Yeast lipases: enzyme purification, biochemical properties and gene cloning Jyoti Vakhlu Avneet Kour *Corresponding author Keywords: Candida, Geotrichum, lipase, Trichosporon, yeast.
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. ( 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 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 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- 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. 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 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
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 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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