty acidsMayordomo et al. (2000)

Ns: Not specified.


illustrated by the high thermosensitivity of the cold active lipasedisplaying at 60 °C a half-life 2 orders of magnitude lower than that ofthe mesophillic enzyme. The widely characterized cold active lipase isfrom yeast (C. antarctica). The two lipases variants are C. antarcticalipase A (CAL A) and C. antarctica lipase B (CAL B) with differentphysiochemical properties (Kirk and Christensen, 2002). CAL B belongsto the α/β-hydrolase fold superfamily (Ollis et al., 1992), whichcontains enzymes that have evolved from a common ancestor(divergent evolution) to catalyze various reactions such as hydrolysisof esters, thioesters, peptides, epoxides, and alkyl halides or cleavage ofcarbon bonds in hydroxynitriles (Holmquist, 2000). CAL B is made upof 317 amino acids and has a molecular weight of 33 kDa. CAL B is lessthermostable, smaller in size, andmore acidic than CAL A (Patkar et al.,1993). CAL B is a stable enzyme that has been used at 150 °C, in organicsolvents of high polarity such as acetonitrile and dimethyl sulfoxide inionic liquids, in solid/gas systems and in supercritical carbon dioxide(Suen et al., 2004). Recently, a cold active lipolytic enzyme wasproduced by cloning the putative lipolytic gene encoding lipo1 fromthe metagenomic library and expressed in Escherichia coli BL21 usingthe pET expression system (Roh and Villatte, 2008). The expressedrecombinant enzyme was purified by Ni-nitrilotriacetic acid affinitychromatography and characterized using general substrates of

Table 6Gene isolation and characterization

Microorganisms Studies conducted/Investigations unde

Moraxella sp. strain TA 144. Analysis of sequence homology of HumPseudomonas fluorescens strain C9 Isolation of lipase-encoding gene lip A

Gene cloning, expression and sequencingPseudomonas fragi Molecular cloning and nucleotide sequP. fragi IFO-12049 Cloning, sequencing and expression ofPseudomonas sp. Strain B11-1 Gene cloning and sequencingMoraxella sp. Strain TA 144 Sequencing of lipase geneMoraxella sp. Strain TA 144. Cloning and expression in E. coli of thrPsychrobacter immobilis B10 Cloning, sequencing and structural feaPseudomonas fluorescens Cloning and sequencing of DNA encodCandida antarctica Protein expression of lipase B in PichiaPseudomonas sp. strain KB700A. Gene cloningP. fragi Heterologous expression, and moleculPsychrobacter sp. Ant 300 Gene cloning, expression and characteP. fragi Molecular properties, mutagenesis andPhotobacterium lipolyticum M37 Isolation of a new cold-adapted lipaseC. antarctica Functional expression of lipase B in EsPsychrobacter sp. 7195 Cloning, expression, and characterizatiMoritella sp. 2-5-10-1 Cloning and expression of lipP, a Gene

lipolytic property. The gene consisted of 972bp encoding a polypeptideof 324 amino acids with a molecular mass of 35.6 kDa. This lipolyticenzyme exhibited the highest activity at pH 7.5 and 10 °C. At thermalstability analysis, lipo1 was more unstable at 40 °C than 10 °C.

5. Biotechnological approaches in cold active lipase

An emerging area of research in the field of enzymology is to developradically different and novel biocatalysts through various molecularapproaches includingrecombinantDNAtechnology, proteinengineering,directed evolution and the metagenomic approach. As a whole, lipasebiotechnology has just reached the end of lag phase and the beginning ofthe exponential phase: it demands extension in termsof bothquality andquantity. Qualitative improvements in restructuring lipase gene and itsprotein can be achieved by employing already established recombinantDNA technology and protein engineering. Quantitative enhancementneeds strain improvement, especially through site-directedmutagenesisand standardizing the nutrient medium for the overproduction of coldactive lipases. Recently, Vakhlu and Kour (2006) compiled the informa-tions on properties of various yeast lipases and genes encoding them.Majority of yeast lipases including C. antactica are extracellular,monomeric glycolproteins with molecular weight ranging between

rtaken References

an HSL gene with Antarctic bacterium Langin et al. (1993)Dieckelmann et al. (1998)

encing of the lipase gene Kugimiya et al. (1986)the lipase gene Aoyama et al. (1988)

Choo et al. (1998)Feller et al. (1991b)

ee lipase-encoding genes Feller et al. (1991a)tures of lipase gene Arpigny et al. (1993)ing Phospholipase C Preuss et al. (2001)pastoris Rotticci-Mulder et al. (2001)

Rashid et al. (2001)ar modeling Alquati et al. (2002)rization Kulakovaa et al. (2004)overexpression of cold active lipase Lafranconi et al. (2005)and gene cloning Ryu et al. (2006)chericha coli Blank et al. (2006)on of a cold-adapted lipase gene Zhang et al. (2007)encoding a cold-adapted lipase Yang et al. (2008)

Table 7Genes/gene fragments encoding cold active lipases

Genes Microorganism References

lip2 Moraxella sp. TA144 Feller et al. (1991c)lip3 Moraxella sp. TA144 Feller et al. (1991b)lipP Moritella sp. 2-5-10-1 Yang et al. (2008)lipo1 metagenomic library Roh and Villatte (2008)lip A Pseudomonas fluorescens

strain C9Dieckelmann et al. (1998)

lipP Pseudomonas sp. B11-1 Choo et al. (1998)lipA1 Psychrobacter sp. 7195 Zhang et al. (2007)KB-lip Pseudomonas sp. Strain

KB700ARashid et al. (2001)

PLC gene Pseudomonas fluorescens Preuss et al. (2001)lip1 Psychrobacter immobilis B10 Arpigny et al. (1993)PsyEst Psychrobacter sp. Ant300 Kulakovaa et al. (2004)

16 S rRNA gene sequenceswp27, wp30, wp32, wp33 Pseudoalteromonas sp. Zeng et al. (2004)wp37 Pseudomonas sp. Zeng et al. (2004)wp17, wp18, wp21,wp24, wp25

Psychrobacter sp. Zeng et al. (2004)


~33 and ~65 kDa. More than 50% reported lipases producing yeast;produce it in the forms of various isozymes. Various lipase-encodinggenes in turn produce these lipase isozymes.

5.1. Gene cloning

To date, a large number of cold active lipase genes were isolatedand the related studies have been carried out (Table 6). Early successesin the production of heterologous proteins were achieved usingEscherichia coli as host and various kinds of proteins were expressed inE. coli. However, expression of eukaryotic proteins in E. coli becamevery difficult due to formation of inclusion bodies, protein misfoldingand safety issues. Other expression systems were developed amongyeasts, fungi, plants and animals. Molecular cloning and nucleotidesequencing of the lipase gene in P. fragi (Kugimiya et al., 1986) andcloning, sequencing and expression of the lipase gene from P. fragiIFO-12049 has been reported (Aoyama et al., 1988). The earlier effortson cloning and expression of the genes coding for cold active lipases inmesophilic organisms such as E. coli did not yield a stable integrationof cold lipase genes within their hosts (Feller et al., 1990; Feller et al.,1991a). The cloning and expression of genes from a psychrotrophicbacterium in a mesophilic host has been described. Three lipase-encoding genes (lip) from the Antarctic psychrotroph, MoraxellaTA144, were cloned by inserting Sau 3AI-generated DNA fragmentsinto the Bam HI site of the pSP73 plasmid vector. To prevent heatdenaturation of the gene product, the screening procedure on agarplates containing an emulsified lipid involved growing of E. colirecombinant colonies at 25 °C followed by incubation at 0 °C. Thethree recombinant lipases (reLip) were cell associated and differed bytheir respective specificity towards p-nitrophenyl esters of variousaliphatic chain lengths. These cloned reLip conserved the main cha-racter of the wild-type enzymes i.e., a dramatic shift of the optimaltemperature of activity towards low temperatures and pronouncedheat lability (Feller et al., 1991a). Cloning and expression of threelipase-encoding genes of Moraxella sp. strain TA 144 in E. coli havebeen reported (Feller et al., 1991a) and the gene has been sequenced(Feller et al., 1991b). Cloning and sequencing of lipase gene P.immobilis B10 and studied the structural features (Arpigny et al.,1993). Isolation of cold active lipase-encoding gene such as lip A fromP. fluorescens strain C9 (Dieckelmann et al., 1998), Pseudomonas sp.strain KB700A (Rashid et al., 2001) and P. lipolyticum M37 (Ryu et al.,2006) were reported. Gene cloning and sequencing of cold-adaptedlipase from Pseudomonas sp. Strain B11-1 has been reported (Chooet al., 1998). DNA encoding Phospholipase C in P. fluorescens has beencloned and sequenced (Preuss et al., 2001) and Rotticci-Mulder et al.(2001) studied protein expression of lipase B in Pichia pastoris from C.antarctica. Studies on the heterologous expression and molecularmodeling of cold-adapted lipase gene from P. fragi and therecombinant lipase retained significant activity at low temperature(Alquati et al., 2002). The three-dimensional structure was built byhomology and compared with homologous mesophilic lipase whichshowed 45% sequential identity with P. aeruginosa lipase and 38%withBurholderia cepacia lipase. A PCRmethodwas designed for the isolationof lipase genedirectly fromenvironmentalDNA, usingprimers, based onlipase consensus (Bell et al., 2002). Gene cloning, expression andcharacterization have been carried out in Psychrobacter sp. Ant 300(Kulakovaa et al., 2004). Recently, a gene (lipP, 837 bp in length) codingfor a cold-adapted lipase of psychrophilic bacterium Moritella sp. 2-5-10-1 was isolated from Antarctic regionwas cloned and sequenced. Thededuced amino acid sequence revealed a protein of 278 amino acidresidueswith amolecularmassof 30,521. Theprimarystructure of lipasededuced from thenucleotide sequence showed consensus pentapeptidecontaining the active serine (Gly-Trp-Ser-Leu-Gly) and a conserved His-Gly dipeptide in the N-terminal part of the enzyme. The gene wassubcloned into pET-28a expression vector to construct a recombinantlipase protein and expressed in E. coli BL21 (DE3) (Yang et al., 2008).

The latest trend in lipase research is the development of novel andimproved lipase through molecular approaches such as directedevolution and exploring natural communities by the metagenomicapproach (Gupta et al., 2004). The main microbial expression systemsare Aspergillus oryzae, Saccharomyces cerevisiae and P. pastoris. Recom-binant DNA technology represents a very attractive technology thatcan be used to increase lipase production mainly in the case ofisoenzymes whose purification leads to a very low yield. This allowsup to 40% decrease in the cost of raw material, water, steam andelectricity compared to the cost of native enzyme production. The firstlipase produced by recombinant DNA technology was Lipolase intro-duced in the market by Novozymes in 1988. Originating from Ther-momyces lanuginosus, formerly Humicola lanuginosa, this lipase wasexpressed in A. oryzae. The growing number of recombinant lipases isattributed to the recent progress in molecular technologies (cloningand sequencing) (Kademi et al., 2005). Functional expression of lipaseB from C. antarctica in E. coli has been studied by Blank et al. (2006).Recently, a novel lipase was isolated from a metagenomic library ofBaltic Sea sediment bacteria (Hardeman and Sjoling, 2007). Prokar-yotic DNA was extracted and cloned into a copy control fosmid vector(pCC1FOS) generating a library of 47,000 clones with inserts of 24–39 kb. Screening for clones expressing lipolytic activity, identified 1%of the fosmids as positive. An insert of 29 kb was fragmented andsubcloned. Subclones with lipolytic activity were sequenced and anopen reading frame of 978 bp encoding a 35.4 kDa putative lipase/esterase h1Lip1 (DQ118648) with 54% amino acid similarity to aPseudomomas putida esterase (BAD07370). Conserved regions, includ-ing the putative active site, GDSAG, a catalytic triad (Ser148, Glu242and His272) and a HGG motif, were identified. The h1Lip1 lipase wasoverexpressed, (pGEX-6P-3 vector), purified and shown to hydrolysep-nitrophenyl esters of fatty acids with chain lengths up to C14.Recently, lipo1 a novel psychrophilic esterase obtained directly fromthe metagenomic DNA was directly extracted from the activatedsludge (Roh and Villatte, 2008). The gene consisted of 972 bp encodinga polypeptide of 324 amino acids with a molecular mass of 35·6 kDa.Typical residues essential for lipolytic activity such as penta-peptide(GXSXG) and catalytic triad sequences (Ser166, Asp221 and His258)were detected. The deduced amino acid sequence of lipo1 showed lowidentity with amino acid sequences of esterase/lipase (32%,ZP_01528487) from Pseudomonas mendocina ymp and esterase (31%,AAY45707) from uncultured bacterium. In addition, few genes/genefragments encoding cold-adapted lipases has also been isolated(Table 7). From the table, it is clear that only a limited number ofstudies were carried out in the isolation of cold active lipase-encodinggene/gene fragments.


5.2. Protein engineering

Cold active lipases could generate avenues for industrial applica-tions, once their specific properties are improved through enzymeengineering. Although lipases carry significant commercial value,biotechnologically produced or engineered cold active lipases mayrepresent the focus of industrial interest in future. Determination ofthree-dimensional structures of more cold active lipases would allowthe detailed analysis of protein adaptation to temperatures at molec-ular level. This may include increased thermolabile nature and/orcatalytic activity at low temperatures, or the modification of pH pro-files. Cold active lipases from microorganisms retaining high catalyticactivity at low temperatures are successfully produced using site-directed mutagenesis and directed evolution. The α/β hydrolase foldof lipase consists of a central hydrophobic, 8-stranded sheet, packedbetween two layers of amphiphilic α-helices. They have a commoncatalytic mechanism involving five subsequent steps: After binding ofthe ester substrate, a first tetrahedral intermediate is formed by nu-cleophilic attack of the catalytic serine, with the oxy anion stabilizedby the 2 or 3 hydrogen bonds, the so called oxy anion hole. The esterbond is cleaved and the alcohol moiety leaves the enzyme. In the laststep the acyl enzyme is hydrolyzed (Pleiss et al., 1998). Analysis ofsequence homology of human hormone sensitive lipase gene withAntarctic bacterium Moraxella sp. strain TA 144 was carried out byLangin et al. (1993). Strictly conserved catalytic centre of the lipasescontain a serine-protease like catalytic triad, consisting of Ser-His-Asp/Glu residues and the active site serine residue is located in a β-Ser-α motif (Jaeger et al., 1994). This motif consists of a six-residue β-strand, a four residue type II turn with serine in the -conformationand buried α-helix packed parallel against strand 4 and 5 of thecentral β-sheet. Unusual and structural feature of the structure oflipases is that the active site is completely buried under a lid/flapcomposed of one or two α helices, so the active site is not accessible tothe substrate unless activation occurs (Lotti et al., 1994). Sequencing,determination of crystal structure and modification of two crystalforms of lipase B from C. antarctica and crystallization and preliminaryX-ray structure of lipase from C. antarctica was studied (Uppenberget al., 1994a,b). Crystallographic and molecular modeling studies oflipase B of C. antarctica was carried out by Uppenberg et al. (1995).Kim et al. (1997) determined the crystal structure of a triacylglycerollipase from Pseudomonas cepacia (PcL) in the absence of a boundinhibitor using X-ray crystallography. The structure shows the lipaseto contain α/β-hydrolase fold and a catalytic triad comprising of re-sidues Ser87, His286 and Asp264. The enzyme shares severalstructural features with homologous lipases from Pseudomonasglumae (PgL) and Chromobacterium viscosum (CvL), including acalcium-binding site. The present structure of PcL reveals a highlyopen conformation with a solvent-accessible active site. This is incontrast to the structures of PgL and PcL in which the active site isburied under a closed or partially opened ‘lid’, respectively. Molec-ular adaptation of cold lipase and 3-dimensional modeling from P.immobilis strain B10 has been reported (Arpigny et al., 1997). A mutantproteinwith a single amino acid substitution, T103G, had an increasedhalf-life at 60 °C, but only 50% of its original activity compared withthe wild-type enzyme (Patkar et al., 1997). From the X-ray structuresof lipases available, it is evident that the so called α/β hydrolasefold (from the secondary structure alignments) with a mixed centralβ-pleated sheet containing the catalytic residues is conserved (Pandeyet al., 1999).

Activity and stability of chemically modified lipase B from C.antarctica has been reported (Koops et al., 1999). Improving toleranceof cold-adapted lipase B of C. antarctica towards irreversible thermalinactivation through directed evolutionwas investigated (Zhang et al.,2003; Cavicchioli and Siddiqui, 2004) and improved activity andthermostability by DNA family shuffling was suggested by Suen et al.(2004). Directed evolution has been reported to be laborious and

costly (Venkatesh and Sundaram, 1998a,b), however, it does providethe means for selecting mutants with improved properties (Tao andCornish, 2002). Circular dichroism measurements, using synchrotronradiation, showed that the secondary structure of C. antarctica lipasedoes not differ significantly when changed from an aqueous to organicsolvent environment (Mc Cabe et al., 2005). Thus, it was concludedthat a major conformational change is not the reason for the differentproducts produced by the enzyme when used in organic solvent.Significant changes in the lipase's α-helix content were found at theextremes of pH 4.2 and 9.0; this is in keeping with the permanent lossof activity of the enzyme at such a pH. Molecular properties, muta-genesis and over expression of cold active lipase in P. fragi have beenreported (Lafranconi et al., 2005). Further, they reported the effect ofmutation in non-consensus Thr-X-Ser-X-Gly on lipase specificity,specific activity and thermostabilty. Effect of mutations on chainlength specificity and thermostability of lipases was studied in thestrain of Pseudomonas IFO3458 (Santarossa et al., 2005). Recently thethree-dimensional model of cold-adapted Alaskan psychrotrophPseudomonas species (Strain B11-1) lipase has been constructed byhomology modeling based on the crystal structure of acetyl esterasefrom Rhodococcus species and refined by molecular dynamicsmethods. The model locates the substrate-binding cavity and furthersuggests that Ser-155, Asp-250, and His-280 are present in thecatalytic triad (Roy and Sengupta, 2007). The crystal structure of theP. lipolyticum M37 lipase at 2.2 Å resolutions was determined andcompared it to that of non-adapted Rhizomucor miehei lipase (Junget al., 2008). Structural analysis revealed that M37 lipase adopted afolding pattern similar to that observed for other lipase structures.However, comparison with RML revealed that the region beneath thelid of the M37 lipase included a significant and unique cavity thatwould be occupied by a lid helix upon substrate-binding. In addition,the oxyanion hole was much wider in M37 lipase than RML. Theyproposed that these distinct structural characteristics of M37 lipasemight facilitate the lateral movement of the helical lid and subsequentsubstrate hydrolysis, which might explain its low activation energyand high activity at low temperatures. These studies may help inunderstanding the structural features and can be used in engineeringlipase with considerable biotechnological potential.

The immobilized form of CAL B is quite thermostable, particularlyunder non-aqueous conditions, where the catalyst remains active formany hours in the presence of high concentrations of reactants oftenwith vigorous agitation (Koops et al., 1999). In aqueous solutions, thelipase denatures relatively quickly at temperatures as low as 40 °C(Homann et al., 2001). However, attempts have been made to improvethe lipase stability via protein engineering have resulted in only amoderate improvement of its thermal properties with a concomitantdecrease in activity. A comprehensive protein database (MELDB) ofmicrobial esterases and lipases was developed by Kang and coworkers(2006). In the database, 883 esterase and lipase sequences derivedfrom microbial sources were deposited and conserved parts of eachprotein were identified. HMM profiles of each cluster were generatedto classify unknown sequences. Contents of the database can bekeyword-searched and query sequences can be aligned to sequenceprofiles and sequences themselves. In MELDB, one can see thegrouping of microbial esterases and lipases based on the TribeMCLtool with conserved patterns within sequences, and with HMM pro-files one can know to which group one's query sequence is related.

6. Industrial applications of cold active lipases

Cold active lipases offer novel opportunities for biotechnologicalexploitation based on their high catalytic activity at low temperatureand low thermostability and unusual specificities. Indeed, the coldenzymes, along with the host microorganisms cover a broad spectrumof biotechnological applications. They include additives in detergents(cold washing), additives in food industries (fermentation, cheese


manufacture, bakery, meat tenderizing), environmental bioremedia-tions (Digesters, composting, oil or xenobiotic biology applications),biotransformation and molecular biology applications, heterologousgene expression in psychrophilic hosts to prevent formation of in-clusion bodies (Feller et al., 1996). Potential applications of cold-activelipases are presented in Table 8. A number of reports mentionedstraightforward reasons why cold-active enzymes have application inbiotechnology (Russell et al., 1998; Margesin and Schinner, 1999;Ohgiya et al., 1999; Gerday et al., 2000; Cavicchioli et al., 2002). Mostof these are appreciated without a detailed knowledge of how cold-active enzymes achieve their performance. The number of presentuses is low and likely to reflect the state of the field, which, forexample has not developed as rapidly as the thermophile field.Nevertheless, despite the difficulties with prediction, important ad-vances have been made (Cui et al., 1999).

6.1. Medical and pharmaceutical applications

Cold active lipases have emerged as an important biocatalyst inbiomedical applications, because of their excellent capability forspecific regioselective reactions in a variety of organic solvents withbroad substrate recognition. Biocatalysis offers a clean and ecologicalway to perform chemical processes, in mild reaction conditions andwith high degree of selectivity. The use of enzymes, especially lipases,in organic solvents proves an excellent methodology for the prepara-tion of single-isomer chiral drugs (Gotor-Fernandez et al., 2006a). Apreparation of optically active amines that was intermediate in thepreparation of pharmaceuticals and pesticides which involvedin reacting stereospecific N-acylamines with lipases, preferably fromC. antarctica or Pseudomonas sp. (Smidt et al., 1996). C. antarcticalipase B (CAL B) is a very effective catalyst for the production of aminesand amides using different enzymatic procedures. Simplicity of use,low cost, commercial availability and recycling possibility make thislipase an ideal tool for the synthesis and resolution of a wide range ofnitrogenated compounds that are for the production of pharmaceu-ticals and manufactures in the industrial sector (Gotor-Fernandezet al., 2006b).

6.2. Synthesis of fine chemicals

Kinetics of acyl transfer reactions in organic media catalyzed bylipase B from C. antarctica has been reported by Martinelle and Hult(1995). Lipase produced by a psychrotroph, P. fluorescens P38, wasfound to catalyze the synthesis of butyl caprylate in n-heptane at lowtemperatures. The optimum yield of ester synthesis was 75% at 20 °C

Table 8Industrial applications of cold active lipases

Field of application Purpose

Medical and pharmaceutical application Synthesis of arylaliphatic glycolipidsEthyl esterification of docosahexaenoic acid to EtSynthesis of citronellol laurate from citronellol an

Fine chemical synthesis Optically active ester synthesisEster synthesis, desymmetrization and productioOrganic synthesis of chiral intermediatesSynthesis of butyl caprylate in n-heptaneSynthesis of butyl lactate by transesterificationSynthesis of amides

Food industry Protein polymerization and gelling in fish, improProduction of fatty acids and interestrification of

Domestic application Detergents and cold water washingProduction of α-butylglucoside lactate by transesConversion of degummed soybean oil to biodieseSynthesis of lipase-catalyzed biodiesel

Environmental application Degradation of lipid wastesBioremediation and bioaugumentation

Removal of solid and water pollution by hydroca

with an organic phase water concentration of 0.25% (v/v). The resultsare discussed in terms of the structural flexibility of psychrotrophderived lipase and the activity of this enzyme within a nearly anhy-drous organic solvent phase (Tan et al., 1996). Applications of lipase Bof C. antarctica in organic synthesis has been reported (Anderson et al.,1998). The ethyl esterification of docosahexaenoic acid (DHA) for theproduction of ethyl docosahexaenoate (EtDHA) in an organic solvent-free system using C. antarctica lipase, which acts strongly on DHA andethanol (Shimada et al., 2001). About 88% esterification was attainedby shaking the mixture of DHA/ethanol (1:1, mol/mol) and 2 wt.%immobilized C. antarctica lipase at 30 °C for 24 h. However, even in thepresence of an excess amount of ethanol, the extent of esterificationcould not be raised above 90%. To attain a higher level of esterification,a two-step reaction was found to be effective. The first step wasperformed in a mixture of DHA/ethanol (1:1, mol/mol), and the re-action mixture was then dehydrated. In the second step, the resultingmixture was shaken at 30 °C for 24 h with 5 M equivalents of ethanolagainst the remaining DHA using 2 wt.% immobilized lipase. By meansof this two-step procedure, 96% esterificationwas attained. Repetitionof the first and second reactions showed that the immobilized lipasewas reusable for at least 50 cycles. In addition, DHA remaining in thesecond-step reaction mixture was removed by a conventional alkalirefining process, giving purified EtDHAwith a high yield. Use of lipaseB from C. antarctica for the preparation of optically active alcohols hasbeen reported (Rotticci et al., 2001).

Enzymatic reactions in non-aqueous solvents offer new possibi-lities for the biotechnological production of many useful chemicalsusing reactions that are not feasible in aqueous media. The use ofenzymes in non-aqueous media has found applications in organicsynthesis, chiral synthesis or resolution, modification of fats and oils,synthesis of sugar-based polymers, etc. The use of lipases in esteri-fication reactions to produce industrially important products such asemulsifiers, surfactants, wax esters, chiral molecules, biopolymers,modified fats and oils, structured lipids, and flavor esters is welldocumented. The interest in using lipases as biotechnological vectorsfor performing various reactions in both macro- and microaqueoussystems has picked up tremendously during the last decade (Krishnaand Karanth, 2002). Crude soybean oil did not undergo methanolysiswith immobilized C. antarctica lipase but degummed oil did(Watanabe et al., 2002). Therefore, the substance that was removedin the degumming step was estimated to inhibit the methanolysis ofsoybean triacylglycerols (TAGs). The main components of soybeangum are phospholipids (PLs), and soybean PLs actually inhibited themethanolysis reaction. Indeed, three-step methanolysis successfullyconverted 93.8% degummed soybean oil to its corresponding methyl

Reference

Ota et al. (2000)hyl docosahexaenoate (EtDHA) Shimada et al. (2001)d lauric acid Ganapati and Piyush (2005)

Anderson et al. (1998)n of peracids Zhang et al. (2003)

Gerday et al. (2000)Tan et al. (1996)Pirozzi and Greco (2004)Slotema et al. (2003)

vement in food texture, flavor modification Cavicchioli and Siddiqui (2004)fats Jaeger and Eggert (2002)

Gerday et al. (2000), Joseph (2006)terification for cosmetics Bousquet et al. (1999)l fuel Watanabe et al. (2002)

Chang et al. (2004)Ramteke et al. (2005)Gerday et al. (2000), Suzuki et al. (2001),Lee et al. (2003)

rbons, oils and lipids Margesin et al. (2002)


esters, and the lipase could be reused for 25 cycles without any loss ofthe activity. Lipase from C. antarctica has been evaluated as a catalystin different reaction media for hydrolysis of tributyrin as reactionmodel by Salis et al. (2003). To introduce polymer to cellulosicmaterial a new approach was developed by Gustavsson et al. (2004),using ability of a cellulose-binding module of C. antarctica lipase Bconjugate to catalyze ring opening polymerization of epsilon-capro-lactone in close proximity to cellulose fiber surface. CAL A posses highthermostability, allowing operation at temperature above 90 °C; theability to accept tertiary and sterically hindered alcohols, which hasrecently been attributed to the existence of a specific aminoacidicsequence in the active site; the sn-2 recognition in hydrolysis oftriglycerides; the selectivity towards trans-fatty acids; the stability inthe acidic pH range. Furthermore, it is an excellent biocatalyst for theasymmetric synthesis of amino acids/amino esters, due to itschemoselectivity towards amine groups (de Maria et al., 2005).Honore and Gerard (2005) reviewed about using lipases as catalysts inorganic synthesis. It provides some specific examples of stereoselec-tive biotransformations used to prepare non-racemic chiral buildingblocks and the utilization of these intermediates to synthesize dif-ferent target molecules by organic transformations. Cold active lipasefrom C. antarctica increased the performance of lipase B in theenantioselective esterification of ketoprofen (Ong et al., 2006).Improvement of the enantioselectivity of lipase from C. antarctica(fraction B) via adsorption on polyethylenimine-agarose has beenreported by Torres et al. (2006). Structure and activity of lipase B fromC. antarctica in ionic liquids has been studied (van Rantwijk et al.,2006). Lipases as a catalyst and t-butanol or acetone, a mixture ofsolvent and ionic liquid as solvents, have been used for the synthesisof ester-based surfactants (Karmee, 2008).

6.3. Applications in food industry

In the food industry, reaction need to be carried out at a lowtemperature in order to avoid changes to food ingredients caused byundesirable side-reaction that would otherwise occur at highertemperatures. Lipases have become an integral part of modern foodindustry. The use of enzymes to improve the traditional chemicalprocesses of food manufacture has been developed in the past fewyears. Stead (1986) and Coenen et al. (1997) stated that thoughmicrobial lipases are best utilized for food processing, a few, especiallypsychrotrophic bacteria of Pseudomonas sp. and a few moulds ofRhizopus sp. and Mucor sp. cause havoc with milk and dairy productsand with soft fruits. An example of the application of a cold-adaptedenzyme in non-aqueous biotransformation is the use of a lipase fromPseudomonas strain P38 for the synthesis in n-heptane of the flavoringcompound, butyl caprylate (Tan et al., 1996). Immobilized lipases fromC. antarctica (CAL B), C. cylindracea AY30, H. lanuginosa, Pseudomonassp. and G. candidumwere used for the esterification of functionalizedphenols for synthesis of lipophilic antioxidants to be used insunflower oil (Buisman et al., 1998; Pandey et al., 1999). Whole-cellbiocatalyst of mutated C. antarctica lipase B (mCAL B) by a yeastmolecular display system and its practical properties were studied(Kato et al., 2007). When mCAL B was displayed on the yeast cellsurface, it showed a preference for short-chain fatty acids, an advant-age for producing flavors.

6.4. Domestic applications

The most commercially important field of application for hydro-lytic lipases is their addition to detergents, which are used mainly inhousehold and industrial laundry and in household dishwashers. C.antarctica lipase was developed into recombinant enzyme used fordetergent formulation (Uppenberg et al., 1994a). Godfrey and West(1996) reported that about 1000 t of lipases are sold every year in thearea of detergents. Enzymes can reduce the environmental load of

detergent products, since they save energy by enabling a lower washtemperature to be used; allow the content of other, often less desir-able, chemicals in detergents to be reduced; are biodegradable,leaving no harmful residues; have no negative impact on sewagetreatment processes; and do not have a risk to aquatic life. Commercialpreparations used for the desizing of denim and other cotton fabrics,contains both α amylase and lipase enzymes. Lipases are stable indetergents containing protease and activated bleach systems. Lipase isan enzyme, which decomposes fatty stains into more hydrophilicsubstances that are easier to remove than similar non-hydrolysedstains (Fuji et al., 1986). The commercial applications of lipasesincludes, detergents is in dish washing, clearing of drains clogged bylipids in food processing or domestic/industrial effluent treatmentplants (Bailey and Ollis, 1986). Further, it is used in liquid leathercleaner (Kobayashi, 1989), a bleaching composition (Nakamura andNasu, 1990), decomposition of lipid contaminants in dry-cleaningsolvents (Abo, 1990), contact lens cleaning (Bhatia, 1990), degradationof organic wastes on the surface of exhaust pipes, toilet bowls, etc.(Moriguchi et al., 1990). Removal of dirt/cattle manure from domesticanimals by lipases and cellulases (Abo,1990), washing, degreasing andwater reconditioning by using lipases along with oxidoreductases,which allows for smaller amounts of surfactants and operation at lowtemperatures (Novak et al., 1990). The lipase component causes anincrease in detergency and prevents scaling. The cleaning power ofdetergents seems to have peaked; all detergents contain similaringredients based on similar detergency mechanisms. To improvedetergency, modern types of heavy-duty powder detergents andautomatic dishwasher detergents usually contain one or moreenzymes (Ito et al., 1998). Lipases show unusual versatile substratespecificity. The tertiary structure of lipases is known, there arepresently significant efforts to improve this class of enzymes byprotein engineering techniques, in view of their use in detergents andother fields of industrial application (Schmid and Verger, 1998). Coldactive lipase from Microbacterium phyllosphaerae and Bacillus sphaer-icus has a remarkable capacity to retain its activity in presence ofcommercially available detergents and exhibited high efficiency forthe removal of lipid stains (kitchen oil stains and used engine oil stain)from fabrics (Joseph, 2006). These reports appear that these coldactive lipolytic enzymes can be used as detergent additive for coldwashing.

6.5. Environmental application

Bioremediation for waste disposal is a new avenue in lipase bio-technology. Cold-adapted organophosphorus acid anhydrolases wascharacterized for application in the efficient detoxification of pesticideand nerve agents (Cheng et al., 1997). Cold-adapted lipases have greatpotential in the field of wastewater treatment, bioremediation in fatcontaminated cold environment and active compounds synthesis incold condition (Buchon et al., 2000). This aspect requires more effortsin identifying and cloning novel lipase genes. Suzuki et al. (2001)identified as a psychrotrophic strain of the genus Acinetobacter strainno. 6 produced extracellular lipolytic enzyme that efficiently hydro-lyzed triglycerides such as soybean oil during bacterial growth even at4 °C; it degraded 60% of added soybean oil (initial concentration,1%w/v) after cultivation in LB medium at 4 °C for 7 days. The bacterium ispotentially applicable to in situ bioremediation or bioaugumentationof fat contaminated cold environments. Belousova and Shkidchenko(2004) isolated 30 strains capable of oil degradation at 4–6 °C.Maximum degradation of masut and ethanol benzene resins wereobserved in Pseudomonas sp. andmaximum degradation of petroleumoils and benzene resins were observed in Rhodococcus sp. Further,they stated that the introduction of psychrotrophic microbial de-graders of oil products into the environment is most important in thecontest of environmental problems in temperate regions. Ramtekeet al. (2005) stated that in temperate regions, large seasonal variations

Table 9Patent details of cold active lipases

Micro organism Patented item/process Patent number Inventor(s) Industrial partner

Process based patentsCandida antarctica Process for the preparation of optically active amines US6387692 Stelzer Uwe (De); Dreisbach Claus (De) Bayer AGC. antarctica Process for preparing esters WO0153511 Christensen Morten Wuertz (dk);

Borch Kim (dk)Christensen Morten Wuertz

C. antarctica Enzymatic synthesis of polyesters US5962624 Hill Karlheinz (De); LagardenMartin (De); (+3)

Hendel Komm andItgesells Chaft A

C. antarctica Process for the enzymatic resolution of N-(alkoxycarbonyl)-4-ketoproline alkyl esters or N-(alkoxycarbonyl)-4-hydroxyproline alkyl esters using lipase B

US5928933 Hong Wonpyo (US);Dicosimo Robert (US)

Du Pont (US)

C. antarctica Process for the preparation of trans-2, cis-4-decadienoicacid ethyl ester

US5753473 Gatfield Ian (De);Kindel Guenter (De)

Haarmann & Reimer Gmbh (De)

C. antarctica Process for producing triglycerides from glycerol andlong-chain polyunsaturated fatty acids using lipase

US5604119 Haraldsson Gudmundur G (Is);Svanholm Hanne (Dk); (+1)

Novonordisk; Lysi Hf

C. antarctica Process for stereoselection of (2R,3S)-3-phenylgycidicester using lipase

US5407828 Kierkels Joannes G T (Nl);Peeters Wijnand P H (Nl)

DSM NV

C. antarctica Immobilization of thermostable microbial lipase byadsorption to macroporous inorganic carrier particles

US5342768 Pedersen Sven (Dk);Hansen Tomas T (Dk)

Novonordisk

C. antarctica Lipase-catalyzed ester hydrolysis WO9218638 Heldt–Hansen Hans Peter (Dk);Awaji Haruo (Jp); (+3)

Novonordisk AS (Dk);Jujo Paper Co Ltd (Jp)

C. antarctica Method for producing optically active s-6-hydroxy-2,5,7,8-tetramethylcumarone-2-carboxylic acid

JP2003144190 Tamura Yutaka Mitsubishi Gas Chem Co Inc

C. antarctica T-24 Method for treating soy sauce oil JP2002101847 Furubayashi Makio;Nakahara Tadaatsu; (+3)

Higashimaru Shoyu Co Ltd

C. antarctica Para-dioxanone-based polymer JP2000044658 Nishida Haruo;Yamashita Mitsuhiro Tokuyama CorpC. antarctica Production of (d)-3(2h)-furanone compounds JP10084988 Suzuki Akio;Nozaki Michio Takasago Internatl CorpC. antarctica Enzymatic production of optically active compound JP7115992 Takagi Naoyuki; Others: 04 Nippon Soda Co LtdC. antarctica Degradation of biologically degradable polymers using

lipase from C. antarctica and a cutinaseNZ337239 Koch Rainhard; Lund Henrik Bayer AG

C. antarctica Enzymatic resolution of benzodiazepine-acetic acid esters(3-oxo-2,3,4,5-1H-tetrahydro-1,4-benzodiazepine-2-aceticacid) with a lipase from C. Antarctica

NZ336376 Wells Andrew Stephen Smithkline Beecham p

C. antarctica Solvent-free method for reacting short-chain alcohols andacids by lipase immobilized to acrylic resin

US5908769 Cho Nam Ryun (Kr);Hwang Soon Ook (Kr); (+1)

Yukong Ltd (Kr)

C. antarctica Process for the esterification of carboxylic acids withtertiary alcohols using a lipase

US5658769 Bosley John Anthony (GB);Casey John (GB); (+2)

Unichem Chemie Bv (Nl)

C. antarctica Resolution of (RS)-ibuprofen catalyzed esterification withlong-chain alcohols while removing water

US5561057 Trani Michael (Ca);Ergan Fran Oise (Fr); (+1)

Canada Nat Res Council (Ca)

Product based patentsC. antarctica C. antarctica lipase and lipase variants US6020180 Egel–Mitani Michi (Dk); Hansen

Mogens Trier (Dk); (+4)Novonordisk As (Dk)

C. Antarctica C. antarctica lipase variants US6074863 Egel–Mitani Michi (Dk); HansenMogens Trier (Dk); (+4)

Novonordisk As (Dk)

C. Antarctica Enzymatic ammonolysis process for the preparationof intermediates

US7223573 Ramesh N. Patel; Ronald L. Hanson;Iqbal Gill; (+6)

NA

Candida sp. Thermally stable and positionally non-specific lipase US5273898 Ishii Michiyo (Jp) Novonordisk As (Dk)

NA: Not available.


in temperature reduce the efficiency of microorganisms in degradingpollutants such as oil and lipids. The enzymes active at low andmoderate temperature may also be ideal for bioremediation process.

6.6. Patents in cold active lipases

Given the high risk and cost concerned in pursuing this largelyunexplored field, it is not surprising that the number of companiesinvolved in funding cold lipase research, screening samples andapplying for Antarctic-based patents is restricted. Eventhough somenoteworthy discoveries based on Antarctic lipases and with potentialcommercial applications were made in collaboration with industrialpartners (Table 9). However, it appears that none of these discoverieshas led to commercialization yet. Patent applicants are largely phar-maceutical, chemical and food companies. Most patents are process,rather than product based and centers on an isolate from an organism(frequently from the yeast C. antarctica), rather than on a syntheticderivative. C. antarctica, one of 154 species of the genus Candida,belongs to the Phylum Ascomycota and to the Class Ascomycetes. It isan alkali-tolerant yeast found in the sediment of Lake Vanda,Antarctica. Two lipase variants from C. antarctica, lipase A and B,have proven of particular interest to researchers.

7. Conclusions and future prospects

Biocatalysis at cold conditions now exist for chemical synthesis andtransformation, bioremediation of contaminants and clean-energyproduction, confirming and reinforcing the potential of this technologyfor environmental purposes. Cold active lipases are promising enzymesto replace the conventional enzyme processes of the biotechnologicalindustries. However, a more extensive effort is required to overcomeseveral bottlenecks: highenzymecost, lowactivityand/or stabilityunderenvironmental conditions, low reaction yields and the low biodiversityof psychrophilic microbes explored so far. The relatively recentintroduction and development of novel recombinant DNA technologiessuch as, metagenomics and site-directed mutagenesis have a profoundpositive effect on the expression and production of greater and greateramounts of recombinant proteins, which means more competitiveprices, by introducingnewor tailoredcatalytic activities of theseproteinsat low temperature. Thus, efforts have to be made in order to achieveeconomical overproduction of cold active lipase in heterologous hostsand their modification by chemical means or protein engineering toobtain more robust and active lipases. Further investigations shouldconsider modeling of such thermostable cold active lipases that can beused for various industrial and biotechnological applications.


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cold active microbial lipases: some hot issues and recent developments

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