Cold active microbial lipases: Some hot issues and recent developments

Biotechnology Advances 26 (2008) 457–470

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Biotechnology Advances j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

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, India b Department of Molecular Biology and Genetic Engineering, College of Biotechnology and Allied Sciences, Allahabad Agricultural Institute-Deemed University, Allahabad 211 007, Uttar Pradesh, India

A R T I C L E

I N F O

Article history: Received 6 November 2007 Accepted 9 May 2008 Available online 18 May 2008 Keywords: Biocatalysts Cold active lipase Enzymes Industrial application Lipolytic Psychrophiles

A B S T R A C T Lipases are glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. Lipases catalyze esterification, interesterification, acidolysis, alcoholysis and aminolysis in addition to the hydrolytic activity on triglycerides. The temperature stability of lipases has regarded as the most important characteristic for use in industry. Psychrophilic lipases have lately attracted attention because of their increasing use in the organic synthesis of chiral intermediates due to their low optimum temperature and high activity at very low temperatures, which are favorable properties for the production of relatively frail compounds. In addition, these enzymes have an advantage under low water conditions due to their inherent greater flexibility, wherein the activity of mesophilic and thermophilic enzymes are severely impaired by an excess of rigidity. Cold-adapted microorganisms are potential source of cold-active lipases and they have been isolated from cold regions and studied. Compared to other lipases, relatively smaller numbers of cold active bacterial lipases were well studied. Lipases isolated from different sources have a wide range 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 processes that closely approach the gentle, efficient processes in nature. Some of these processes using cold active lipase from C. antarctica have been patented by pharmaceutical, chemical and food industries. Cold active lipases cover a broad spectrum of biotechnological applications like additives in detergents, additives in food industries, environmental bioremediations, biotransformation, molecular biology applications and heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies. Cold active enzymes from psychrotrophic microorganisms showing high catalytic activity at low temperatures can be highly expressed in such recombinant strains. Thus, cold active lipases are today the enzymes of choice for organic chemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, microbiologists and biochemists. © 2008 Elsevier Inc. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Cold active lipases . . . . . . . . . . . . . . . . . . . . . . Structural features of lipase . . . . . . . . . . . . . . . . . . 3.1. General lipase structure . . . . . . . . . . . . . . . . 3.2. Modifications of lipase structure for cold adaptation . . . 3.3. Structure of Candida antarctica lipase . . . . . . . . . . Production of cold active lipases . . . . . . . . . . . . . . . 4.1. Psychrophiles as sources of cold active lipases . . . . . . 4.2. Fermentation conditions for cold active lipase production 4.3. Purification and characterization of cold active lipases . . Biotechnological approaches in cold active lipase. . . . . . . . 5.1. Gene cloning . . . . . . . . . . . . . . . . . . . . . 5.2. Protein engineering . . . . . . . . . . . . . . . . . .

⁎ Corresponding author. Tel.: +91 532 2684296; fax: +91 532 2684593. E-mail address: [email protected] (B. Joseph). 0734-9750/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2008.05.003

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Industrial applications of cold active lipases . . 6.1. Medical and pharmaceutical applications 6.2. Synthesis of fine chemicals . . . . . . . 6.3. Applications in food industry . . . . . . 6.4. Domestic applications . . . . . . . . . 6.5. Environmental application . . . . . . . 6.6. Patents in cold active lipases . . . . . . 7. Conclusions and future prospects . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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1. Introduction Lipase belongs to the enzyme class of hydrolases (E.C.3). It acts on ester bonds (E.C.3.1) of carboxylic esters (E.C.3.1.1). They hydrolyze triacylglycerols 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 lipolytic enzymes play an important role in the turnover of these water insoluble compounds. Lipases break and or modify the carboxyl ester bonds of lipids and its derivatives. Hydrolysis of fat is the primary reaction of lipases (Khare et al., 2000). Lipolytic enzymes are involved in breakdown and thus in the mobilization of lipids within cells of individual organism as well as in the transfer of lipids from one organism to another (Beisson et al., 2000). Lipases catalyze esterification, interesterification, acidolysis, alcoholysis and aminolysis in addition to the hydrolytic activity on triglycerides. As hydrolases, they do not require cofactors. They usually exhibit good chemioselectivity, regioselectivity and enantioselectivity. Finally, lipases possess broad substrate specificity and found with optimum activities over a wide range of temperatures. These interesting properties make lipases the most versatile biocatalyst (Kademi et al., 2005). Lipases have emerged as one of the leading bio-catalyst/bio-accelerators with proven potential for contributing to the multibillion dollar under exploited lip-tech bio-industry and are used both in situ lipid metabolism and ex situ multi-faceted industrial application (Benjamin and Pandey, 1998; Pandey et al., 1999). Few reports reveal that lipases have emerged as key enzymes in swiftly growing biotechnology, owing to their multi-faceted properties, which find usage in a wide array of industrial applications, such as food technology, detergent, chemical industry and biomedical sciences (Jaeger et al., 1999; Pandey et al., 1999). The commercial use of lipases is a billion dollar business that comprises a wide variety of different applications including synthesis of biopolymer and biodiesel, production of pharmaceuticals, agrochemicals, cosmetics and flavors (Haki and Rakshit, 2003). The global market for industrial enzymes was estimated nearly US $ 2 billion in 2004. As a result, the market is expected to rise at an average annual growth rate (AAGR) of a little over 3% over the next four years, and the total industrial enzyme market in 2009 is expected to reach nearly US $ 2.4 billion (Rajan, 2004; Hasan et al., 2006). Through the number of review articles (Sharma et al., 2001; Gupta et al., 2004; Akai and Kita, 2007), importance of lipases can be easily envisioned. Over the last few years, there has been a dramatic increase in the number of publications in the field of lipase-catalyzed reactions performed in common organic solvents, ionic liquids or even non-conventional solvents. Considerable research has shown that reactions catalyzed by enzymes are more selective and efficiently performed than many of their analogues in the organic chemistry laboratory (Ghanem, 2007). The present review adopts 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, which function effectively at cold temperatures with high rates of catalysis in

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comparison to the lipases from mesophiles or thermopiles, which shows little or no activity at low temperature. These lipases have evolved a range of structural features that confer a high level of flexibility, particularly around the active site are translated into low activation enthalpy, low-substrate affinity, and high specific activity at low temperatures. Moreover, the maximum level of activity of these lipases is shifted towards lower temperatures with a concomitant decrease in thermal stability. The knowledge of cold active lipolytic enzymes is increasing at a rapid and exciting rate. Unfortunately, the studies 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 important enzyme and its characteristics has been collected and compiled from the information available in the literature. From the limited number of available reports on cold active lipases, it is clear that most of the studies were focused on isolation, purification and characterization of these enzymes followed by gene cloning, expression and sequencing. The genes encoding for cold active lipases were isolated and cloned into mesophilic bacteria (E. coli) as host organism and used for their expression. However, the review of Gerday et al. (1997) revealed an extremely unstable condition for the expression of coldadapted lipases within their host (Feller et al., 1990, 1991a). In other reports related to expression studies, the stability of gene encoding lipase production in the host is not clear. A worldwide initiative has taken up for exploring cold active lipase producing microorganisms and their industrial applications. 3. Structural features of lipase 3.1. General lipase structure The lipase consists of a single domain molecule and all lipases conform to a common structural organization, viz., the alpha/beta hydrolase fold (Ollis et al., 1992; Nardini and Dijkstra, 1999). The active site of lipase contains the catalytic triad, Ser105-His224-Asp187, common to all serine hydrolases where Ser as the nucleophile, His as the basic residue, and Asp or Glu as the acidic residue (Uppenberg et al., 1994a,b). Such a catalytic triad exists in enzymes with different folding, including trypsin and subtilisin, and is an example of convergent evolution (Holmquist, 2000). Access to the active site, consisting a serine, histidine, carboxylic acid triad may be shielded by a mobile lid, whose position closed or open determines the enzyme in an inactive or active conformation. The activation can be explained by the opening of a lid (flap) structure of the enzyme at an interface. The lipase with open lid is the active form of the enzyme and gives the substrate access to the active site. The substrate-binding site is located inside 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 to substrate specificity (Pleiss et al., 1998) and residues that contact the substrate have been identified by crystallography and docking. However, it is recognized that other protein regions, such as the lid itself (Brocca et al., 2003) and the reaction conditions (Verger, 1980) may play a role in lipases specificity.

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3.2. Modifications of lipase structure for cold adaptation Cold active lipases are structurally modified by an increasing flexibility of the polypeptide chain enabling an easier accommodation of substrates at low temperature. The fundamental issues concerning molecular basis of cold activity and the interplay between flexibility and catalytic efficiency are important in the study of structure– function relationships in enzymes. Such issues are often approached through comparison with the mesophilic or thermophilic counterparts, by site-directed mutagenesis and 3D crystal structures (Narinx et al., 1997; Wintrode et al., 2000). The molecular modelling of Pseudomonas immobilis lipase revealed several features of cold-adapted lipases (Arpigny et al., 1997). A very low proportion of arginine residues as compared to lysines, a low content in proline residues, a small hydrophobic core, a very small number of salt bridges and of aromatic–aromatic interactions are the possible features of lipase for cold adaptation. Similarly the weakening of hydrophobic clusters, the dramatic 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 Xanthobacter autotrophicus, the cold active lipase displays a very small number of aromatic–aromatic interactions and of salt bridges. The location of some salt bridges which are absent in the cold lipase seems to be crucial for the adaptation to cold. A large amount of charged residues exposed at the protein surface, have been detected in the cold active lipase from Pseudomonas fragi (Alquati et al., 2002). They also observed a reduced number of disulphide bridges and of Prolines in loop structures. Arginine residues were distributed differently than in mesophilic enzymes, with only a few residues involved in stabilizing intramolecular salt bridges and a large proportion of them exposed at the protein surface that may contribute to increased conformational flexibility of the cold-active lipase. In addition to this, the structural factors possibly involved in cold adaption are increased number and clustering of glycine residues (providing local mobility), lower number of ion pairs and weakening of charge-dipole interactions in α helices (Georlette et al., 2004; Gomes and Steiner, 2004). The substitution of Glycine with Proline by mutation caused a shift of the acyl chain length specificity of the enzyme towards short-chain fatty acid esters and enhanced themostability of the enzyme (Kulakovaa et al., 2004). A mutation in the lid region of catalytic triad of cold active lipases from P. fragi improved substrate selectivity and thermostability (Santarossa et al., 2005). Introduction of polar residues in the surface of exposed lid might be involved in improved substrate specificity and protein flexibility. The sequence alignment study of cold active lipase from Photobacterium 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). It is understood that the catalytic cavity of the psychrophillic lipase is characterized by high plasticity. These structural adaptations may confer on the enzyme a more flexible structure, in accordance with its low activation energy and its low thermal stability. The above discussions may help to obtain information for insights into the molecular mechanisms of cold adaption and thermolability of cold active lipases. 3.3. Structure of Candida antarctica lipase Literature reviews reveal that the Antarctic yeast (C. antarctica) is the most extensively studied microorganism with respect to its lipase secretion. The psychrophilic yeast, C. antarctica, originally isolated from Antarctic habitat expresses two lipase variants viz., C. antarctica lipase A (CAL A) and C. antarctica lipase B (CAL B) with different physiochemical properties (Kirk and Christensen, 2002). Many of the lipases show interfacial activation; their activity is much higher when acting on substrates at a water–micelle interface compared to the dissolved substrates. CAL A shows interfacial activation, while CAL B

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does not show such behavior and is therefore not considered to be a true lipase (Martinelle and Hult, 1995). CAL B was found to be selective for position sn-3 and CAL A could preferentially cleave sn-2 ester bond (Rogalska et al., 1993). The interfacial activation can be explained by the opening of a lid (flap) structure of the enzyme at an interface. This lid structure covering the active site of true lipases is absent or very small in CAL B. The structure of CAL B was solved in 1994 (Uppenberg et al., 1994a,b, 1995). The CAL B contains the sequence, Thr-x-Ser-x-Gly around the active-site Ser, whereas in all other microbial and mammalian lipases, Thr is replaced by Gly. When Thr was replaced by a Gly in a CAL B site-directed mutant (expected to create local flexibility), thermostability increased (rather than decreased) with a concomitant decrease in specific activity. The unexpected increase in thermostability and the decrease in specific activity were thought to arise from the replacement of Thr side-chains with Gly enhancing contacts between secondary structural elements around the active site (Patkar et al., 1997). The active site of CAL B possesses an oxyanion hole that stabilizes the transition state and the oxyanion in the reaction intermediate. This oxyanion hole is a spatial arrangement of three hydrogen-bond donors, one from the side chain of Thr40 and two from the back-bone amides of Thr40 and Gln106. The active site also contains a small cavity called the stereospecificity pocket (Uppenberg et al., 1995), in which secondary alcohols have to orient one substituent during catalysis. This gives CAL B a high enantioselectivity towards chiral secondary alcohols. The enantioselectivity of CAL B towards secondary alcohols is determined by the steric requirements of the stereospecificity pocket. The fast-reacting enantiomer of the secondary alcohols orients its large substituent towards the activesite entrance and its medium-sized substituent in the stereospecificity pocket. To react, the slow-reacting enantiomer has to have the opposite orientation for its substituents compared to the fast-reacting enantiomer. The large substituent is not easily accommodated in the stereospecificity pocket, which explains the low reaction rate of this enantiomer (Rotticci et al., 1998). Enzyme variants were created where amino acids predicted to play key roles for the lipase activity in the different models were replaced by an inert amino acid (alanine). Kasrayan et al. (2007) studied activity measurements of the overproduced and purified mutant CAL A. Moreover, found that the active site consists of amino acid residues Ser184, His366, and Asp334 and in which there is no lid. They suggested that this model could be used for future targeted modifications of the enzyme to obtain new substrate acceptance, 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 surviving at low temperatures near 5 °C. Although a number of lipase producing sources are available, only a few bacteria and yeast were exploited for the production of cold active lipases. Attempts have been made from time to time to isolate cold active lipases from these microorganisms having high activity at low temperatures. A list of various cold active lipase producing psychrophillic and psychrotrophic bacteria is presented in Table 1. These bacterial strains were isolated mostly from Antarctic and Polar regions which represents a permanently cold (0 ± 2 °C) and constant temperature habitat. Various studies shows that a high bacterial count has been recorded as high as 105 ml− 1 and 106 ml− 1 in water column and in the sea ice respectively (Sullivan and Palmisano, 1984; Delille, 1993). Another potential source of cold active lipases is deep-sea bacteria. A marine bacterium Aeromonas hydrophila growing at a temperature range between 4 and 37 °C produced cold active lipolytic enzyme (Pemberton et al., 1997). Few bacterial genera have been isolated and characterized from deepsea sediments where temperature is below 3 °C. They include Aeromonas sp. (Lee et al., 2003); Pseudoalteromonas sp. and Psychrobacter

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Table 1 Bacteria producing cold active lipases Microorganism

Sources

Acinetobacter sp. strain no. 6 Siberian tundra soil Acinetobacter sp. strain no. O16 Ns Achromobacter lipolyticum Ns Aeromonas sp. strain no. LPB 4 Sea sediments Aeromonas hydrophila Marine habitat Bacillus sphaericus MTCC 7526 Gangothri Glacier (Western Himalaya) Microbacterium phyllosphaerae MTCC 7530 Corynebacterium Naukuchiatal lake paurametabolum MTCC 6841, Uttaranchal (Western Himalaya) Moraxella sp. Antarctic habitat Morexella sp TA144 Antarctic habitat Photobacterium Marine habitat lipolyticum M37 Pseudoalteromonas sp. Deep-sea sediments wp27 Pseudoalteromonas sp. Antarctic marine Psychrobacter sp. Vibrio sp. Pseudomonas sp. Subterranean strain KB700A environment Pseudomonas sp. B11-1: Alaskan soil Pseudomonas P38 Ns Pseudomonas fluorescens Refrigerated milk samples Pseudomonas fluorescens Refrigerated food Pseudomonas fluorescens Refrigerated human placental extracts Pseudomonas fragi BCCM™/LMG2191T strain no. IFO3458 BCUG, Belgium Pseudomonas fragistrain no. Ns IFO 12049 strain no. IFO 12049 Psychrobacter okhotskensis sp. Sea coast Psychrobacter sp. wp37 Deep-sea sediments Psychrobacter sp. Ant300 Antarctic habitat Psychrobacter immobilis Antarctic habitat strain B 10 Psychrobacter sp. 7195 Psychrobacter sp. Serratia marcescens Staphylococcus aureus Staphylococcus epidermidis

Antarctic habitat Antarctic habitat Raw milk Ns Frozen fish samples

References Suzuki et al. (2001) Breuil and Kushner (1975) Khan et al. (1967) Lee et al. (2003) Pemberton et al. (1997) Joseph (2006)

Joshi et al. (2006)

Feller et al. (1990) Feller et al. (1991a) Ryu et al. (2006) Zeng et al. (2004) Giudice et al. (2006)

Rashid et al. (2001) Choo et al. (1998) Tan et al. (1996) Dieckelmann et al. (1998) Andersson (1980) Preuss et al. (2001) Alquati et al. (2002)

Aoyama et al. (1988)

Yumoto et al. (2003) Zeng et al. (2004) Kulakovaa et al. (2004) Arpigny et al. (1997)

Zhang et al. (2007) Parra et al. (2007) Abdou (2003) Alford and Pierce (1961) Joseph et al. (2006)

Ns: Not specified.

sp. (Zeng et al., 2004) P. lipolyticum (Ryu et al., 2006). Bacterial genera including P. fragi (Aoyama et al., 1988; Alquati et al., 2002), Pseudomonas fluorescens (Dieckelmann et al., 1998) and S. marcescens (Abdou, 2003) which produces cold active lipases were isolated from refrigerated milk and food samples. Permanently cold regions such as glaciers and mountain regions are another habitat for cold active lipase producing microorganisms. The soil and ice in Alpine region also harbor psychrophillic microorganisms, which produces lipases. In addition to all these permanently cold regions, there are many other accessible and visible soil and water which become cold both diurnally and seasonally from which cold active lipase producing microbes can be isolated using appropriate low temperature techniques. The widespread use of refrigeration to store fresh and preserved foodstuffs provides a great diversity of nutrient rich habitat for some wellknown psychrotolerant food spoilage microorganisms. Cold active lipases were also reported in psychrophilic fungi and yeast. They include Candida lipolytica, Geotrichum candidum and Pencillium roqueforti isolated from frozen food samples for the cold active lipases production (Alford and Pierce, 1961). Aspergillus nidulans (Mayordomo

et al., 2000) and C. antarctica have been reported to produce cold active lipolytic enzymes. However, a deep research has been done on the C. antarctica (Table 2). 4.2. Fermentation conditions for cold active lipase production Cold active lipases are mostly extra cellular and are highly influenced by nutritional and physicochemical factors such as temperature, agitation, pH, nitrogen source, carbon source, inducers, inorganic sources and dissolved oxygen. Submerged fermentation is the most common method used for cold active lipase production (Dieckelmann et al., 1998; Lee et al., 2003). A list of various production parameters for different cold active lipase producing microorganisms is given in Table 3. Cold-adapted microorganisms tend to have good growth rate at low temperature. The production of cold active lipase is considered temperature dependent and thermolabile (Rashid et al., 2001). Moraxella sp. isolated from Antarctican habitat grows well at 25 °C and produced cold active lipolytic enzyme (Feller et al., 1990). Pseudomonas sp. strain B11-1 utilized yeast extract and tryptone as best carbon and nitrogen sources for growth and production of lipases. Tween 80 and Tributyrin induced production of cold active lipases at 4 °C with an optimum pH 7.6 (Choo et al., 1998). A. nidulans WG312 produced cold active lipase by utilizing olive oil as an inducer at 30 °C (Mayordomo et al., 2000). Soybean oil induced the production of cold active lipases from Acinetobacter sp. strain no. 6 at 4 °C within four days (Suzuki et al., 2001). Aeromonas sp. LPB 4 produced lipase at 10 °C in eight days time duration by using tryptone and yeast extract as carbon and nitrogen source and trybutylin as an inducer (Lee et al., 2003). Serratia marcescens produced cold active lipase in presence of skim milk as energy source at 6 °C in 6 days of incubation. Tween 80 and Tween 20 were the best inducers for cold-adapted lipase production with yeast extract as carbon source in 14 days at 25 °C for Psychrobacter sp. wp37. Another isolate of Pseudoalteromonas sp. wp27 produced lipases at 25 °C in 14 days with yeast extract as carbon source 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 strategies. However, in these years new techniques have been developed that may yield high recovery. Based on the nature of lipase produced by the organism one has to design the protocol for purification (Saxena et al., 2003). The significance of cold active lipases is extensively recognized in a number of applications (Houde et al., 2004). The purified lipase is needed for the synthesis of fine chemicals, cosmetics and in pharmaceutical industries. However, homogenous preparation of cold-adapted lipases is not required for all industrial applications. A list of purified cold active lipases and results obtained during purification studies are given in Table 4. Lipases from Antarctic coldadapted bacteria have been the subject of several studies, all of them failing to obtain purified forms due to the difficulty of eliminating Table 2 Fungi producing cold active lipases Microorganism

Sources

References

Aspergillus nidulans Candida antarctica

Ns Antarctic habitat

C. lipolytica Geotrichum candidum Pencillium roqueforti

Frozen food

Mayordomo et al. (2000) 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) Alford and Pierce (1961)

Ns: Not specified.

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Table 3 Overview of production parameters for cold active lipase Microorganism

pH

Temp.

Incubation period

C source

N source

References

(°C) Bacteria Aeromonas sp. LPB 4 Acinetobacter sp. strain no. 6 Bacillus sphaericus MTCC 7526 Corynebacterium paurometabolum MTCC 6841 Microbacterium phyllosphaerae MTCC 7530 Moraxella sp. Pseudoalteromonas sp. wp27 Pseudomonas sp. strain KB700A Pseudomonas sp. strain B11-1 Psychrobacter sp. wp37 Serratia marcescens

Ns Ns 8 8.5 8 Ns Ns 7.2 7.6 Ns Ns

10 4 15 25 15 25 25 −5 4 25 6

8 days 7 days 48 h Ns 36 h Ns 14 days Ns Ns 14 days 3 days

Trybutylin Soybean oil Lactose/Tributyrin Soybean oil/olive oil Tributyrin/Lactose Ns Olive oil, Tween80 Tributyrin Tween80, Tributyrin Tween80, Tween20 Ns

Tryptone, Yeast extract Ns Peptone NaNO3 and KNO3 Peptone Ns Yeast extract Tryptone, Yeast extract Yeast extract, Tryptone Yeast extract Skim milk

Lee et al. (2003) Suzuki et al. (2001) Joseph (2006) Joshi et al. (2006) Joseph (2006) Feller et al. (1990) Zeng et al. (2004) Rashid et al. (2001) Choo et al. (1998) Zeng et al. (2004) Abdou (2003)

Fungus Aspergillus nidulans WG312

Ns

30

Ns

Ns

Olive oil

Mayordomo et al. (2000)

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

lipopolysaccharides produced by Antarctic microorganisms, and found strongly associated with the lipid hydrolases (Gerday et al., 1997). The investigations on cold active lipases from Psychrobacter immoblis B10 were perused on semi-purified preparations, the nucleotide sequence of which is also available (Arpigny et al., 1995). Lipase purity is evaluated after each purification step by measuring the overall activity and specific activity. The purification efficiency is determined 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 the protein containing lipases by ammonium sulphate precipitation, ultrafiltration or extraction with organic solvents. Since lipases are known to be hydrophobic in nature having large hydrophobic surfaces around the active site, the purification may be achieved by opting for affinity chromatographic techniques. The widely used chromatographic technique involves columns packed with QAE sephadex, CM cellulose, DEAE cellulose, phenyl-sepharose etc. However in certain applications, further purification can be achieved by gel filtration chromatography. The usual procedures for purification of lipases are troublesome, time consuming and results in low yield. Low thermostability of these cold active lipases is also a major problem in purification. Novel purification steps are therefore needed to increase the overall enzyme yield and purification fold. The effective catalytic properties of enzymes have led to introduction 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 as protein engineering (Kim and Choi, 1984; Joo et al., 1998; Eijsink et al., 2004) and directed evolution, have provided important tools for the efficient development of new enzymes. The characterization and

kinetic study of cold active lipase were studied in terms of optimum pH and stability, optimum temperature, thermo-stability and effect of chelating agents, inhibitors, solvents and metal ions. The cold active lipases from microorganisms have an optimum activity at 20 °C and are stable at a wide range of temperatures. However, these cold active enzymes are unstable above 65 °C (Table 5). These cold active lipases possessing stability at various physical and chemical conditions may have potentials in biotechnological and industrial applications at low temperatures. The molecular weight of these purified proteins varied from 50–85 kDa and they have broad substrate specificity. Lipases are widely used as industrial catalyst; there are several advantages and disadvantages for industrial application of cold active lipases. They are easily deactivated when subjected to heat, extreme pH range or in organic solvents (Jensen, 1983; Longo and Combes, 1999; Matsumoto et al., 2001; Noel and Combes, 2003). Numbers of strategies have been proposed to overcome such a limitation including the use of soluble additives, immobilization, protein engineering, and chemical modification (Kwon and Rhee, 1984; Chae et al.,1998; Park et al., 2001b; Lee et al., 2002). The modification of protein surface with modifiers by chemical binding appears to be a good strategy to improve biocatalyst performance. Modified enzymes were typically macroscopic catalysts that retained in the reactor; therefore, continuous replacement of the enzyme is not necessary. The activity of the cold enzyme presents an apparent optional activity around 35 °C and retains about 20% of its activity at 0 °C. The activity of mesophilic lipase is close to zero below 20 °C and still increases at temperatures above 60 °C. Properties of cold lipase from P. immobilis strain B10 was compared with lipase from mesophillic bacterium, Pseudomonas aeruginosa (Arpigny et al., 1997). The activation energies evaluated from the Arrhenius plots are 63 and 110 kJ/mol for the cold and mesophilic enzymes, respectively underlining the cold character of lipase produced by the Antarctic bacterium. This characteristics also

Table 4 Purification of cold active lipases Organism

Purification technique

Fold increase/yield (%)

References

Aeromonas sp. LPB 4 Bacillus sphaericus MTCC 7526 Microbacterium phyllosphaerae MTCC 7530 Moraxella sp. Pseudomonas sp. Strain B11-1: — [gene cloned into E. coli and purified] Psychrobacter sp. 7195 Serratia marcescens

QAE Sephadex column DEAE cellulose DEAE cellulose AcA 34 column, Ultrafiltration DEAE Cellulofine

53.5/7.5 17.74/4.70 22.03/7.50 Ns 38/17

Lee et al. (2003) Joseph (2006) Joseph (2006) Feller et al. (1990) Choo et al. (1998)

DEAE Sepharose CL-4B, and Sephadex G-75 CM Cellulose, DEAE cellulose Sephadex G-150 phenyl-sepharose chromatography, linolenic acid-agarose

Ns 20/45

Zhang et al. (2007) Abdou (2003)

Ns

Mayordomo et al. (2000)

Aspergillus nidulans WG312 Ns: Not specified.

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Table 5 Characterization of cold active lipases Organism

Optimum

Stability

Temp./pH

Temp./pH

MW (kDa)

Comments

References

Acinetobacter sp. strain no. 6

20 °C/Ns

Ns/Ns

Ns

Suzuki et al. (2001)

35 °C/Ns

50 °C/Ns

50

Bacillus sphaericus MTCC 7526

15 °C/8.0

30 °C/8

40

Microbacterium phyllosphaerae MTCC 7530 Pseudo-alteromonas sp. wp27 Pseudomonas sp. strain KB700A Pseudomonas sp. strain B11-1: [recombinant] Psychrobacter sp. wp37 Psychrobacter sp 7195 [recombinant] Serratia marcescens Aspergillus nidulans WG312

20 °C/8.0

35 °C/8

42

20–30 °C/7.0–8.0 35 °C/8.0–8.5 45 °C/8.0

Ns/Ns Ns/Ns 5–35 °C/6.0–9.0

85 49.9 33.7

Broad specificity towards the acyl group (C8−C16) of ethyl esters Medium chain acyl group p-nitrophenyl esters seemed to be good substrate; Increased activity with detergents Stable in presence of organic solvents and compatible with detergents Presence of organic solvents activity compatible with detergents Enzymes were 60% active at 4 °C Highest activity with p-nitrophenyl caprate Strongly inhibited by Zn2+, Cu2+, Fe3+, Hg2+

Aeromonas sp. LPB 4

20–30 °C/7.0–8.0 30 °C/9.0 37 °C/8.0 40 °C/6.5

Ns/Ns Ns/7.0–10.0 65 °C/6.6 Low thermal stability/Ns

85 Ns 52 29

Enzymes were 60% active at 4 °C Ca2+ and Mg2+ enhanced activity Observed 90% activity at 5 °C preference toward esters of short- and middle-chain fatty acids

Lee et al. (2003) Joseph (2006) Joseph (2006) Zeng et al. (2004) Rashid et al. (2001) Choo et al. (1998) Zeng et al. (2004) Zhang et al. (2007) Abdou (2003) Mayordomo et al. (2000)

Ns: Not specified.

illustrated by the high thermosensitivity of the cold active lipase displaying at 60 °C a half-life 2 orders of magnitude lower than that of the mesophillic enzyme. The widely characterized cold active lipase is from yeast (C. antarctica). The two lipases variants are C. antarctica lipase A (CAL A) and C. antarctica lipase B (CAL B) with different physiochemical properties (Kirk and Christensen, 2002). CAL B belongs to the α/β-hydrolase fold superfamily (Ollis et al., 1992), which contains enzymes that have evolved from a common ancestor (divergent evolution) to catalyze various reactions such as hydrolysis of esters, thioesters, peptides, epoxides, and alkyl halides or cleavage of carbon bonds in hydroxynitriles (Holmquist, 2000). CAL B is made up of 317 amino acids and has a molecular weight of 33 kDa. CAL B is less thermostable, smaller in size, and more acidic than CAL A (Patkar et al., 1993). CAL B is a stable enzyme that has been used at 150 °C, in organic solvents of high polarity such as acetonitrile and dimethyl sulfoxide in ionic liquids, in solid/gas systems and in supercritical carbon dioxide (Suen et al., 2004). Recently, a cold active lipolytic enzyme was produced by cloning the putative lipolytic gene encoding lipo1 from the metagenomic library and expressed in Escherichia coli BL21 using the pET expression system (Roh and Villatte, 2008). The expressed recombinant enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography and characterized using general substrates of

lipolytic property. The gene consisted of 972 bp encoding a polypeptide of 324 amino acids with a molecular mass of 35.6 kDa. This lipolytic enzyme exhibited the highest activity at pH 7.5 and 10 °C. At thermal stability 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 develop radically different and novel biocatalysts through various molecular approaches including recombinant DNA technology, protein engineering, directed evolution and the metagenomic approach. As a whole, lipase biotechnology has just reached the end of lag phase and the beginning of the exponential phase: it demands extension in terms of both quality and quantity. Qualitative improvements in restructuring lipase gene and its protein can be achieved by employing already established recombinant DNA technology and protein engineering. Quantitative enhancement needs strain improvement, especially through site-directed mutagenesis and standardizing the nutrient medium for the overproduction of cold active lipases. Recently, Vakhlu and Kour (2006) compiled the informations 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

Table 6 Gene isolation and characterization Microorganisms

Studies conducted/Investigations undertaken

References

Moraxella sp. strain TA 144. Pseudomonas fluorescens strain C9

Analysis of sequence homology of Human HSL gene with Antarctic bacterium Isolation of lipase-encoding gene lip A

Langin et al. (1993) Dieckelmann et al. (1998)

Gene cloning, expression and sequencing Pseudomonas fragi P. fragi IFO-12049 Pseudomonas sp. Strain B11-1 Moraxella sp. Strain TA 144 Moraxella sp. Strain TA 144. Psychrobacter immobilis B10 Pseudomonas fluorescens Candida antarctica Pseudomonas sp. strain KB700A. P. fragi Psychrobacter sp. Ant 300 P. fragi Photobacterium lipolyticum M37 C. antarctica Psychrobacter sp. 7195 Moritella sp. 2-5-10-1

Molecular cloning and nucleotide sequencing of the lipase gene Cloning, sequencing and expression of the lipase gene Gene cloning and sequencing Sequencing of lipase gene Cloning and expression in E. coli of three lipase-encoding genes Cloning, sequencing and structural features of lipase gene Cloning and sequencing of DNA encoding Phospholipase C Protein expression of lipase B in Pichia pastoris Gene cloning Heterologous expression, and molecular modeling Gene cloning, expression and characterization Molecular properties, mutagenesis and overexpression of cold active lipase Isolation of a new cold-adapted lipase and gene cloning Functional expression of lipase B in Eschericha coli Cloning, expression, and characterization of a cold-adapted lipase gene Cloning and expression of lipP, a Gene encoding a cold-adapted lipase

Kugimiya et al. (1986) Aoyama et al. (1988) Choo et al. (1998) Feller et al. (1991b) Feller et al. (1991a) Arpigny et al. (1993) Preuss et al. (2001) Rotticci-Mulder et al. (2001) Rashid et al. (2001) Alquati et al. (2002) Kulakovaa et al. (2004) Lafranconi et al. (2005) Ryu et al. (2006) Blank et al. (2006) Zhang et al. (2007) Yang et al. (2008)

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

~33 and ~65 kDa. More than 50% reported lipases producing yeast; produce it in the forms of various isozymes. Various lipase-encoding genes in turn produce these lipase isozymes. 5.1. Gene cloning To date, a large number of cold active lipase genes were isolated and the related studies have been carried out (Table 6). Early successes in the production of heterologous proteins were achieved using Escherichia coli as host and various kinds of proteins were expressed in E. coli. However, expression of eukaryotic proteins in E. coli became very difficult due to formation of inclusion bodies, protein misfolding and safety issues. Other expression systems were developed among yeasts, fungi, plants and animals. Molecular cloning and nucleotide sequencing of the lipase gene in P. fragi (Kugimiya et al., 1986) and cloning, sequencing and expression of the lipase gene from P. fragi IFO-12049 has been reported (Aoyama et al., 1988). The earlier efforts on cloning and expression of the genes coding for cold active lipases in mesophilic organisms such as E. coli did not yield a stable integration of cold lipase genes within their hosts (Feller et al., 1990; Feller et al., 1991a). The cloning and expression of genes from a psychrotrophic bacterium in a mesophilic host has been described. Three lipaseencoding genes (lip) from the Antarctic psychrotroph, Moraxella TA144, were cloned by inserting Sau 3AI-generated DNA fragments into the Bam HI site of the pSP73 plasmid vector. To prevent heat denaturation of the gene product, the screening procedure on agar plates containing an emulsified lipid involved growing of E. coli recombinant colonies at 25 °C followed by incubation at 0 °C. The three recombinant lipases (reLip) were cell associated and differed by their respective specificity towards p-nitrophenyl esters of various aliphatic chain lengths. These cloned reLip conserved the main character of the wild-type enzymes i.e., a dramatic shift of the optimal temperature of activity towards low temperatures and pronounced heat lability (Feller et al., 1991a). Cloning and expression of three lipase-encoding genes of Moraxella sp. strain TA 144 in E. coli have been 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 from P. 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-adapted lipase from Pseudomonas sp. Strain B11-1 has been reported (Choo et al., 1998). DNA encoding Phospholipase C in P. fluorescens has been cloned 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 molecular modeling of cold-adapted lipase gene from P. fragi and the recombinant lipase retained significant activity at low temperature (Alquati et al., 2002). The three-dimensional structure was built by homology and compared with homologous mesophilic lipase which showed 45% sequential identity with P. aeruginosa lipase and 38% with Burholderia cepacia lipase. A PCR method was designed for the isolation of lipase gene directly from environmental DNA, using primers, based on lipase consensus (Bell et al., 2002). Gene cloning, expression and characterization have been carried out in Psychrobacter sp. Ant 300 (Kulakovaa et al., 2004). Recently, a gene (lipP, 837 bp in length) coding for a cold-adapted lipase of psychrophilic bacterium Moritella sp. 2-510-1 was isolated from Antarctic region was cloned and sequenced. The deduced amino acid sequence revealed a protein of 278 amino acid residues with a molecular mass of 30,521. The primary structure of lipase deduced from the nucleotide sequence showed consensus pentapeptide containing the active serine (Gly-Trp-Ser-Leu-Gly) and a conserved HisGly dipeptide in the N-terminal part of the enzyme. The gene was subcloned into pET-28a expression vector to construct a recombinant lipase protein and expressed in E. coli BL21 (DE3) (Yang et al., 2008).

463

The latest trend in lipase research is the development of novel and improved lipase through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach (Gupta et al., 2004). The main microbial expression systems are Aspergillus oryzae, Saccharomyces cerevisiae and P. pastoris. Recombinant DNA technology represents a very attractive technology that can be used to increase lipase production mainly in the case of isoenzymes whose purification leads to a very low yield. This allows up to 40% decrease in the cost of raw material, water, steam and electricity compared to the cost of native enzyme production. The first lipase produced by recombinant DNA technology was Lipolase introduced in the market by Novozymes in 1988. Originating from Thermomyces lanuginosus, formerly Humicola lanuginosa, this lipase was expressed in A. oryzae. The growing number of recombinant lipases is attributed to the recent progress in molecular technologies (cloning and sequencing) (Kademi et al., 2005). Functional expression of lipase B from C. antarctica in E. coli has been studied by Blank et al. (2006). Recently, a novel lipase was isolated from a metagenomic library of Baltic Sea sediment bacteria (Hardeman and Sjoling, 2007). Prokaryotic 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 and subcloned. Subclones with lipolytic activity were sequenced and an open reading frame of 978 bp encoding a 35.4 kDa putative lipase/ esterase h1Lip1 (DQ118648) with 54% amino acid similarity to a Pseudomomas putida esterase (BAD07370). Conserved regions, including the putative active site, GDSAG, a catalytic triad (Ser148, Glu242 and His272) and a HGG motif, were identified. The h1Lip1 lipase was overexpressed, (pGEX-6P-3 vector), purified and shown to hydrolyse p-nitrophenyl esters of fatty acids with chain lengths up to C14. Recently, lipo1 a novel psychrophilic esterase obtained directly from the metagenomic DNA was directly extracted from the activated sludge (Roh and Villatte, 2008). The gene consisted of 972 bp encoding a 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 low identity 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/gene fragments encoding cold-adapted lipases has also been isolated (Table 7). From the table, it is clear that only a limited number of studies were carried out in the isolation of cold active lipase-encoding gene/gene fragments. Table 7 Genes/gene fragments encoding cold active lipases Genes

Microorganism

References

lip2 lip3 lipP lipo1 lip A

Moraxella sp. TA144 Moraxella sp. TA144 Moritella sp. 2-5-10-1 metagenomic library Pseudomonas fluorescens strain C9 Pseudomonas sp. B11-1 Psychrobacter sp. 7195 Pseudomonas sp. Strain KB700A Pseudomonas fluorescens Psychrobacter immobilis B10 Psychrobacter sp. Ant300

Feller et al. (1991c) Feller et al. (1991b) Yang et al. (2008) Roh and Villatte (2008) Dieckelmann et al. (1998)

Pseudoalteromonas sp. Pseudomonas sp. Psychrobacter sp.

Zeng et al. (2004) Zeng et al. (2004) Zeng et al. (2004)

lipP lipA1 KB-lip PLC gene lip1 PsyEst 16 S rRNA gene sequences wp27, wp30, wp32, wp33 wp37 wp17, wp18, wp21, wp24, wp25

Choo et al. (1998) Zhang et al. (2007) Rashid et al. (2001) Preuss et al. (2001) Arpigny et al. (1993) Kulakovaa et al. (2004)

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5.2. Protein engineering Cold active lipases could generate avenues for industrial applications, once their specific properties are improved through enzyme engineering. Although lipases carry significant commercial value, biotechnologically produced or engineered cold active lipases may represent the focus of industrial interest in future. Determination of three-dimensional structures of more cold active lipases would allow the detailed analysis of protein adaptation to temperatures at molecular level. This may include increased thermolabile nature and/or catalytic activity at low temperatures, or the modification of pH profiles. Cold active lipases from microorganisms retaining high catalytic activity at low temperatures are successfully produced using sitedirected mutagenesis and directed evolution. The α/β hydrolase fold of lipase consists of a central hydrophobic, 8-stranded sheet, packed between two layers of amphiphilic α-helices. They have a common catalytic mechanism involving five subsequent steps: After binding of the ester substrate, a first tetrahedral intermediate is formed by nucleophilic attack of the catalytic serine, with the oxy anion stabilized by the 2 or 3 hydrogen bonds, the so called oxy anion hole. The ester bond is cleaved and the alcohol moiety leaves the enzyme. In the last step the acyl enzyme is hydrolyzed (Pleiss et al., 1998). Analysis of sequence homology of human hormone sensitive lipase gene with Antarctic bacterium Moraxella sp. strain TA 144 was carried out by Langin et al. (1993). Strictly conserved catalytic centre of the lipases contain a serine-protease like catalytic triad, consisting of Ser-HisAsp/Glu residues and the active site serine residue is located in a βSer-α motif (Jaeger et al., 1994). This motif consists of a six-residue βstrand, a four residue type II turn with serine in the -conformation and buried α-helix packed parallel against strand 4 and 5 of the central β-sheet. Unusual and structural feature of the structure of lipases is that the active site is completely buried under a lid/flap composed of one or two α helices, so the active site is not accessible to the substrate unless activation occurs (Lotti et al., 1994). Sequencing, determination of crystal structure and modification of two crystal forms of lipase B from C. antarctica and crystallization and preliminary X-ray structure of lipase from C. antarctica was studied (Uppenberg et al., 1994a,b). Crystallographic and molecular modeling studies of lipase B of C. antarctica was carried out by Uppenberg et al. (1995). Kim et al. (1997) determined the crystal structure of a triacylglycerol lipase from Pseudomonas cepacia (PcL) in the absence of a bound inhibitor using X-ray crystallography. The structure shows the lipase to contain α/β-hydrolase fold and a catalytic triad comprising of residues Ser87, His286 and Asp264. The enzyme shares several structural features with homologous lipases from Pseudomonas glumae (PgL) and Chromobacterium viscosum (CvL), including a calcium-binding site. The present structure of PcL reveals a highly open conformation with a solvent-accessible active site. This is in contrast to the structures of PgL and PcL in which the active site is buried under a closed or partially opened ‘lid’, respectively. Molecular adaptation of cold lipase and 3-dimensional modeling from P. immobilis strain B10 has been reported (Arpigny et al., 1997). A mutant protein with a single amino acid substitution, T103G, had an increased half-life at 60 °C, but only 50% of its original activity compared with the wild-type enzyme (Patkar et al., 1997). From the X-ray structures of lipases available, it is evident that the so called α/β hydrolase fold (from the secondary structure alignments) with a mixed central β-pleated sheet containing the catalytic residues is conserved (Pandey et al., 1999). Activity and stability of chemically modified lipase B from C. antarctica has been reported (Koops et al., 1999). Improving tolerance of cold-adapted lipase B of C. antarctica towards irreversible thermal inactivation through directed evolution was investigated (Zhang et al., 2003; Cavicchioli and Siddiqui, 2004) and improved activity and thermostability 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 provide the means for selecting mutants with improved properties (Tao and Cornish, 2002). Circular dichroism measurements, using synchrotron radiation, showed that the secondary structure of C. antarctica lipase does not differ significantly when changed from an aqueous to organic solvent environment (Mc Cabe et al., 2005). Thus, it was concluded that a major conformational change is not the reason for the different products produced by the enzyme when used in organic solvent. Significant changes in the lipase's α-helix content were found at the extremes of pH 4.2 and 9.0; this is in keeping with the permanent loss of activity of the enzyme at such a pH. Molecular properties, mutagenesis and over expression of cold active lipase in P. fragi have been reported (Lafranconi et al., 2005). Further, they reported the effect of mutation in non-consensus Thr-X-Ser-X-Gly on lipase specificity, specific activity and thermostabilty. Effect of mutations on chain length specificity and thermostability of lipases was studied in the strain of Pseudomonas IFO3458 (Santarossa et al., 2005). Recently the three-dimensional model of cold-adapted Alaskan psychrotroph Pseudomonas species (Strain B11-1) lipase has been constructed by homology modeling based on the crystal structure of acetyl esterase from Rhodococcus species and refined by molecular dynamics methods. The model locates the substrate-binding cavity and further suggests that Ser-155, Asp-250, and His-280 are present in the catalytic triad (Roy and Sengupta, 2007). The crystal structure of the P. lipolyticum M37 lipase at 2.2 Å resolutions was determined and compared it to that of non-adapted Rhizomucor miehei lipase (Jung et al., 2008). Structural analysis revealed that M37 lipase adopted a folding pattern similar to that observed for other lipase structures. However, comparison with RML revealed that the region beneath the lid of the M37 lipase included a significant and unique cavity that would be occupied by a lid helix upon substrate-binding. In addition, the oxyanion hole was much wider in M37 lipase than RML. They proposed that these distinct structural characteristics of M37 lipase might facilitate the lateral movement of the helical lid and subsequent substrate hydrolysis, which might explain its low activation energy and high activity at low temperatures. These studies may help in understanding the structural features and can be used in engineering lipase with considerable biotechnological potential. The immobilized form of CAL B is quite thermostable, particularly under non-aqueous conditions, where the catalyst remains active for many hours in the presence of high concentrations of reactants often with vigorous agitation (Koops et al., 1999). In aqueous solutions, the lipase denatures relatively quickly at temperatures as low as 40 °C (Homann et al., 2001). However, attempts have been made to improve the lipase stability via protein engineering have resulted in only a moderate improvement of its thermal properties with a concomitant decrease in activity. A comprehensive protein database (MELDB) of microbial esterases and lipases was developed by Kang and coworkers (2006). In the database, 883 esterase and lipase sequences derived from microbial sources were deposited and conserved parts of each protein were identified. HMM profiles of each cluster were generated to classify unknown sequences. Contents of the database can be keyword-searched and query sequences can be aligned to sequence profiles and sequences themselves. In MELDB, one can see the grouping of microbial esterases and lipases based on the TribeMCL tool with conserved patterns within sequences, and with HMM profiles 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 biotechnological exploitation based on their high catalytic activity at low temperature and low thermostability and unusual specificities. Indeed, the cold enzymes, along with the host microorganisms cover a broad spectrum of biotechnological applications. They include additives in detergents (cold washing), additives in food industries (fermentation, cheese

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

manufacture, bakery, meat tenderizing), environmental bioremediations (Digesters, composting, oil or xenobiotic biology applications), biotransformation and molecular biology applications, heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies (Feller et al., 1996). Potential applications of cold-active lipases are presented in Table 8. A number of reports mentioned straightforward reasons why cold-active enzymes have application in biotechnology (Russell et al., 1998; Margesin and Schinner, 1999; Ohgiya et al., 1999; Gerday et al., 2000; Cavicchioli et al., 2002). Most of these are appreciated without a detailed knowledge of how coldactive enzymes achieve their performance. The number of present uses is low and likely to reflect the state of the field, which, for example has not developed as rapidly as the thermophile field. Nevertheless, despite the difficulties with prediction, important advances have been made (Cui et al., 1999). 6.1. Medical and pharmaceutical applications Cold active lipases have emerged as an important biocatalyst in biomedical applications, because of their excellent capability for specific regioselective reactions in a variety of organic solvents with broad substrate recognition. Biocatalysis offers a clean and ecological way to perform chemical processes, in mild reaction conditions and with high degree of selectivity. The use of enzymes, especially lipases, in organic solvents proves an excellent methodology for the preparation of single-isomer chiral drugs (Gotor-Fernandez et al., 2006a). A preparation of optically active amines that was intermediate in the preparation of pharmaceuticals and pesticides which involved in reacting stereospecific N-acylamines with lipases, preferably from C. antarctica or Pseudomonas sp. (Smidt et al., 1996). C. antarctica lipase B (CAL B) is a very effective catalyst for the production of amines and amides using different enzymatic procedures. Simplicity of use, low cost, commercial availability and recycling possibility make this lipase an ideal tool for the synthesis and resolution of a wide range of nitrogenated compounds that are for the production of pharmaceuticals and manufactures in the industrial sector (Gotor-Fernandez et al., 2006b). 6.2. Synthesis of fine chemicals Kinetics of acyl transfer reactions in organic media catalyzed by lipase B from C. antarctica has been reported by Martinelle and Hult (1995). Lipase produced by a psychrotroph, P. fluorescens P38, was found to catalyze the synthesis of butyl caprylate in n-heptane at low temperatures. The optimum yield of ester synthesis was 75% at 20 °C

465

with an organic phase water concentration of 0.25% (v/v). The results are discussed in terms of the structural flexibility of psychrotroph derived lipase and the activity of this enzyme within a nearly anhydrous organic solvent phase (Tan et al., 1996). Applications of lipase B of C. antarctica in organic synthesis has been reported (Anderson et al., 1998). The ethyl esterification of docosahexaenoic acid (DHA) for the production of ethyl docosahexaenoate (EtDHA) in an organic solventfree system using C. antarctica lipase, which acts strongly on DHA and ethanol (Shimada et al., 2001). About 88% esterification was attained by 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 the presence of an excess amount of ethanol, the extent of esterification could not be raised above 90%. To attain a higher level of esterification, a two-step reaction was found to be effective. The first step was performed in a mixture of DHA/ethanol (1:1, mol/mol), and the reaction mixture was then dehydrated. In the second step, the resulting mixture was shaken at 30 °C for 24 h with 5 M equivalents of ethanol against the remaining DHA using 2 wt.% immobilized lipase. By means of this two-step procedure, 96% esterification was attained. Repetition of the first and second reactions showed that the immobilized lipase was reusable for at least 50 cycles. In addition, DHA remaining in the second-step reaction mixture was removed by a conventional alkali refining process, giving purified EtDHA with a high yield. Use of lipase B from C. antarctica for the preparation of optically active alcohols has been reported (Rotticci et al., 2001). Enzymatic reactions in non-aqueous solvents offer new possibilities for the biotechnological production of many useful chemicals using reactions that are not feasible in aqueous media. The use of enzymes in non-aqueous media has found applications in organic synthesis, chiral synthesis or resolution, modification of fats and oils, synthesis of sugar-based polymers, etc. The use of lipases in esterification reactions to produce industrially important products such as emulsifiers, surfactants, wax esters, chiral molecules, biopolymers, modified fats and oils, structured lipids, and flavor esters is well documented. The interest in using lipases as biotechnological vectors for performing various reactions in both macro- and microaqueous systems has picked up tremendously during the last decade (Krishna and Karanth, 2002). Crude soybean oil did not undergo methanolysis with immobilized C. antarctica lipase but degummed oil did (Watanabe et al., 2002). Therefore, the substance that was removed in the degumming step was estimated to inhibit the methanolysis of soybean triacylglycerols (TAGs). The main components of soybean gum are phospholipids (PLs), and soybean PLs actually inhibited the methanolysis reaction. Indeed, three-step methanolysis successfully converted 93.8% degummed soybean oil to its corresponding methyl

Table 8 Industrial applications of cold active lipases Field of application

Purpose

Reference

Medical and pharmaceutical application

Synthesis of arylaliphatic glycolipids Ethyl esterification of docosahexaenoic acid to Ethyl docosahexaenoate (EtDHA) Synthesis of citronellol laurate from citronellol and lauric acid Optically active ester synthesis Ester synthesis, desymmetrization and production of peracids Organic synthesis of chiral intermediates Synthesis of butyl caprylate in n-heptane Synthesis of butyl lactate by transesterification Synthesis of amides Protein polymerization and gelling in fish, improvement in food texture, flavor modification Production of fatty acids and interestrification of fats Detergents and cold water washing Production of α-butylglucoside lactate by transesterification for cosmetics Conversion of degummed soybean oil to biodiesel fuel Synthesis of lipase-catalyzed biodiesel Degradation of lipid wastes Bioremediation and bioaugumentation

Ota et al. (2000) Shimada et al. (2001) Ganapati and Piyush (2005) Anderson et al. (1998) Zhang et al. (2003) Gerday et al. (2000) Tan et al. (1996) Pirozzi and Greco (2004) Slotema et al. (2003) Cavicchioli and Siddiqui (2004) Jaeger and Eggert (2002) Gerday et al. (2000), Joseph (2006) Bousquet et al. (1999) Watanabe et al. (2002) Chang et al. (2004) Ramteke et al. (2005) Gerday et al. (2000), Suzuki et al. (2001), Lee et al. (2003) Margesin et al. (2002)

Fine chemical synthesis

Food industry Domestic application

Environmental application

Removal of solid and water pollution by hydrocarbons, oils and lipids

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esters, and the lipase could be reused for 25 cycles without any loss of the activity. Lipase from C. antarctica has been evaluated as a catalyst in different reaction media for hydrolysis of tributyrin as reaction model by Salis et al. (2003). To introduce polymer to cellulosic material a new approach was developed by Gustavsson et al. (2004), using ability of a cellulose-binding module of C. antarctica lipase B conjugate to catalyze ring opening polymerization of epsilon-caprolactone in close proximity to cellulose fiber surface. CAL A posses high thermostability, allowing operation at temperature above 90 °C; the ability to accept tertiary and sterically hindered alcohols, which has recently been attributed to the existence of a specific aminoacidic sequence in the active site; the sn-2 recognition in hydrolysis of triglycerides; the selectivity towards trans-fatty acids; the stability in the acidic pH range. Furthermore, it is an excellent biocatalyst for the asymmetric synthesis of amino acids/amino esters, due to its chemoselectivity towards amine groups (de Maria et al., 2005). Honore and Gerard (2005) reviewed about using lipases as catalysts in organic synthesis. It provides some specific examples of stereoselective biotransformations used to prepare non-racemic chiral building blocks and the utilization of these intermediates to synthesize different target molecules by organic transformations. Cold active lipase from C. antarctica increased the performance of lipase B in the enantioselective esterification of ketoprofen (Ong et al., 2006). Improvement of the enantioselectivity of lipase from C. antarctica (fraction B) via adsorption on polyethylenimine-agarose has been reported by Torres et al. (2006). Structure and activity of lipase B from C. antarctica in ionic liquids has been studied (van Rantwijk et al., 2006). Lipases as a catalyst and t-butanol or acetone, a mixture of solvent and ionic liquid as solvents, have been used for the synthesis of ester-based surfactants (Karmee, 2008). 6.3. Applications in food industry In the food industry, reaction need to be carried out at a low temperature in order to avoid changes to food ingredients caused by undesirable side-reaction that would otherwise occur at higher temperatures. Lipases have become an integral part of modern food industry. The use of enzymes to improve the traditional chemical processes of food manufacture has been developed in the past few years. Stead (1986) and Coenen et al. (1997) stated that though microbial lipases are best utilized for food processing, a few, especially psychrotrophic bacteria of Pseudomonas sp. and a few moulds of Rhizopus sp. and Mucor sp. cause havoc with milk and dairy products and with soft fruits. An example of the application of a cold-adapted enzyme in non-aqueous biotransformation is the use of a lipase from Pseudomonas strain P38 for the synthesis in n-heptane of the flavoring compound, butyl caprylate (Tan et al., 1996). Immobilized lipases from C. antarctica (CAL B), C. cylindracea AY30, H. lanuginosa, Pseudomonas sp. and G. candidum were used for the esterification of functionalized phenols for synthesis of lipophilic antioxidants to be used in sunflower oil (Buisman et al., 1998; Pandey et al., 1999). Whole-cell biocatalyst of mutated C. antarctica lipase B (mCAL B) by a yeast molecular display system and its practical properties were studied (Kato et al., 2007). When mCAL B was displayed on the yeast cell surface, it showed a preference for short-chain fatty acids, an advantage for producing flavors. 6.4. Domestic applications The most commercially important field of application for hydrolytic lipases is their addition to detergents, which are used mainly in household and industrial laundry and in household dishwashers. C. antarctica lipase was developed into recombinant enzyme used for detergent formulation (Uppenberg et al., 1994a). Godfrey and West (1996) reported that about 1000 t of lipases are sold every year in the area of detergents. Enzymes can reduce the environmental load of

detergent products, since they save energy by enabling a lower wash temperature to be used; allow the content of other, often less desirable, chemicals in detergents to be reduced; are biodegradable, leaving no harmful residues; have no negative impact on sewage treatment processes; and do not have a risk to aquatic life. Commercial preparations used for the desizing of denim and other cotton fabrics, contains both α amylase and lipase enzymes. Lipases are stable in detergents containing protease and activated bleach systems. Lipase is an enzyme, which decomposes fatty stains into more hydrophilic substances that are easier to remove than similar non-hydrolysed stains (Fuji et al., 1986). The commercial applications of lipases includes, detergents is in dish washing, clearing of drains clogged by lipids in food processing or domestic/industrial effluent treatment plants (Bailey and Ollis, 1986). Further, it is used in liquid leather cleaner (Kobayashi, 1989), a bleaching composition (Nakamura and Nasu, 1990), decomposition of lipid contaminants in dry-cleaning solvents (Abo, 1990), contact lens cleaning (Bhatia, 1990), degradation of organic wastes on the surface of exhaust pipes, toilet bowls, etc. (Moriguchi et al., 1990). Removal of dirt/cattle manure from domestic animals by lipases and cellulases (Abo, 1990), washing, degreasing and water reconditioning by using lipases along with oxidoreductases, which allows for smaller amounts of surfactants and operation at low temperatures (Novak et al., 1990). The lipase component causes an increase in detergency and prevents scaling. The cleaning power of detergents seems to have peaked; all detergents contain similar ingredients based on similar detergency mechanisms. To improve detergency, modern types of heavy-duty powder detergents and automatic dishwasher detergents usually contain one or more enzymes (Ito et al., 1998). Lipases show unusual versatile substrate specificity. The tertiary structure of lipases is known, there are presently significant efforts to improve this class of enzymes by protein engineering techniques, in view of their use in detergents and other fields of industrial application (Schmid and Verger, 1998). Cold active lipase from Microbacterium phyllosphaerae and Bacillus sphaericus has a remarkable capacity to retain its activity in presence of commercially available detergents and exhibited high efficiency for the removal of lipid stains (kitchen oil stains and used engine oil stain) from fabrics (Joseph, 2006). These reports appear that these cold active lipolytic enzymes can be used as detergent additive for cold washing. 6.5. Environmental application Bioremediation for waste disposal is a new avenue in lipase biotechnology. Cold-adapted organophosphorus acid anhydrolases was characterized for application in the efficient detoxification of pesticide and nerve agents (Cheng et al., 1997). Cold-adapted lipases have great potential in the field of wastewater treatment, bioremediation in fat contaminated cold environment and active compounds synthesis in cold condition (Buchon et al., 2000). This aspect requires more efforts in identifying and cloning novel lipase genes. Suzuki et al. (2001) identified as a psychrotrophic strain of the genus Acinetobacter strain no. 6 produced extracellular lipolytic enzyme that efficiently hydrolyzed triglycerides such as soybean oil during bacterial growth even at 4 °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 is potentially applicable to in situ bioremediation or bioaugumentation of 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 were observed in Pseudomonas sp. and maximum degradation of petroleum oils and benzene resins were observed in Rhodococcus sp. Further, they stated that the introduction of psychrotrophic microbial degraders of oil products into the environment is most important in the contest of environmental problems in temperate regions. Ramteke et al. (2005) stated that in temperate regions, large seasonal variations

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

467

Table 9 Patent details of cold active lipases Micro organism

Patent number

Inventor(s)

Industrial partner

Process based patents Candida antarctica Process for the preparation of optically active amines C. antarctica Process for preparing esters

US6387692 WO0153511

Bayer AG Christensen Morten Wuertz

C. antarctica

Enzymatic synthesis of polyesters

US5962624

C. antarctica

Process for the enzymatic resolution of N-(alkoxycarbonyl)4-ketoproline alkyl esters or N-(alkoxycarbonyl)-4hydroxyproline alkyl esters using lipase B Process for the preparation of trans-2, cis-4-decadienoic acid ethyl ester Process for producing triglycerides from glycerol and long-chain polyunsaturated fatty acids using lipase Process for stereoselection of (2R,3S)-3-phenylgycidic ester using lipase Immobilization of thermostable microbial lipase by adsorption to macroporous inorganic carrier particles Lipase-catalyzed ester hydrolysis

US5928933

Stelzer Uwe (De); Dreisbach Claus (De) Christensen Morten Wuertz (dk); Borch Kim (dk) Hill Karlheinz (De); Lagarden Martin (De); (+3) Hong Wonpyo (US); Dicosimo Robert (US)

C. antarctica C. antarctica C. antarctica C. antarctica C. antarctica C. antarctica C. antarctica T-24 C. C. C. C.

antarctica antarctica antarctica antarctica

C. antarctica

C. antarctica C. antarctica C. antarctica

Patented item/process

US5753473 US5604119 US5407828 US5342768 WO9218638

Method for producing optically active s-6-hydroxy2,5,7,8-tetramethylcumarone-2-carboxylic acid Method for treating soy sauce oil

JP2003144190

Para-dioxanone-based polymer Production of (d)-3(2h)-furanone compounds Enzymatic production of optically active compound Degradation of biologically degradable polymers using lipase from C. antarctica and a cutinase Enzymatic resolution of benzodiazepine-acetic acid esters (3-oxo-2,3,4,5-1H-tetrahydro-1,4-benzodiazepine-2-acetic acid) with a lipase from C. Antarctica Solvent-free method for reacting short-chain alcohols and acids by lipase immobilized to acrylic resin Process for the esterification of carboxylic acids with tertiary alcohols using a lipase Resolution of (RS)-ibuprofen catalyzed esterification with long-chain alcohols while removing water

JP2002101847

Haarmann & Reimer Gmbh (De) Novonordisk; Lysi Hf DSM NV Novonordisk Novonordisk AS (Dk); Jujo Paper Co Ltd (Jp) Mitsubishi Gas Chem Co Inc Higashimaru Shoyu Co Ltd

JP2000044658 JP10084988 JP7115992 NZ337239

Furubayashi Makio; Nakahara Tadaatsu; (+3) Nishida Haruo;Yamashita Mitsuhiro Suzuki Akio;Nozaki Michio Takagi Naoyuki; Others: 04 Koch Rainhard; Lund Henrik

NZ336376

Wells Andrew Stephen

Smithkline Beecham p

US5908769

Cho Nam Ryun (Kr); Hwang Soon Ook (Kr); (+1) Bosley John Anthony (GB); Casey John (GB); (+ 2) Trani Michael (Ca); Ergan Fran Oise (Fr); (+1)

Yukong Ltd (Kr)

US5658769 US5561057

Product based patents C. antarctica C. antarctica lipase and lipase variants

US6020180

C. Antarctica

C. antarctica lipase variants

US6074863

C. Antarctica

Enzymatic ammonolysis process for the preparation of intermediates Thermally stable and positionally non-specific lipase

US7223573

Candida sp.

Gatfield Ian (De); Kindel Guenter (De) Haraldsson Gudmundur G (Is); Svanholm Hanne (Dk); (+1) Kierkels Joannes G T (Nl); Peeters Wijnand P H (Nl) Pedersen Sven (Dk); Hansen Tomas T (Dk) Heldt–Hansen Hans Peter (Dk); Awaji Haruo (Jp); (+3) Tamura Yutaka

Hendel Komm and Itgesells Chaft A Du Pont (US)

US5273898

Egel–Mitani Michi (Dk); Hansen Mogens Trier (Dk); (+4) Egel–Mitani Michi (Dk); Hansen Mogens Trier (Dk); (+4) Ramesh N. Patel; Ronald L. Hanson; Iqbal Gill; (+6) Ishii Michiyo (Jp)

Tokuyama Corp Takasago Internatl Corp Nippon Soda Co Ltd Bayer AG

Unichem Chemie Bv (Nl) Canada Nat Res Council (Ca)

Novonordisk As (Dk) Novonordisk As (Dk) NA Novonordisk As (Dk)

NA: Not available.

in temperature reduce the efficiency of microorganisms in degrading pollutants such as oil and lipids. The enzymes active at low and moderate 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 largely unexplored field, it is not surprising that the number of companies involved in funding cold lipase research, screening samples and applying for Antarctic-based patents is restricted. Eventhough some noteworthy discoveries based on Antarctic lipases and with potential commercial applications were made in collaboration with industrial partners (Table 9). However, it appears that none of these discoveries has led to commercialization yet. Patent applicants are largely pharmaceutical, 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 synthetic derivative. C. antarctica, one of 154 species of the genus Candida, belongs to the Phylum Ascomycota and to the Class Ascomycetes. It is an 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 and transformation, bioremediation of contaminants and clean-energy production, confirming and reinforcing the potential of this technology for environmental purposes. Cold active lipases are promising enzymes to replace the conventional enzyme processes of the biotechnological industries. However, a more extensive effort is required to overcome several bottlenecks: high enzyme cost, low activity and/or stability under environmental conditions, low reaction yields and the low biodiversity of psychrophilic microbes explored so far. The relatively recent introduction and development of novel recombinant DNA technologies such as, metagenomics and site-directed mutagenesis have a profound positive effect on the expression and production of greater and greater amounts of recombinant proteins, which means more competitive prices, by introducing new or tailored catalytic activities of these proteins at low temperature. Thus, efforts have to be made in order to achieve economical overproduction of cold active lipase in heterologous hosts and their modification by chemical means or protein engineering to obtain more robust and active lipases. Further investigations should consider modeling of such thermostable cold active lipases that can be used for various industrial and biotechnological applications.

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