Electronic Journal of Biotechnology ISSN: 0717-3458
  © 2006 by Universidad Católica de Valparaíso -- Chile
Vol. 9 No.1, Issue of January 15, 2006 
 
Microbial Biotechnology

Biotechnology Industry

Electronic Journal of Biotechnology ISSN: 0717-3458  
© 2006 by Pontificia Universidad Católica de Valparaíso -- Chile  
BIP REVIEW ARTICLE

Yeast lipases: enzyme purification, biochemical properties and gene cloning 

Jyoti Vakhlu
Department of Biotechnology
University of Jammu
Jammu-180006 ( J&K)
India
Tel: 094191-17624
E-mail: jyotivakhlu@yahoomail.com

Avneet Kour
Department of Biotechnology
University of Jammu
Jammu-180006( J&K)
India
E-mail: avneetneeti@rediffmail.com

*Corresponding author

Keywords: Candida, Geotrichum, lipase, Trichosporon, yeast.

Abstract Reprint (PDF)

Enzymes have been used by man since biblical times, either as vegetables rich in enzymes, or as microorganisms and their products (in brewing processes, in baking, and in the production of alcohol). Modern enzyme technology really began in 1874 when the Danish chemist Christian Hansen produced the first specimen of rennet by extracting dried calves stomach with saline solution. Apparently this was the first enzyme preparation of relatively high purity used for industrial purposes. It is quite recently that industrial importance of enzymes was realized. Earlier enzymatic processes, particularly fermentation, were the focus of numerous studies in the 19th century and many valuable discoveries in this field were made. Today, enzymes are major contributors to clean industrial products and processes. Enzymes show numerous advantages over chemical technology as far as their specificity, efficiency and compatibility with environment is concerned. At present the three major giants in industrial enzyme production are Novozymes A/S (North America), Genencor International Inc. (California, USA) and DSM N.V. Enzymes (Netherlands). Overall global market for enzymes (in baking, beverage, dairy, feed, paper and pulp) is estimated at around US $ 1.5 billion and it is set to grow by 5-10% per year. The market for enzymes in detergent industry alone is around 0.5 billion US $. Whilst lipases at present account for less than 5% of the market, this share has the potential to increase dramatically via a wide range of different applications.

Lipases (E.C.3.1.1.3) are a class of serine hydrolases which belongs to the α/β hydrolases super family. They are ubiquitous enzymes of considerable physiological significance and industrial potential. They catalyse the hydrolysis of triglyceride to give di- and mono- glycerides, glycerol and free fatty acids. Lipases can be divided generally into the following four groups according to their specificity in hydrolysis reaction: substrate specific lipases, regio-selective lipases, fatty acid specific lipases, and stereo -specific lipases. Industrial uses of lipases is in areas such as: hydrolysis of tallow for laundry detergent; synthesis of esters; trans-esterification for fragrances, flavours, and cocoa butter substitutes; synthesis of structured lipids for infant formula and neutracieuticals; improve PUFA content in fish oil; and enantio-resolution of esters for chemical and drug intermediates to name a few. However the last quarter of the 20th century has witnessed unprecedented use of lipases in biotechnology, manufacture of pharmaceuticals & pesticides, single cell protein production, biosensor preparation and in waste managementetc. Lipases have become an integral part of the modern food industry and are used in the preparation of a variety of products including fruit juices, baked food, vegetable fermentation and dairy enrichment. They are also used in leather industry for processing hides and skins  (bating) and for treatment of activated sludge and other aerobic waste products where they remove the thin layer of the fats and by so doing provide for oxygen transport. The lipid digesting preparation is employed in sewage disposal plants in USA under the trade name lipase M-Y (Meito Sangyo Co., Nagoya Japan). Lipases may also assist in the regular performance of anaerobic digesters. Nearly 1000 tones of lipase are used annually in detergent industry, primarily as lipid stain digesters. They also are used as flavour development agents in the preparation of cheese, butter and margarine. These hydrolases are endowed with substrate specificity that surpasses any known enzyme. This property confers to them the potential that is literally boundless. The growing interest in lipases is reflected by publication of an average of 1000 research papers per year, on different aspects of these enzymes.

They are obtained from a variety of sources like plants, animals, yeast, bacteria, but among all microbial lipases are the most popular for industrial use as they are easy to produce and are stable comparatively. Pancreatic lipase of porcine origin is one of the earliest recognized  lipases and is still the best-known lipase. Plant lipases are not used commercially; the animal and microbial lipases are used extensively. The most important source of animal lipase is the pancreas of cattle, sheep, hogs and pigs. The disadvantage with pancreatic (/animal) lipases is that they cannot be used in the processing of vegetarian or kosher food. Also, that these extracts contain components which have undesirable effect. The pig pancreatic extract contains trypsin, which produces bitter tasting amino acids. They are also likely to contain residual animal viruses, hormones etc.

Microbes are major source of the 100 or so enzymes produced industrially. Yeast has been used in food and other industries since ages. They have earned acceptability since long and are considered natural. Yeasts are also considered to be easy to handle and grow, in comparison to bacteria. Lipases produces from number of yeasts have been studied and Table 1 enumerates some of them. The lipase produced by Candida rugosa is fast becoming one of the most frequently  used enzyme industrially. This is because of its use in avariety of processes due to its high activity, both in hydrolysis as well as synthesis (Redondo et al. 1995). A Japanese company has used the Candida rugosa lipase for production of fatty acids from castor bean long back in 1985. Pandey et al. 1999 investigated the production of flavour in concentrated milk and creams by using microbial lipases. Organolephtically each lipase develops a characteristic flavour. The Candida rugosa lipase is rated the most suitable lipase. Candida antarctica AY30 immobilized lipase has been used for esterification of functional phenols for synthesis of lipophillic antioxidants subsequently used in sunflower oil. Uppenberg and co workers (1994) developed C. antarctica lipase into recombinant enzyme used for detergent formulation. The extra-cellular lipase produced by the asporogenic C. cylindracea ATCC 14830 (CCl/CRL) hydrolyses triglycerides without specificity, both in attacked position of the glycerol molecule and in the nature of fatty acid released. This relaxed specificity vis-à-vis other lipases makes CCl/CRL particularly useful for industrial application.

In detergent industry, lipases find use as lipid stain digesters. Lipases from Candida cylindracea and C. lypolytica (now Yarrowia lipolytica) are choice enzymes for the purpose. Polyglycerol and carbohydrate fatty acid esters are widely used as industrial detergents and as emulsifiers in variety of food formulations (low fat spreads, ice creams, mayonnaise). Enzymatic synthesis of functionally similar surfactants has been carried out at moderate temperature (60ºC-80ºC) with excellent regioselectivity. Recently, Unichem International has launched production of isopropyl myristate, isopropyl palmitate and 2-ethylpalmitate for use of emollient in personal care products. Presently these compounds are being manufactured enzymatically using C. cylindracea lipase in batch bioreactor.

A promising new field is the use of microbial lipase as biosensors. Biosensors can be chemical or electronic in nature. An important analytical use of lipases is determination of lipids for clinical purpose. The basic concept is to utilize a lipase to generate glycerol from triacylglycerol and quantify the released glycerol or alternatively the non-esterified fatty acid by chemical and enzymatic method. This principal enables physicians precisely to diagnose patients with cardiovascular complaints. Non-specific lipases, especially of C. rugosa with high specific activity has been selected to allow rapid liberation of glycerol. C. rugosa lipase biosensor, which optically conjugates to biorecognition group in DNA, has been developed as probe.

The application of lipases in organic synthesis is tremendous. Stereoselectivity of lipases for resolution of racemic acid mixture in immiscible biphasic system has been demonstrated. Efficient kinetic resolution processes are in vogue for the synthesis of Niknomycin-B, non-steroid anti-inflammatory drugs Naproxen, ibuprofen, suprofen and ketoprofen, the potential antiviral agent lamividine (that can also be used against HIV) and enantiospecific synthesis of anti-tumour agents alkaloids, antibiotics and vitamins. Workers have isolated two iso-forms, labeled A and B from C. rugosa that are stereoselective.

Preparations of optically active amines that are intermediate in preparation of pharmaceuticals and pesticides have been standardised.This involved reacting stereo specific N-acylamines with lipase preferably from C. antarctica. In an attempt to determine substrate specificity of lipases, alkyl esters of 2 aryl- propionic acid, a class of non-steroid anti- inflammatory drugs were hydrolysed with C. rugosa lipase. All transformations were found to be highly selective. Lipases are also used for enantio-specific catalysis. The stereo selective enatio-discrimination of C. rugosa lipase yielded optically pure propionic acid derivative in S-form. The S-form was then converted to corresponding R form, which was effective against the insect pest Tetramuchus.

Triglycerides, steryl esters, resin acids, free fatty acids and sterols which are lipophylic extractives (/extracts) of wood (commonly referred to as pitch or wood resin) have negative impact on paper machine run ability and quality of paper. Kontkanen and his group (2004) in their study tested 19 commercial lipase preparations able to show degradation of steryl esters. They found lipase preparations of Pseudomonas sp. Chromobacteriumviscosum and Candida rugosa were shown to have highest sterile esterase activity. All the three enzymes were able to hydrolyse sterile esters totally to completion in presence of a surfactant. Preliminary characterization of enzymatic activity revealed that the lipase preparation of Pseudomonas sp. could be the most potential industrial enzyme but among yeast Candida rugosa lipase ( CRL) ruled the roost.  To introduce polymer to cellulosic material a new approach was developed by Gustavsson and his co workers (2004) using ability of a cellulose binding module of Candida antarctica lipase B conjugate to catalyze ring opening polymerization of epsilon-caprolactone in close proximity to cellulose fibber surface. Wang and co workers (2003) demonstrated effective biocatalysis also by C. antarctica Lipase (CAL B) in resolution of several 1-or 2-hydroxyalkanephosphonates.

Many yeast lipases have already been developed into a commercial processes and are available in the market. Table 2 enumerates some selected yeast lipases, which are already being produced commercially along with the companies that produce them. Microbial lipases are identified as an important field in enzyme biotechnology and in microbial lipases yeast lipases hold great

References

GUSTAVSSON, M.T.; PERSSON, P.V.; IVERSEN, T.; HULT, K. and MARTINELLE, M. Polyester coating of cellulose fiber surfaces catalyzed by a cellulose-binding module-Candida antarctica lipase B fusion protein.Biomacromolecules, January 2004, vol. 5, no. 1, p. 106-112. [CrossRef]

KONTKANEN, H.; TENKANEN, M.; FAGERSTROM, R. and REINIKAINEN, T. Characterisation of steryl esterase activities in commercial lipase preparations. Journal of Biotechnology, February 2004, vol. 108, no. 1, p. 51-59. [CrossRef]

PANDEY, Ashok; BENJAMIN, Sailas; SOCCOL, Carlos R.; NIGAM, Poonam; KRIEGER, Nadia and SOCCOL, Vanete T.The realm of microbial lipases in biotechnology. Biotechnology and Applied Biochemistry, April 1999, vol. 29, no. 2, p. 119-131.

REDONDO, O.; HERRERO, A.; BELLO, J.F.; ROIG, M.G.; CALVO, M.V.; PLOU, F.J. and BURGUILLO, F.J. Comparative kinetic study of lipases A and B from Candida rugosa in the hydrolysis of lipid P-nitrophenyl esters in mixed micells with triton X-100. Biochimica et BiophysicaActa (BBA)-General Subjects, January 1995, vol. 1243, no. 1, p. 15-24. [CrossRef]

UPPENBERG, Jonas; PATKAR, Shamkant; BERGFORS, Terese and JONES, T. Alwyn.Crystilization and preliminary X-ray studies of lipase B from Candida Antarctica. Journal of Molecular Biology, January 1994,vol. 235, no. 2, p. 790-792. [CrossRef]

WANG, K.; ZHANG, Y. and YUAN, C. Enzymatic synthesis of phosphocarnitine, phosphogabob and fosfomycin. Organic and Biomolecular Chemistry, October 2003, vol. 1, no. 20, p. 3564-3569.

 
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