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Isolation of lipase organisms from soil sample

INDUSTRIAL IMPORTANCE OF LIPASES Lipases are a class of enzymes which catalyze the hydrolysis of long chain triglycerides and constitute the most important group of biocatalysts for biotechnological applications. Lipolytic enzymes are currently attracting an enormous attention because of their biotechnological potential. They constitute the most important group of biocatalysts for biotechnological applications further more, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agro-chemicals, and flavour compounds. The chemo- regio- and enantio-specific behavior of these enzymes caused tremendous interest among scientists and industrialists.The knowledge of lipolytic enzymes in industrial applications is increasing at a rapid and exciting rate. Lipases in organic synthesis For synthetic chemists, the application of lipases as catalysts in organic synthesis is of much advantage. These enzymes can show stereoand regiospecificity, and tolerate organic solvents in the incubation mixture. Both the activity of the enzyme and the identity of the product depend upon the solvents used, which may vary from aqueous buffer systems through biphasic emulsions and microemulsions, to organic solvents. For synthetic purposes, crude enzyme preparations are often convenient. Because the catalyst is always an expensive factor in a chemical reaction, strategies for enzyme recycling are being developed. Synthetic strategies involving microbial lipases can be used to prepare molecules of high positional and configurational purity. Lipases can be used to create biologically active

Isolation of lipase organisms from soil sample


analogues of naturally occurring messenger molecules as antagonists or inhibitors in biological systems.

Lipase catalyses the hydrolysis of triglycerides at the waterlipid interface. Under given experimental conditions, the amount of water in the reaction mixture will determine the direction of the lipase-catalysed reac-tion. While in absence of water or its presence in trace quantities esterification and transesterification are favoured; in presence of excess water hydrolysis occurs.

Medical and pharmaceutical application Enantioselective interesterification and transesterification have great significance in pharmaceutical for selective acylation and deacylation (Stinson, 1995). Lipases are important in application in pharmaceuticals in transesterificatrion and hydrolysis reaction. They play a prime role in production of specialty lipids and digestive aids (Vulfson, 1994). The alteration of temperature during the esterification reaction drastically changes the

Isolation of lipase organisms from soil sample


enantiomeric values and also the stereopreference (Yasufuku et al., 1996). Lipases play an important role in modification of monoglycerides for use as emulsifiers in pharmaceutical applications (Sharma et al., 2001). Psychrophiles producing cold active lipases may be a good source for polyunsaturated fatty acids for the pharmaceutical industry. It is because of their excellent capability for specific regioselective reactions in a variety of organic solvents with broad substrate recognition makes lipases as an important biocatalyst in biomedical applications (Margesin et al., 2002). 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 Candida antarctica or Pseudomonas sp. (Smidt etal., 1996). In an attempt to determine the substrate specificity for lipases, alkyl esters of 2-arypropionic acids, a class of non-steroidal anti-inflammatory drugs, were hydrolyzed with Caenorhabditis rugosa lipase in which all transformations were highly enantioselective (Botta et al.1997). Synthesis of fine chemicals Some of the industrially important chemicals manufactured from fats and oils by chemical processes could be produced by lipases with greater rapidity and better specificity under mild conditions (Sih and Wu, 1989; Vulfson, 1994). The use of industrial enzymes allows the technologists to develop processes that more closely approach the gentle, efficient process in nature. Some of these processes using cold active lipase from C. antarctica have been patented by pharmaceutical, chemical and food industries. C. antarctica, one of 154 species of the genus Candida belongs to the phylum Ascomycota. It is 044 Biotechnol. Mol. Biol. Rev. alkali tolerant yeast found in the sediment of Lake Vanda, Antarctica (Joseph, 2006). The two lipase variants from this organism viz., Lipase A and Lipase B have proven of particular interest to the researchers. Martinelle and Hult (1995) conducted

Isolation of lipase organisms from soil sample


the comparative studies on the interfacial activation of these lipases A and B with Humicola lanuginose lipase. The characterization of lipase A for substrate specificity and its utility as biocatalyst was reported (Kirk and Christensen, 2002). Further, the kinetics of acyl transfer reactions in organic media catalyzed by lipase B were studied (Martinelle and Hult, 1995). Andersonet al. (1998) determined the applications of lipase B in organic synthesis and the enantioselectivity of lipase for some synthetic substrates. Rotticcietal. (1998) proposed the molecular recognition of alcohol enantiomers by lipase B. The use of lipase B for the preparation of optically active alcohols was also determined (Rotticci etal., 2001). Studies revealed that size as a parameter for solvent effects on lipase B enantioselectivity. The evaluation of lipase as catalyst in different reaction media for hydrolysis of tributyrin as reaction model has been reported (Salis et al., 2003). The amidase activity of lipase B and structural feature of the substrates were reported (Torres et al., 2006). The performance of lipase B in the enantioselectivity esterifiction of ketoprofen (Ong et al., 2006) and the improvement of enantioselectivity of lipase (fraction B) via adsorption on polyethylenimineagarose (Torres et al., 2006) were studied recently. The structure and activity of lipase B of C. antarctica in ionic liquids (van Rantwijk et al., 2006) and the applications of lipase B in organic synthesis has been reported (Anderson et al., 1998). Shimada et al. (2001) attempted the ethyl esterification of docosahexaenoic acid (DHA) in an organicsolventfree system using C. antarctica lipase, which acts strongly on DHA and ethanol. About 88% esterification was attained by shaking the mixture of DHA / ethanol (1:1, mol/mol) and 2 %wt immobilized C. antarctica lipase B at 30C for 24 h. Use of lipase B from C. antarctica for the preparation of optically active alcohols has been reported (Rotticci et al., 2001). 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

Isolation of lipase organisms from soil sample


yield of ester synthesis was 75% at 20C with an organic phase water concentration of 0.25% (v/v). The results are discussed in terms of the structural flexibility of psychrotrophderived lipase and the activity of this enzyme within a nearly anhydrous organic solvent phase (Tan et al.,1996). Watanabe et al. (2002) found that the crude soybean oil did not undergo methanolysis with immobilized C. antarctica lipase but degummed oil did. The substance that was removed in the degumming step was estimated to inhibit the methanolysis of soybean triacylglycerols (TAGs). Methanolysis is the displacement of alcohol from an ester by methanol in a process similar to hydrolysis, except that methanol is employed instead of water. Methanol is widely used because of its low cost and its physical and chemical advantages. 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 esters, and could be reused for 25 cycles without any loss of the activity. This process widely reduced viscosity of triglycerides, thereby enhancing the physical properties the lipase of renewable fuels to improve engine performance. Lipase from C. antarctica has been evaluated as catalyst in different reaction media for hydrolysis of tributyrin as reaction model (Salis et al., 2003). Applications in food Industry In the food industry, reaction needs to be carried out at low temperature in order to avoid changes in food ingredients caused by undesirable side-reaction that would otherwise occur at higher temperatures. Lipases have become an integral part of the 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.

Isolation of lipase organisms from soil sample


and a few moulds of Rhizopus sp. and Mucor sp. caused havoc with milk and dairy products and soft fruits. Cold active lipase from Pseudomonas strain P38 is widely used in non-aqueous biotransformation for the synthesis of nheptane 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 Geotrichum candidum were used for the esterification of functionalized phenols for synthesis of lipophilic antioxidants in sunflower oil Buisman et al. (1998).

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. Godfrey and West (1996) reported that about 1000 t of lipases are sold every year in the area of detergents. The use of cold active lipase in the formulation of detergents would be of great advantage for cold washing that would reduce the energy consumption and wear and tear of textile fibers (Feller and Gerday, 2003). The industrial dehairing of hides and skin at low temperature using psychrophilic protease or keratinase would not only save energy but also reduce the impacts of toxic chemicals used in dehairing. 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. Addition of cold active lipsase in detergent become biodegradable, leaving no harmful residues, have no negative impact on sewage treatment processes and do not present a risk to aquatic life. Commercial preparations used for the desizing of denim and other cotton fabrics contain both alpha amylase and lipase enzymes. Lipases are stable in detergents containing protease and activated bleach systems.

Isolation of lipase organisms from soil sample


Lipase is an enzyme, which decomposes fatty stains into more hydrophilic substances that are easier to remove than similar non-hydrolyzed stains (Fujietal., 1986). The low temperature active lipase can be added to detergents to hydrolyze oily stains at the temperature of tap water to reduce energy consumption and protect the color of fabrics (Feller and Gerday, 2003). The other common commercial applications for detergents is in dish washing, clearing of drains clogged by lipids in food processing or domestic/industrial effluent treatment plants (Bailey and Ollis, 1986). The use of cold active lipase as a liquid leather cleaner (Kobayashi, 1989) and as an ingredient in bleaching composition (Nakamura and Nasu, 1990) has been reported. Similarly its use in decomposition of lipid contaminants in drycleaning 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) have been reported. The 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) have been proposed. 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 and are 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, such as protease, amylase, cellulase and lipase (Ito et al., 1998).

Lipases in dairy industry Lipases are used extensively in the dairy industry for the hydrolysis of milk fat. Current applications include flavour enhancement of cheese, acceleration of cheese ripening, manufacture of cheese-like products,

Isolation of lipase organisms from soil sample


and lipolysis of butter fat, and cream. While the addition of lipases primarily releases short-chain (C4 and C6) fatty acids that leads to the development of sharp, tangy flavour, the release of medium-chain (C 12 and C14) fatty acids tends to impart a soapy taste to the product. In addition, the free fatty acids take part in simple chemical reactions where they initiate the synthesis of other flavour ingredients such as aceto-acetate, -keto acids, methyl ketones, flavour esters, and lactones. More recently, a whole range of microbial lipase preparations have been developed for the cheese manufacturing industry, such as those of Mucor miehei, Aspergillus niger and A. oryzae. A range of cheese of good quality were produced by using individual microbial lipases or mixtures of several preparations. Lipases are widely used for imitation of cheese made from ewes or goats milk. Addition of lipases to cows milk, generates a flavour rather similar to that of ewes or goats milk. This is used for producing cheese or the so-called enzyme-modified cheese (EMC). EMC is a cheese that has been incubated in the presence of enzymes at elevated temperatures in order to produce a concentrated flavour for use as an ingredient in other products such as dips, sauces, soups, and snacks.

Lipases in detergents The usage of enzymes in washing powders still remains the single biggest market for industrial enzymes. The world-wide trend towards lower laundering temperatures has led to much higher demand for household detergent formulations. Recent intensive screening programmes, followed by genetic manipulations, have resulted in the introduction of several suitable preparations, for example, Novo Nordisks Lipolase (Humicola lipase expressed in Aspergillus oryzae).

Isolation of lipase organisms from soil sample


Lipases in oleochemical industry The scope for application of lipases in the oleochemical industry is enormous as it saves energy and minimizes thermal degradation during hydrolysis, glycerolysis, and alcoholysis. Miyoshi Oil and Fat Co., Japan, reported commercial use of Candida cylindracea lipase in production of soap. The introduction of the new generation of cheap and very thermostable enzymes can change the economic balance in favour of lipase use. The current trend in the oleochemical industry is a movement away from using organic solvents and emulsifiers. The various reactions involving hydrolysis, alcoholysis, and glycerolysis have been carried out directly on mixed substrates, using a range of immobilized lipases. This has resulted in high productivity as well as in the continuous running of the processes. Enzymatic hydrolysis perhaps offers the greatest hope to successful fat splitting without substantial investment in expensive equipment as well as in expenditure of large amounts of thermal energy.

Lipases in synthesis of triglycerides The commercial value of fats depends on the fatty acid composition within their structure. A typical example of a high-value asymmetric triglyceride mixture is cocoa butter. The potential of 1,3regiospecific lipases for the manufacture of cocoa-butter substitutes was clearly recognized by Unilever and Fuji Oil. Comprehensive reviews on this technology, including the analysis of the product composition, are available. In principle, the same approach is applicable to the synthesis of many other structured triglycerides possessing valuable dietic or nutritional properties, for example, human milk fat. This triglyceride and functionally similar fats are readily obtained by acidolysis of palm oil fractions which are rich in 2-palmitoyl glyceride with unsaturated fatty acid(s). Acidolysis, catalysed by 1,3-specific

Isolation of lipase organisms from soil sample


lipases, is used in the preparation of nutritionally important products which generally contain medium-chain fatty acids. Lipases are being investigated extensively with regard to the modification of oils rich in high-value polyunsaturated fatty acids such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acids. Substantial enrichment in the polyunsaturated fatty acid content of mono-glyceride fraction has been achieved by lipasecatalysed alcoholysis or hydrolysis.

Lipases in synthesis of surfactants Polyglycerol and carbohydrate fatty acid esters are widely used as industrial detergents and as emulsifiers in a great variety of food formulations (low-fat spreads, sauces, ice-creams, mayonnaises). Enzymic synthesis of functionally similar surfactants has been carried out at moderate temperature (6080C) with excellent regioselectivity. Adelhorst etal. have carried solvent-free esterification of simple alkyl-glycosides using molten fatty acids and immobilized Candida antarctica lipase. Fregapane etal. obtained mono- and diesters of monosaccharides in high yields, using sugar acetals as starting materials. Lipase from A. terreus synthesizes a biosurfactant by transesterification between natural oils and sugar alcohols. Lipases may also replace phospholipases lipase has in the production used for of the lysophospholipids. Mucor miehei been

transesterification of phospholipid in a range of primary- and secondary alcohols. Lipases may also be useful in the synthesis of a whole range of amphoteric bio-degradable surfactants, namely amino acid-based esters and amides.

Lipases in synthesis of ingredients for personal care products

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Unichem International has recently launched the production of isopropyl myristate, isopropyl palmitate, and 2-ethylhexyl palmitate for use as emollient in personal care products like skin and sun-tan creams, and bath oils. Wax esters have similar application in personal care products and are being manufactured enzymatically, using C. cylindracea lipase, in a batch bioreactor.

Lipases in pharmaceuticals and agrochemicals The utility of lipases in the preparation of chiral synthons is well recognized and documented. Several processes have recently been commercialized which have been described by Sainz-Diaz etal. and Davis etal.. The resolution of 2-halopropionic acids, the starting materials for the synthesis of phenoxypropionate herbicides, is a process based on the selective esterification of (S)-isomers with butanol, which is catalysed by porcine pancreatic lipase in anhydrous hexane. Another impressive example of the commercial application of lipases in the resolution of racemic mixtures is the hydrolysis of epoxyester alcohols. The reaction products, (R)-glycidyl esters and (R)-glycidol are readily converted to (R)- and (S)-glycidyltosylates which are attractive intermediates for the preparation of optically active blockers and a wide range of other products. A similar technology has been commercialized to produce 2(R),3(S)-methylmethoxyphenyl glycidate, the key intermediate in the manufacture of the optically pure cardiovascular drug Diltiazem. Lipases have applications as industrial catalysts for the resolution of racemic alcohols in the preparation of some prostaglandins, steroids, and carbocyclic nucleoside analogues. Regioselective modification of polyfunctional organic compounds is yet another rapidly expanding area of lipase application, particularly in the field of AIDS treatment. Lipases from A. carneus and A. terreus show chemo- and regiospecificity in the hydrolysis of

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peracetates of pharmaceutically important polyphenolic compounds. Lipases are also useful in the synthesis of the artificial sweetner sucralose by regioselective hydrolysis of octa-acetylsucrose.

Bioconversions in aqueous media A typical lipase-catalysed reaction in aqueous media is ester hydrolysis. This enzymic conversion can be used for the synthesis of triglycerides as shown for the preparation of platelet-activating factor. Another application of the hydrolytic specificity of lipases is the partial hydrolysis of triglycerides to di- and monoglycerides in the food industry, where di- and monoglycerides serve as biocompatible emulsifiers and food additives. These and other applications of lipases in industry and research have been discussed in the review by Iwai and Tsujisaka.

Bioconversions in organic media The synthetic potential of lipases in organic solvents has been widely recognized and is well documented, particularly on the basis of activity of lipases in organic solvents containing low water content. The main application of lipases in organic chemistry is the resolution of enantiomeric compounds, making use of the enantioselectivity of these enzymes. The use of organic solvents for lipase-catalysed resolutions has four main advantages in comparison with water as the solvent: (i) racemic mixtures of alcohols or acids need not be esterified before resolution into enantiomers, (ii) these enzymes are more stable in organic solvents than in water, (iii) lipases used need not be immobilized for recovery, owing to their insolubility in organic solvents; they can be collected by filtration in their active state, and (iv) substrates and products may be unstable in aqueous solution. In this case, reaction in organic solvents is essential for formation and isolation of the

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products. The three main areas of lipase-catalaysed reactions in organic solvents are:

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Applications

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In the present day industry, lipases have made their potential realized owing to their involvement in various industrial reactions either in aqueous or organic systems, depending on their specificity (Table 2).

Lipase on various environmental pollutants There are number of uses of the cold active enzymes, presently it is conceivable that they could be used for environmental bioremediation

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e.g., as a biodegradable means of treating an oil spill such as that which occurred by the Exon Valdese in Arctic water. Bioremediation for waste disposal is a new avenue in lipase biotechnology. Cheng et al. (1997) characterized cold-adapted organophosphorus acid anhydrolases for application in the efficient detoxification of pesticide and nerve agents. According to Buchoetal. (2000), cold adapted lipases have great potential in the field of wastewater treatment, bioremediation in fat contaminated cold environment, active compounds synthesis in cold condition. Further, more efforts are needed in identifying and cloning of nov Babu et al 045 el lipase genes for environmental applications. Suzuki et al. (2001) identified a psychrotrophic strain of the genus Acinetobacter strain No. 6, producing an extracellular lipolytic enzyme that efficiently hydrolyzed triglycerides, such as soybean oil during bacterial growth even at 4C.The strain degraded 60% of added soybean oil (initial concentration, 1% w/v) after cultivation in LB medium at 4C for 7 days. The psychrophilic microorganisms as well as their enzymes have been proposed as alternative to physicochemical methods for bioremediation of solids and waste waters polluted by hydrocarbons, oils and lipids (Margesin et al., 2002). Belousova and Shkidchenko (2004) isolated 30 strains including Pseudomonas sp. and Rhodococcus sp. capable of oil degradation at 4-6C and 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 in temperature reduce the efficiency of microorganisms in degrading pollutants such as oil and lipids. The lipase active at low and moderate temperature may also be ideal for bioremediation process.

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Isolation of lipase organisms from soil sample

Isolation and molecular confirmation of lipase producing organisms from oil spills The aim of this study is to characterize the microbes in genetic level. Lipases are the very commercially important enzymes which have the wide application in degradation studies. Once if the microorganism confirmed in molecular level, based on modifying the cultural characteristics we can increase the production of lipases enzyme expression. Its very important for knowing the cultural characteristics of organisms, because the cultural characteristics and expression of desired products varies from organism to organisms. Here we aimed to isolate and molecular characterize the microbes which have the lipids degrading activity. By using the PCR based amplification of 16s rDNA gene we can classify the microbes in species and strain level. The species and strain level information provides the much information about the organisms and its lipase production. After getting the much information about the organism we used to assay it both biological and enzyme kinetic assays for lipase production.

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The demand for industrial enzymes, particularly of microbial origin, is ever increasing owing to their applications in a wide variety of processes. Enzyme-mediated reactions are attractive alternatives to tedious and expensive chemical methods. Enzymes find great use in a large number of fields such as food, dairy, pharmaceutical, detergent, textile, and cosmetic industries. In the above scenario, enzymes such as proteases and amylases have dominated the world market owing to their hydrolytic reactions for proteins and carbohydrates. However, with the realization of the biocatalytic potential of microbial lipases in both aqueous and nonaqueous media in the last one and a half decades, industrial fronts have shifted towards utilizing this enzyme for a variety of reactions of immense importance. It is in the last decade that lipases have gained importance to a certain extent over proteases and amylases, specially in the area of organic synthesis. The enantioselective and regioselective nature of lipases have been utilized for the resolution of chiral drugs, fat modification, synthesis of cocoa butter substituents, biofuels, and for synthesis of personal care products and flavour enhancers. Thus, lipases are today the enzymes of choice for organic chemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, microbiologists and biochemists. Lipases (triacylglycerol acylhydrolases) belong to the class of serine hydrolases and therefore do not require any cofactor.

Bacterial lipases A relatively smaller number of bacterial lipases have been well studied compared to plant and fungal lipases. Bacterial lipases are glycoproteins, but some extracellular bacterial lipases are lipoproteins. Winkler et al. reported that enzyme production in most of the bacteria is affected by certain polysaccharides. Most of the bacterial lipases reported so

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far are constitutive and are nonspecific in their substrate specificity, and a few bacterial lipases are thermostable. Among Arthrobacter sp., bacteria, Achromobacter sp., sp., Alcaligenes sp., sp., and

Pseudomonas

Staphylococcus

Chromobacterium sp. have been exploited for the production of lipases. Staphylococcal lipases are lipoprotein in nature. Lipases purified from S.aureus and S. hyicus show molecular weights ranging between 3446 kDa. They are stimulated by Ca++ and inhibited by EDTA. The optimum pH varies between 7.5 and 9.0. The lipase genes from S. hyicus and S. aureus have been cloned, sequenced, and compared with other lipases. This revealed two conserved domains separated by 100 amino acids which are likely to form active site. Putative active site residues around His 269 and Ser 369 of the S. hyicus lipase are highly conserved in the two S. aureus lipases and in several eukaryotic lipases. Lipases from different species of Psuedonomas were purified by acidification of the culture supernatant, ammonium sulphate precipitation, sepharose CL-6B chromatography, and isoelectric focussing using CHAPS 47. The purified lipase of P. fragi, P. fluorescens, and P. aeruginosa were monomeric with molecular weight of 33 kDa, 45 kDa, and 29 kDa, respectively. The lipase was inhibited by Zn++, Fe++, and Al+++ and activa-ted by Ca++ (ref. 48). The lipase gene of P. fragi has been cloned and sequenced.

Fungal lipases Fungal lipases have been studied (Lawrence etal., in 1950) have presented comprehensive reviews. These lipases are being exploited due to their low cost of extraction, thermal and pH stability, substrate specificity, and activity in organic solvents. The chief producers of commercial lipases are Aspergillus niger, Candida cylindracea, Humicola lanuginosa, Mucor miehei, Rhizopus arrhizus, R. delemar, R. japonicus, R. niveus and R. oryzae.

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Lipids are insoluble in water and need to be broken down extracellularly into their more polar components to facilitate absorption if they are to function as nutrients for the cell. Therefore majority of the lipases are secreted extracellularly.

Optimization parameters Varying the following parameters one at a time with optimization of fermentation media. The parameters varied were i. incubation period, ii. temperature, iii. pH and iv. lipid substrates. The influence of growth period on biomass and lipase production of Bacillus sps (B1 B5) was assessed by culturing it on production media for different time duration (24, 48 and 72 h). The effect of medium pH on lipase production was assessed at different pH ranged from 4 to 9 by culturing the isolated Bacillus sps (B1 B5) on medium with different temperatures (27, 37 and 47C) was also assessed.

Optimization of growth parameters A number of reports exist on influences of various environmental factors such as temperature, pH, nitrogen, carbon and lipid sources, agitation, and dissolved oxygen concentration on lipase production. Lipase production is generally stimulated by lipids. The lipase activity steadily increases to a peak and declines. Lipase production is usually coordinated with, and dependant on the availability of triglycerides. Besides this, free fatty acids, hydrolysable esters, bile salts, and glycerol also stimulate lipase production. High production of lipase in case of P. fragi occurs in peptone-supplemented medium, although different peptones vary in their effective-ness. Though Pseudomonas sp. grow in a basal medium with ammonium sulphate, glucose, citrate or pyruvate, it requires an organic nitrogen source for lipase production. A mixture of arginine, lysine and glutamic acid in medium was observed to be effective for lipase production. A strain of Penicillium

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roqueforti produces maximum amount of lipase when grown in 0.5% casitone and 1% proflobroth. Growth and lipase production by Micrococcus sp. were unaffected by peptone of 0.5% to 2%, but lipase production by Pseudomonas sp, A. wentii, M. hiemalis, R. nigricans, and M. racemosus were stimulated by peptone. Soybean meal extract in Rhizopus oligosporus culture medium supported good growth and lipase production. Physiological regula-tion of lipase activity by thermotolerant strain of P. aeruginosa EF2 under various conditions in batch, fed-batch, and continuous cultures support the contention that nitrate generally stimulates production of lipase. Milk is a good medium for growth of psychrotrophic bacteria and for lipase production which was found to be susceptible to catabolite repression by glucose. While glucose is essential for production of lipase by P. fragi, A. wentii, M. hiemalis, R. nigricans, and M. racemosus. P. aeruginosa EF2 (ref. 78) showed no such requirement. Lipase activity per milligram dry weight of mycelium was much higher on lactose, mannose, xylose, fructose, dextrin, and rhamnose in case of Talaromyces emersonii. Mannitol, galactose, sucrose, fructose, lactose, maltose, raffinose or ribose produced less amount of lipase caused decreased growth with corresponding reduction in lipase activity in M. racemosus. Polysaccharides such as glycogen, hyaluronate, laminarin, gum arabic, and pectin stimulated production of lipase in Serratia marcescens and Saccharomycopsis lipolytica. This might probably be due to the detachment of lipase from the oil surface. A similar mechanism may explain the stimulating effect of lecithin on lipase production, as investigated in R. japonicus. Triglyceride is important for lipase production as it can act both as an inducer and inhibitor. Among the triglycerides, olive oil was observed to be effective in inducing lipase. Salts of unsaturated fatty acids inhibited lipase production by P. fragi, whereas tributyrin and trioctanoin had no effect on lipase production by P. fragi and M. freudenreichii. Butter oil, corn oil or olive oil inhibited lipase production by P. roqueforti, Saccharomycopsis sp., B.

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licheniformis, M. caseolyticus and Staphylococcus sp.. Triglycerides such as olive oil, groundnut oil and cotton seed oil, and fatty acids such as oleic acid, linoleic acid and linolenic acid stimulated lipase production by P. mephitica. Lipids are considered not to be true inducers. A. wentii showed reduced growth and lipase production when the synthetic and natural lipids were added to the growth medium. Emulsification of culture media containing oil by gum acacia supported good growth and lipase production in R. oligosporus. Triolein, olive oil, tributyrin, and oleic acid butylester were able to induce lipase in immobilized protoplasts, whereas Tween 80 enhanced lipase activity. The initial pH of the growth medium is also important for lipase production. Maximum activity was observed at pH > 7.0 for P. fragi and at pH 9.0 for P. aeruginosa wherein development of acidity in media reduced lipase activity. In contrast, maximum growth at acidic pH (4.07.0) was reported for S. lipolytica, M. caseolyticus, B. licheniformis, A. wentii, M. hiemalis, R. nigricans., Mucor racemosus, R. oligosporus and P. aeruginosa EF2 (ref. 78). It has been observed that increasing the temperature above 8C resulted in a depressing effect on lipase production by P. fluorescens and P. fragi. However, the rapid inactivation of the P. fluorescens lipase by subtilisin at 20C (ref. 90) indicated that bacterial lipases could potentially be inactivated by simultaneously secreted proteinases and that this effect is likely to be greater in cultures grown at relatively higher temperatures (3040C) (ref. 36). Oso determined 45C to be the best temperature for lipase production by T. emersonii. Temperatures in the range of 2235C were however observed to be optimum for maximum lipase production for A. wentii, M. heimalis, R. nigricans, M. racemosus R. oligosporus, and P. aeruginosa. Aeration has variable effect on lipase production by different organisms. The degree of aeration appears to be critical in some cases since shallow layer cultures (moderate aeration) produced much more lipase than shake cultures (high aeration). Vigorous aeration greatly reduced lipase production by R. oligosporus, P. fragi, P. aeruginosa, and M. racemosus

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Isolation of lipase organisms from soil sample


resulted in increased lipase production in static culture conditions. However, high aeration was needed for high lipase activity by P. mephitica var. lipolytica, A. wentii and M. hiemalis. Changing the ratio of surface area to volume and hence, aeration of cultures of P. fragi had no effect on the quantity of lipase produced per cell; but increasing aeration by shaking resulted in both increased growth and lipase production, followed by a rapid decrease of lipase activity as shaking continued. Oso reported that stationary conditions in T. emersonii favoured maximum lipase production. Row and Gilmour observed that lipase synthesis, by two strains of P. fluorescens (psychrotroph), stimulated in milk medium at 7C, was immediately preceded by a decrease in O2 tension which resulted in earlier production of lipase. The influence of the concentration of O2 on lipase productivity by R. delemar has been studied in different fermenters. Giuseppin suggested that oxygen is the limiting factor in shake-flask cultures. He also reported that low oxygen concentration negatively affects the metabolism of R. delemar, which explains that low oxygen concentra-tion is a useful tool to scale down fermentation processes in cases where a transient or local oxygen limitation occurs.

Lipase purification Lipases have been purified from animal, plant, fungal and bacterial sources by different methods involving ammonium sulphate precipitation, gel filtration, and ion exchange chromatography. In recent years, affinity chromatographic techniques have come into use as this technique decreases the number of steps necessary for lipase purification as well as increases specificity. Currently, reversed-micellar and two-phase systems, membrane processes, and immunopurification are being used for purification of lipases. Lipase assay

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Isolation of lipase organisms from soil sample


Lipases are known to hydrolyse triglycerides and give rise to free fatty acids and glycerol. Therefore, the assay methods involve spectrophotometry or titrimetry, radiolabelling assay, fluorimetry, surface tension method, and estimation of free fatty acids by high performance liquid chromatography (HPLC). Tributyrin plate assay and titrimetry are the most commonly used methods for screening of lipase producers and estimation of lipase activity, respectively. Lipase properties pH optima The lipases that have been studied show profound stability around pH 6.07.5 with considerable stability at acidic pH up to 4 and at alkaline pH up to 8. Extracellular lipase of A. niger, Chromobacterium viscosum and Rhizopus sp. are active at acidic pH (refs 38, 63, 140). An alkaline lipase active at pH 11.0 has been isolated from P. nitroreducens.

Temperature optima and thermal stability The pancreatic lipases loose activity on storage at temperatures above 40C, but some microbial lipases are more resistant to heat inactivation. While lipases of A. niger, R. japonicus, and C. viscosum are stable at 50C, lipases of thermotolerant H. lanuginosa and P. sp. nitroreducens are stable at 60C and 70C (ref. 141), respectively. C. gigantea lipase had half life for inactivation of 35.7, 46.4 and 22.9 min. at 45C, 50C and 55C (ref. 12), similar to lipases of R. japonicus. In our laboratory, we observed that purified lipase from A. terreus retained 100% of its activity at 60C after 24 h. But, the maximum activities of C. gigantea and other lipases from mesophiles were at 3035C (ref. 144). Thermophilic bacterial lipases obtained from Icelandic hot spring showed higher lipase activity at 40 to 60C (ref. 145).

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Isolation of lipase organisms from soil sample

Substrate specificity Specificity of lipases is controlled by the molecular properties of the enzyme, structure of the substrate, and factors affecting binding of the enzyme to the substrate. Substrate specificity of lipases is often crucial to their application for analytical and industrial purposes. Specificity is shown both with respect to either fatty acyl or alcohol parts of their substrates. Many microbes produce two or more extracellular lipases with different fatty acid specificities. Tributyrin is hydrolysed slowly by some microbial lipases. In contrast, M. miehei lipase preferentially releases butyric acid from milk fat especially at low pH (ref 150). Geotrichum candidum produces a lipase, which shows pronounced specificity for the hydrolysis of esters of a particular type of long-chain fatty acid. Substrate specificity of this lipase has been summarized by Jensen, Jensen and Pitas, and Macrae. Lipases show both regio- and stereospecificity with respect to the alcohol moeity of their substrates. Lipases can be divided into two groups on the basis of the regiospecificity exhibited acylglycerol substrates. Lipases in the first group catalyse the complete breakdown of triacylglycerol to glycerol and free fatty acids together with diacylglycerols and monoacylglycerol as intermediates in the reaction. These intermediates do not accumulate since they are hydrolysed more rapidly than the triacylglycerol. Examples of the first group of lipases include lipase from C. cylindracea. The second group of lipases release fatty acids regiospecifically from the outer 1 and 3 positions of acylglycerols. These lipases hydrolyse triacylglycerol to give free fatty acids, 1,2-diacylglycerols, and 2monoacylglycerol. Many extracellular microbial lipases, such as those from A. niger and R. arrhizus, show 1,3-(regio)-specificity. Lipases excreted by R. japonicus, M. miehei, H. lanuginosa, C. viscosum, and P. fluorescens are also 1,3-(regio)-specific. Till date, there are no authentic reports of lipases which

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Isolation of lipase organisms from soil sample


catalyse the release of fatty acids selectively from the central 2-position of acylglycerols, except for a report of Asahara et al.. Partial stereospecificity in the hydrolysis of triacyl glycerols has been observed in R. arrhizus, R. delemar, C. cylindracea, and P. aeruginosa. Owing to this property, these enzymes can be used to isolate optically pure esters and alcohols. Production of an extracellular microbial lipase possessing pronounced stereospecificity in the hydrolysis of triacylglycerols, would be of considerable commercial interest. Most lipases attack triglycerides as readily as partially esterified glycerides, but an enzyme from a specific P. cyclopium strain has been shown to attack monoglycerides most rapidly followed by di- and triglycerides, respectively, and it has been described as a partial glycerol ester hydrolase. Several kinds of microbial lipases have already been introduced commercially (Table 1) and exploited for their potential to catalyse a large number of hydrolytic and synthetic reactions in both aqueous and organic media as mentioned below. As the net energy resulting from transesterification reactions is zero, these reactions can be easily carried out. However, the other two types of reactions are also gaining significant industrial importance. In particular, esterification reactions are industrially important in the synthesis of valueadded esters used in the cosmetic industry; the reaction is carried out by controlling the water content in the reaction mixture. When lipases are incubated with triglycerides, hydrolysis and resynthesis result in acyl migration between glyceride molecules. By controlling the quantity of water in the reaction system, it is possible to restrict the hydrolysis. The transesterification reactions can thus be made to dominate. If a nonspecific lipase is used, the rearranged triglycerides become more or less the same as obtained by chemical transesterification using sodium alkoxides as catalysts. However, by the use of 1,3-specific lipases, a mixture of triglycerides is obtained which is not obtained by chemical methods.

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Isolation of lipase organisms from soil sample


Lipase-catalysed trans-esterification reactions that are 1,3specific are utilized for making cocoa butter substitutes, using cheaper midfraction of palm oil and stearic acid. While the mid-fraction of palm oil contains 1,3-dipalmitoyl-2-monoolein (POP) as the major triglyceride, 1-(3)-palmitoyl-3(1)-stearoyl-2-monoolein (POS) and 1-(3)-distearyl-2-monoolein (TOS) are the main constituents of cocoa butter. Therefore, by transesterification between POP and stearic acid or POP and tristearin, valuable equivalents of cocoa butter have been made. The mid-fraction of palm oil is obtained by double-stage fractionation of the oils from n-hexane. The transesterification of the midfraction is carried out at 37C for 20 h using one part of the mid-fraction, 0.7 parts of stearic acid dissolved in water-saturated n-hexane (2.8 to 3 parts), and 0.1 parts of celite-immobilized lipase (Rhizopus arrhizus lipase from Sigma). The composition of the transesterified product approaches that of the cocoa butter. Sunflower and safflower seed oils have also been used for the production of cocoa butter substitutes in Japan. There is a large volume of literature on hydrolysis of fats and oils by lipases used either in the pure form or in the immobilized form or in the cellbound form. The hydrolysis is carried out using the conventional emulsion systems. The enzymatic hydrolysis has not however replaced the conventional colgate emery process. The saving in energy costs is perhaps not adequate enough to attract adoption of lipase-catalysed fat-splitting process over the conventional chemical process. However, some companies have reported use of lipases, for example use of lipase from Candida cylindracea for the splitting of oils, and the use of resulting fatty acids for the production of soaps. It has been claimed that the enzymatic method yielded soaps with better colour and odour, and resulted in an overall cost saving. Oils containing highly unsaturated or conjugated fatty acids are considered particularly amenable to enzymatic-hydrolysis processes. Currently, the focus of researches are on investigating the continuous hydrolysis of oils into fatty

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Isolation of lipase organisms from soil sample


acids, using membrane bioreactors or hollow fibre reactors. The enzymatic method of fat splitting will gain industrial importance with increasing high energy cost or with requirements for large-scale production of special grades of susceptible fatty acids. Manufacture of esters, through lipase-catalysed synthesis as well as alcoholysis is gaining industrial importance. Several high-purity esters have been manufactured for use in the cosmetic industry. Many of the esters, derived by using these reactions, resemble naturally occurring waxes of commercial importance. While the esters produced from short-chain fatty acids have applications as flavour constituents in the food industry, methyl- or ethyl esters produced from long-chain acids have a potential application for diesel fuels. Esters derived from long-chain fatty acids and long-chain fatty alcohols are referred to as waxes which have uses as lubricants or in the cosmetic industry; kilogram-scale synthesis of such esters has been described by Olivecrona and Bengtsson. Lipase-catalysed ester synthesis requires the maintenance of low concentration of water. The available literature indicates a variation in water concentration from 0.75% to 4% (w/v) in different types of ester synthesis. This condition is satisfied by using nonaqueous solvents like hexane. Certain lipases also selectively esterify alcohols. Thus,

Rhizopus arrhizus lipase of Novo Nordisk converts selectively geraniol into its acetate ester, when a mixture of geraniol and nerol is subjected to esterification in hexane. Thus, a reaction between 0.1 M each of geraniol and nerol mixed with acetic acid in hexane for 2 days produced 80% of the total esters as geranyl acetate, and the remaining 20% as nerol acetate. Laboratory-scale studies on an enzyme dose of 1% w/v for the synthesis of geranyl butyrate gave a conversion of almost 100% after 1.5 days at 30C when 0.1 M geraniol and 0.1 M butyric acid were used as reactants in hexane.

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Isolation of lipase organisms from soil sample


Lipases in polymer synthesis The stereoselectivity of lipase is useful for synthesis of optically active polymers252. These polymers are asymmetric reagents, and are used as absorbents. In the field of liquid crystals, suitable monomers can be prepared by lipase-catalysed transesterification of alcohols253, which with racemic alcohols may be accompanied by resolution254. The use of chiral glycidyltosylates for the preparation of ferroelectric liquid crystals246 has also been reported. Thus, this enzyme has diversified commercial use, both in terms of scale and processes. Lipases have been employed successfully in the food industry as well as in hightech production of fine chemicals and pharmaceuticals. Furthermore, this enzyme has potentials in newer fields, for example lipases have successfully been used in paper manufacturing apparently, the treatment of pulp with lipase leads to a higher quality product and reduced cleaning requirement. Similarly, the enzyme has also been used in association with a microbial cocktail for the treatment of fatrich effluents from an ice-cream plant. This could also be utilized in waste processing of many food industries294.

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Isolation of lipase organisms from soil sample

Lipase degradation current research going for lipase Industrial significance lipases have been widely studied with main focus on enzyme action, kinetics, sequencing and cloning of lipase genes and structural characterization but the studies on using RSM for validating and optimizing nutritional parameters for R. oryzae have been left relatively unexplored. Development of novel bioprocess is based on the

advancements in optimization of process which is a tedious and time taking process because effects of multivariable process parameters. For such purposes screening nutritional factors of importance initially carried out and selected factors are then optimized on priority basis by different available techniques. Response surface methodology (RSM) is an advanced tool now a days commonly applied involves three factorial designs giving number of input (independent) factors and their corresponding relationship between one or more measured dependent responses. RSM is advantageous over conventional methods available and it includes less experiment numbers, its suitability for multiple factor experiments and search for common relationship between various factors towards finding the most suitable production conditions for the bioprocess and forecast response . In this, linear or quadratic effects of experimental variables construct contour plots and a model equation fitting the experimental data. This facilitates the determination of optimum value of factors under investigation and prediction of response

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Isolation of lipase organisms from soil sample


under optimized condition [37]. The objective of the present study was to optimize the nutrients for lipase production by indigenous hyperlipolytic R. oryzae KG-10 isolated from Jharkhand, India using shake flask method. Response surface methodology was used here to optimize important nutritional factors screened by PlackettBurman design. Response Surface methodology (RSM) is a collection of statistical techniques for designing experiments, building models, evaluating the effects of variables and searching for the optimum conditions. Nowadays RSM widely used in optimization of different types of fermentations and bioprocesses [38-39]. The present work was an innovative step towards evaluating the industrial relevance and utilization of our indigenous strains R. oryzae KG-10 for the evaluation of physicochemical parameters. Particularly in this work we have applied RSM to evaluate the effect of physical as well as nutritional variables on lipase production by R.oryzae KG-10 and search optimal condition to attain a higher lipase yield.

Current status of lipase research in India Researches on microbial lipases in India date back to late seventies when a few reports on screening and production of lipase from a few fungi and bacteria appeared. The initial emphasis on screening exercises was followed by process optimization for maximum lipase production. Scientists at the National Diary Research Institute, Karnal113,115,117 investigated physicochemical conditions of lipases produced by M. racemosus, A. wentii, and P. chrysogenum. In 1981, one group highlighted the lipolytic activity of thermophilic fungi of paddy straw compost295. Systematic screening strategies were employed by Bhaduria296. This study reported A. niger, A. flavus, A. fumigatus and Penicillium glaucum as the potential lipase producers isolated from the kernels of chironji and walnut.

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Isolation of lipase organisms from soil sample


Owing to the industrial applications of lipases, the Department of Biotechnology, New Delhi, promoted research activities in this important area and consequently the momentum of research on lipases picked up in India. We have carried out large-scale process optimization for lipase production using A. terreus, A. carneus and B. stearothermophilus76,182,278. Chakrabarty et al.297 utilized extracellular microbial lipases for transesterification reactions for producing valuable transformed edible oils which cannot be obtained by chemical interesterification methods297. Chand et al.298 carried out fat splitting using castor-bean lipase. Lipases from H. lanuginosa and Y. lipolytica have also been reported for the synthesis of geranyl esters299. Kundu et al.160 isolated and characterized an extracellular lipase from the conidia of N. crassa, with an apparent molecular weight of 54 kDa and 27 kDa, determined by gel filtration and SDS-PAGE, suggesting thereby the presence of two identical subunits. Since 1988, extensive work on various aspects of lipase research, starting from production and purification to characterization and industrial applications, has been carried out on various fungi and bacteria in South Campus, Delhi University43,76,151,182,278,288,289. Novel thermostable and alkaline lipases from A. terreus and A. carneus are being developed for the production of biosurfactants, glycerides, and pharmaceutically important compounds. These lipases show regio- and chemoselective cleavage of polyphenolic compounds in a novel manner. Lipase from a strain of B. stearothermophilus shows remarkable activity even at 100C. Besides this, a rapid zymogram for lipase activity in polyacrylamide gels was developed which is of immense use to investigators in this field151. The ability of lipases to show increased stability and selectivity in organic solvents has been exploited by various researchers: Parmar et al.300, Gupta301 at Indian Institue of Technology, Delhi, Qazi and his group302, and SPIC Science Foundation. Biotransformations on polyacetoxy arylmethyl ketones, benzylphenylketone peracetates, esters of polyacetoxy aromatic

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Isolation of lipase organisms from soil sample


acids, and peracetylated benzopyranones, using commercial lipases have been carried out by Parmar et al.300. Qazi and his group263 have exploited the enantioselective behaviour of microbial lipases for the resolution of racemic drugs. Gupta and his group301 have investigated enzyme activity in organic solvents. They noted enhancement of enzyme activity in aqueous-organic solvent mixtures. Sridhar et al.303 employed lipase-catalysed ester interchanges for the modification of selected Indian vegetable oils into cocoa butter substitutes and high oleic oils. Satyanarayana and Johri 295, Sharath and Kamat305, and scientists at SPIC Anna University Bioprocess Laboratory have started work on the production of novel microbial lipases, which is yet to be exploited at commercial level. Scientists at the Central Leather Research Institute, Madras, and at the Central Food Technology Research Institute, Mysore, are using microbial lipases in the treatment of leather, and the production of flavour esters, respectively. The work being carried out in Indian laboratories has made considerable progress. Novel lipases with properties of chemo-, regioand enantioselectivity have been isolated, which may be eligible for exploitation at commercial level for industrial applications in course of time. But, some of the indigenously developed technologies for the production of lipases are already in the commercial production stage. Furthermore, comparison of some of the lipases produced by microorganisms indigenously is at par or even better than the well-known commercially available imported lipases. Thus, utilizing these lipases will greatly boost many biotechnology-based industries with the ushering of the 21st century.

Future outlook In spite of the importance of cold active lipases, studies on the mechanisms of production of microbial lipases and the role of lipidic substances used as inducers in lipase production are scanty. Cold active lipases represent an extremely versatile group of bacterial extracellular

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Isolation of lipase organisms from soil sample


enzymes that are capable of performing a variety of important reactions, thereby presenting a fascinating field for future research. The understanding of structure-function relationships will enable researchers to tailor new lipases active at low temperatures for biotechnological applications. Developments in research are expected from interchange of experiences between biochemists, geneticists and biochemical engineers. Wide and constant screening of new microorganisms for their lipolytic enzymes at low temperature will open novel and simpler routes for the synthetic processes. Consequently, this may pave new ways to solve biotechnological and environmental problems. The natural substrates of lipases are triacylglycerols, having very low solubility in water. Under natural conditions, they catalyse the hydrolysis of ester bonds at the interface between an insoluble substrate phase and the aqueous phase in which the enzyme is dissolved (Figure 1). Under certain experimental conditions, such as in the absence of water, they are capable of reversing the reaction. The reverse reaction leads to esterification and formation of glycerides from fatty acids and glycerol. The occurrence of the lipase reaction at an interface between the substrate and the aqueous phase causes difficulties in the assay and kinetic analysis of the reaction42. The usual industrial lipases are special classes of esterase enzymes that act on fats and oils, and hydrolyse them initially into the substituted glycerides and fatty acids, and finally on total hydrolysis into glycerol and fatty acids43-45.

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Isolation of lipase organisms from soil sample

In nature, the lipases available from various sources have considerable variation in their reaction specificities: this property is generally referred as enzyme specificity. Thus, from the fatty acid side, some lipases have affinity for short-chain fatty acids (acetic, butyric, capric, caproic, caprylic, etc.), some have preference for unsaturated fatty acids (oleic, linoleic, linolenic, etc.), while many others are nonspecific and randomly split the fatty acids from the triglycerides. From the glycerol side of the triglycerides, the lipases often show positional specificity and attack the fatty acids at 1 or 3 carbon position of glycerol or at both the positions but not the fatty acid at the 2 position of the glycerol molecule. However, through random acyl migration, the 2-fatty acid monoglyceride undergoes rearrangement pushing the fatty acid to the 1 or 3 position of the glycerol molecule; as acyl migration is a slow process and as the available lipases do not act on glycerol 2-mono fatty acid esters, the hydrolysis slows down and awaits the acyl migration to complete for enabling the lipase to attack the glyceride at the 1 and/or the 3 position. Interestingly, lipases function at the oilwater interface (Figure 2). The amount of oil available at the interface determines the activity of the lipases46. This interface area can be increased substantially to its

35

Isolation of lipase organisms from soil sample


saturation limit by the use of emulsifier as well as by agitation. The saturation limit depends on the ingredients used as well as the physical conditions deployed. Thus, the activities of lipases can be pronouncedly increased by use of emulsifying agents as well as by methods that increase the size of the emulsion micelles.

Enzyme assay and enzyme activity. The lipase triacylglycerol acylhydrolases, EC 3.1.1.3) assay was carried out by using modified lipase assay media (Peptone: 15g, NaCl: 5g, CaCl2: 1g, Tween 20: 10mL, Agar: 15g, Water: 1000 mL; pH: 7.0). The lipase activity was detected due to occurrence of a zone of clearance around the colony and subsequent formation of white precipitate of calcium monolaurate around the colony [25-26].

Enzyme activity was measured by titrimetric method. The oilwater emulsion and enzyme extract in a ratio 0.1 mL : 9.9 mL : 1 mL was titrated at constant temperature against 0.1 N NaOH using Phenolphthalein as indicator. A blank (9.9mL water, 0.1mL Tween20, 1mL sterilized broth media) was previously run to find the standard deduction in titer value. The principle behind this method is that Lipase release free fatty acid from the Tween 20 and cause acidic pH, which is neutralized by titrating with NaOH in constantly stirring vessel. Each reading was taken in triplicate. The activity is measured as amount of enzyme required liberating one micromole equivalent fatty acid per mL min-1.

Lipid optimization by substrates By using different substrates sources such as olive oil, coconut oil and sunflower oil, their effect on lipase production by the selected Bacillus

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Isolation of lipase organisms from soil sample


sp. was assessed at optimum pH (pH 7) and temperature (37C). Further the influence of substrate concentration (0.5 to 2.5%) on lipase production was also assessed on the optimized substrate, which maximized the lipase production.

Enzyme assay Isolated Bacillus sps (B1 B5) were assayed for extracellular lipase production using titrimetric method (Sadasivam and Manickam, 1996).

Lipase activity One unit of lipase activity was defined as the amount of enzyme releasing one mole of free fatty acid in one minute under standard assay condition. Volume of alkali consumed x Normality of NaOH Lipase activity = ( g/ml/min) Time of incubation x Volume of enzyme solution Mohan et al. 2729

Table 1. Biochemical identification of lipase-producing Bacillus strains from oil mill waste. Test Results Catalase + V-P +

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Isolation of lipase organisms from soil sample


Starch + Casein + Gelatin + Citrate + Nitrate + Acid production + Glucose + Arabinose + Xylose + Mannitol + MYP Yellow Statistical analysis The results obtained were subjected to relevant statistical analysis described by Zar (1974). Enrichment culture technique enabled the isolation of strains with lipolytic activity in tributyrin media plates. In total, 28 isolates were collected from the soil sample and among them; five isolates (B1 to B5) showed high lipolytic activity. The lipolytic microbes were further screened and characterized by their features and reactions and then identified as Gram positive, rod shaped motile organisms (Table 1). Finally the morphological and biochemical test indicated that the suspected organisms were Bacillus sp. The efficiency of lipolytic Bacillus sps. (B1 - B5) was assayed with different substrates like coconut oil, sunflower oil, olive oil and also at varied medium pH, temperatures and substrate concentration at different time intervals. Among the tested substrates, Bacillus sp. Showed maximum activity (0.0029 g/ml/min) in coconut oil at pH 7 (Figure 1). Among the Bacillus sp. tested, maximum lipase activity was achieved by the following Bacillus sp. B1, B4 and B5. Two way ANOVA test indicated that the influence

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Isolation of lipase organisms from soil sample


of various Bacillus strains on lipase production was statistically more significant (P < 0.01) than the independent influence (P > 0.05) of substrates. The results on the effect of medium pH on the tested sun flower oil indicated that the lipase production were maximum (0.026 g/ml/min) at pH 7. In low (4, 5 and 6) and also at high (8 and 9) medium pH, the lipase activity was less for all the tested Bacillus strains. Similar to that of recorded in the first experiment, here also the Bacillus strain B5 showed maximum lipase production at the opti2730 Afr. J. Biotechnol.

1a. At pH 4.0 1b. At pH 5.0 1c. At pH 6.0 1d. At pH 7.0 1e. At pH 8.0 1f. At pH 9.0 Figure 1. Effect of coconut oil on lipase activity of Bacillus sp. (B1 - B5) cultured for different time intervals (24 72 h) at different medium pH 4 9. mum medium pH (7.0), (Table 2) (Figure 2) Two-way analysis of variance for the data on lipase production indicated that the influence of different Bacillus strains was statistically more significant (P < 0.01) than the independent influence (P < 0.05) of medium pH. The result of the effect of medium pH on the tested olive oil indicated that the lipase production was maximum (0.026 g/ml/min) at pH 7. In low 4, 5, and also at high 8 and 9 medium pH the lipase activity was less for all the tested bacillus strains similar to that recorded in the first experiment has also the bacillus strain B5 showed maximum lipase production at the optimum me.

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Isolation of lipase organisms from soil sample

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40

Isolation of lipase organisms from soil sample

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Isolation of lipase organisms from soil sample

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Mechanism of lipid degradation enzymes Lipases are not involved in any anabolic processes. Since this enzyme acts at the oilwater interface, it can be used as a catalyst for the preparation of industrially important compounds. Lipases catalyse the hydrolysis of triglycerides into diglycerides, monoglycerides, glycerol and fatty acids, and under certain conditions the reverse reaction leads to esterification and formation of glycerides from glycerol and fatty acids. As lipases act on ester bonds, they have been used in fat splitting, interesterification (transesterification), development of different flavours in cheese, improving pallatability of beef fat for making dog food, etc. A current application involves using lipases in water-deficient organic solvents for synthesizing different value-added esters from organic acids and alcohols. Lipases which are stable and work at alkaline pH, say 8 to 11, which are usually the suitable wash conditions for enzymated-detergent powders and liquids, have also been found, and these hold good potential for use in the detergent industry.

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Isolation of lipase organisms from soil sample

Figure 2. Lipolytic reaction at the oilwater interface. Fungal lipases which degrade lipids from palm oil were investigated by Turner (cited by Lazer and Schroder, and listed by Sztajer and Zboinska). Among Mucorales, the lipolytic enzymes of the moulds Mucor hiemalis, M. miehei, M. lipolyticus, M. pusillus, Rhizopus japonicus, R. arrhizus, R. delemar. R. nigricans, R. nodosus, R. microsporus, and R. chinesis have been studied in great detail50. The thermophilic M. pusillus is well known as a producer of thermostable extracellular lipase. From a lipaseproducing strain of M. miehei, two isoenzymes with slightly different isoelectric points but a high degree of antigenic identity could be isolated92. A lipase of M. miehei, immobilized on a resin (Lipozyme TM) has been commercialized by Novo Industries. Due to the 1,3-(regio)-specificity of Rhizopus, lipases that are especially suited for the conversion of triglycerides to their corresponding monoglycerides, and interesterification reactions of fats and oils, have food and pharmaceutical applications. R. japonicus lipase has been used to produce hard butter suitable for chocolate manufacture by interesterification of palm oil with methyl stearate54. The lipases (40 to 45 kDa) of various Rhizopus species show maximum activity towards medium-chain fatty acids (C8C10). In case of R. delemar, extracellular94 and intracellular95 lipase isoenzymes have been isolated.

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Isolation of lipase organisms from soil sample


Lipase producers within the order Entomophthorales include Entomophthora apiculata, E. coronata, E. thaxteriana, E. virulenta, Basidiobolus spp. and Conidiobolus spp. The genera Pichia, Hansenula, and Saccharomyces are also reported to produce lipase82. Two kinds of cell-bound lipases were purified from Saccharomyces lipolytica89. Lipases are reported from Candida curvata, C. tropicalis, C. valida, and C. pellioculosa50 and are nonspecific towards the different ester bonds in triglycerides, with the exception of C. deformans89. H. lanuginosa lipases show a high degree of hydrolytic activity with coconut oil and oils having a high content of lauric acid. The two lipases differ in their positional specificity101. Newly produced olive oil has an opalescent appearance due to the presence of solid particles and micro-drops of vegetation water from the fruits. Some of our recent microbiological research has shown that a rich micro-flora is present in the suspended fraction of the freshly produced olive oil capable of improving the quality of the oil through the hydrolysis of the oleuropein. Present research however has, for the first time, demonstrated the presence of lipase-positive yeasts in some samples of extra virgin olive oil which can lower the quality of the oil through the hydrolysis of the triglycerides. The tests performed with yeasts of our collection, previously isolated from olive oil, demonstrated that two lipase-producing yeast strains named Saccharomyces cerevisiae 1525 and Williopsis californica 1639 were able to hydrolyse different specific synthetic substrates represented by pnitrophenyl stearate, 4-nitrophenyl palmitate, tripalmitin and triolein as well as olive oil triglycerides. The lipase activity in S. cerevisiae 1525 was confined to the whole cells, whereas in W. californica 1639 it was also detected in the extracellular fraction. The enzyme activity in both yeasts was influenced by the ratio of the aqueous to the organic phase reaching its maximum value in S. cerevisiae 1525 when the water added to the olive oil was present in a ratio of 0.25% (v/v), whereas in W. californica 1639 the

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Isolation of lipase organisms from soil sample


optimal ratio was 1% (v/v). Furthermore, the free fatty acids of olive oil proved to be good inducers of lipase activity in both yeasts. The microbiological analysis carried out on commercial extra virgin olive oil, produced in four different geographic areas, demonstrated that the presence of lipase-producing yeast varied from zero to 56% of the total yeasts detected, according to the source of oil samples. The discovery of lipasepositive yeasts in some extra virgin olive oils leads us to believe that yeasts are able to contribute in a positive or negative way towards the organological quality of the olive oil.. Oily substances reaching the sublittoral or surf zone soon become coated on or mixed with solids. In this form the oil is less likelly to be carried back to sea by ebbing tides or by the back rush of water from waves. Some oil may get buried on sandy beaches or under other debris. Its adsorption by solids renders the oil more susceptible to auto oxidation and particularly to microbial oxidation. It is also known that emulsions of oil or thin films are much more susceptible to auto oxidation than large coherent masses. In the first phases of auto oxidation, a free radical chain reaction results in the formation of hydroperoxide and in the second phase, the hydroperoxide is oxidized further with the formation of alcohols, organic acids, esters and ketones. One of the biggest problems in employing microorganisms to oxidize the emulsions of oil or thin films is that the bacteria only exist in small amounts and in order to be effective, large quantities had to be developed. Once the bacteria metabolize the oil, they die. The end product produces just as in auto or chemical oxidation, alcohols, fatty acids, ketones and other emulsified products which have no harmful effects on the existing marine environment. In the prior art, microorganisms have been used to convert oil to protein. For example, U.S. Pat. No. 2,769,750 discloses admixing particulate anhydrous inert adsorbents with hydrocarbons and/or oxygenated hydrocarbons.

45

Isolation of lipase organisms from soil sample


This homogenous mixture is further mixed with a nutrient and inoculated with hydrocarbon-consuming microorganisms. The adsorbents are normally added in sufficient amounts so that the adsorbent hydrocarbon mixture is a dry powder. The adsorbents are preferably naturally occurring clays, such as kaolinic clay. Further bacterial cultures have been freeze-dried, one technique being disclosed in U.S. Pat. No. 3,261,761. That process involves growing a culture in the presence of a metal salt such as NaCl; diluting the culture with buffer and sugar solutions; pre-cooling it to 0C to 1C, reducing its temperature to -5C or lower by applying a vacuum and subliming off the moisture by maintaining it at -5C or below. Concerning other techniques of drying microorganisms, U.S. Pat. No. 2,919,194 discloses suspending fine particles of wet yeast in a liquid and drying the suspended yeast by evaporating the water. The water evaporation is done by contact with a stream of inert gas. This patent teaches this type of drying as preferred when yeast cells are being processed. U.S. Pat. No. 3,224,946 relates to the use of synthetic or natural zeolites in microbial conversion of hydrocarbons to other products. The hydrocarbons are absorbed on the molecular sieves or within their crystalline pore structure. The hydrocarbon containing zeolites are contacted with the water containing the necessary nutrients and hydrophilic microbes are added. This allows the microbes to retain contact with its nutrient source and yet attack the hydrocarbons While the present invention is applicable to a broad scope of operable microorganisms, there are a number of microorganisms which are especially suitable for dispersing and degrading oil spilled as well as preventing the accumulation of oil on beaches, rocks, jetties and the like. These species were specially selected by elective cultures and screening techniques upon a wide variety of hydrocarbons. The hydrocarbon dispersing and degrading microorganisms useful in this invention include bacteria, yeasts, actinomyces and filamentous fungi. Examples of specific useful aerobic species of microorganisms are: Corynebacterium,

46

Isolation of lipase organisms from soil sample


Brevibacterium Job 5, Alcaligines entrophus, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas Oleovorans, Pseudomonas putida, Pseudomonas paraffinae, Mycobacterium desmolyticum, Acetobacter thodochrous, Pseudomonas Achromobacter methanica, agile, Micrococcus smegmatis, Achromobacter peroxydans, Mycobacterium

centropunctatum, Arthrobacter, Bacillus hexacarbovorum, Candida lipolytica, Candida tropicalis, Candida utilis, Candida parasilosis, Candida guilliermondii, Candida rugosa, Trichosporan cutaneum, Aureobasidium pullulans, The preferred carrier materials when used include clays such as kaolin, zeolites and other microporous silicaalumina materials, silica gels, vermiculities and perlites, and particularly these in hydrophillic forms. The operable materials, however, include microporous materials of the class into which microorganisms and nutrients or microorganisms alone can be absorbed and freeze-dried, and which will subsequently adsorb oil so as to bring this oil into a close relationship with the microorganisms for digestion. A particularly preferred material is vermiculite and ideally an exfoliated vermiculite. Vermiculite as used herein refers to the group of rock-forming mineral species characterized by a layer of latticized structure 10A which the silicatelayer units have a thickness of approximately 10a (Angstrom units). The main elements present in the layer are magnesium, aluminum, silica, iron and oxygen with the latter being separated by one or two sheets of water molecules associated with cations, such as magnesium, calcium, sodium and hydrogen. The layers have considerably lateral extent relative to the thickness of the basic 10 Angstrom-unit layer. Further, vermiculite belongs to the phyllosilicate group, which are characterized by the presence of Si--O sheets formed by the linkage of three corners of each SiO The type formula is Am(B X )
4

tetrahedron to

neighbors so that each tetrahedron has three shared and one free oxygen.
2 5 n

. Vermiculite has monoclinic hexagonal

plates and a hardness of one. The term "vermiculite" as used herein therefore

47

Isolation of lipase organisms from soil sample


includes minerals consisting wholly or largely of vermiculite, or minerals of a mixed-layer type containing vermiculite layers as an important constituent, such as hydrobiotites and chlorite-vermiculites. Unexfoliated vermiculite is an expanded vermiculite. For very effective use, the pore diameters of the microporous carriers should be in the range of 10A or greater, although smaller pore sizes in the range of 5A can be used. In regard to particle size this is not a critical factor, with sizes of from within the micron range up to a centimeter being useful. The particle size will be dependent to a degree on the particular microporous carrier being used, that is, for example, it will be different for an exfoliated and an unexfoliated vermiculite. After admixing, the mixture is then dried at a temperature ranging from 25 to 55C and preferably from 30 to 50C so as to form an anhydrous powder. The drying temperature must be maintained within this range in order to ensure that the full biological activity and viability of the final product is maintained. Alternatively, the mixture can be lyophilized (freezedried) in order to form the said anhydrous powder. In this form, the microorganisms can be stored until they are required for use in dispersing and degrading various oil spills. At the time of use, the dry matter is reconstituted with an aqueous phase such as seawater so as to maintain the viability (Table VII) and biological activity of the microorganisms (Table VII). The reconstitution step requires vigorous mixing to avoid caking. The reconstituted liquid is then sprayed over an area containing an oil spill rapidly and evenly.

The microorganisms act on the film of oil and on the oil so as to break it up into smaller globules in a period of over four hours or less, the globule particle size in diameter decreases from 200 microns to approximately 20 to 40 microns. After the globules reach the size of 6 to 20 microns, the oxidation products are realized in that the hydroxides, alcohols, aldehydes, and acids are formed by the microorganisms, then die. When the assimiable nutrients are exhausted, there is in fact, no contamination of the sea water by

48

Isolation of lipase organisms from soil sample


the microorganisms themselves.

It has also been found that when sand, rocks, concrete, metals, plastics and so forth are treated with a water slurry of the above-described microorganisms such materials are not readily contaminated by oil. It was found that plastic in contact with oil acquires a rather tenacious oil film in the absence of treatment with the described microorganisms. However, after the plastic is treated with a water slurry of the microorganisms by dipping the plastic into the slurry followed by draining, oil no longer stick to the plastic. Similar tests were conducted on metals, gears, rocks and sand.

The concentration of the viable cells per mole of slurry ranges from 10 5 to 10 8 . These microorganisms also can be employed for recovering petroleum oil from oil-bearing earth formations and can be useful in the secondary and tertiary recovery of petroleum oil from such formations. The process of this invention of preparing freeze-dried cultures of selected bacterial species of hydrocarbon consuming microorganisms involves growing the mixed population in a suitable aqueous medium containing high inorganic phosphate concentration and a suitable easily assimiable inorganic nitrogen source, trace metals and a hydrocarbon containing C 8 to C
18

carbons as the

only carbon source. Generally the mixed culture is produced in gram quantities in 20 to 50 liter quantities under vigorously aerated and agitated conditions. After the cell density has reached the desirable concentration, it is then preferably centrifuged to collect the cells and lyophilized or directly freeze-dried or adsorbed on to a suitable carrier selected from the inert supports described in this invention. The latter procedure is preferred since the desired amounts of nutrients are already present in this spent broth. In use, the preparation is generally reconstituted in suitable aqueous medium

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Isolation of lipase organisms from soil sample


such as water or sea water and applied onto the oil or hydrocarbon surfaces or sprayed on the solid surfaces such as beaches, rocks, shore lines, to prevent oil contaminating the solid surfaces.

Table VII sets forth the viability of the bacterial pool during storage at -18C prior to lyophylization and also the viability of lyophilized preparations after a week of storage at ambient temperatures. In addition, Table VII also sets forth the ability of the lyophylized preparations to disperse No. 4H.O heating oil in laboratory TABLE ____________________________________________________________ ______________ VIABILITY OF LYOPHYLIZED BACTERIAL POOL EM-30 AND INDIVIDUAL ISOLATES DAYS* VIABILITY % ** TIME FOR 100% DISPERSION LOT No. TYPE STORED CELLS/GRAM VIABILITY OF No. 4H.O HOURS ____________________________________________________________ ______________ 1. EM-30 5 1 10 10 1 1 Bacterial pool 2. EM-30 12 3 10 10 3 1 3. EM-30 20 2 10
8 10

tests. VII

2.5 1 4. EM-30 90 9 10 8 0.1 1 5. EM-30 90 9 10


10

0.1 1 6. Arthrobacter 3 4 10

5.0 0.5 ATCC No. 21908 7. Micrococcus 6 9 21910

10 9 4.0 0.5 ATCC No. 21909 8. Achromobacter 5 5 10 9 0.5 0.75 ATCC. No. ____________________________________________________________ ______________ * Wet cell paste stored at -18C prior to lyophylization. ** Viability tested after a week of storage at ambient temperatures. By employing the microorganisms in the manner shown and described hereinabove they produce a solution to the problems of not getting a large enough quantity into an oil slick in order to effectively accomplish its dispersion and degradation. The above procedure is inexpensive to prepare,

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Isolation of lipase organisms from soil sample


easy to store and convenient to transport wherever needed. By following this procedure a high degree of cell viability is maintained with no loss of biological activity when stored at ambient temperatures in sealed drums under an atmosphere of an inert gas such as nitrogen.

Mechanism of Microbial Oil Dispersion In order to determine the mechanism(s) adopted by these hydrocarbonophilic microorganisms a series of experiments were conducted. The affinity of the bacterial pool for oil and the initial changes in the physical nature of the oil were determined by examining a sample of oil from control and reaction flasks at 0, 10 and 20 minutes under phase contrast microscope. The results recorded are that initially the single oil globule is surrounded by a thin film of water and the oil-water interface is coated by bacteria. After the 20 minutes of exposure to the microorganisms the oil droplets are further reduced in size and after further incubation the continuous film forming property of the oil is lost and the inherent property of the oil to adhere to the solid surfaces such as glass, paper, wood and polyethylene, polypropylene, sand and rocks. Finally, the system becomes homogeneous and all oil washes away.

EXAMPLE 4: To determine whether the property of the bacterial pool herein referred to an EM-30 to disperse and degrade No. 4 Heating oil was unique, additional tests were conducted using several typical crude oils and two oil blends, heating oils No. 4 and No. 6. The rate of dispersion of total crudes and heating oils was determined by inoculating calculated amounts of EM-30 into a Basal Salts aqueous medium supplemented with 1% V/V of the crude oils and heating oils listed in Table X. The inoculated flasks were incubated on a

51

Isolation of lipase organisms from soil sample


New Brunswick rotary shaker describing 100 RPM and maintained at 24C. After periods specified, percent oil dispersed in each system was determined. The results are tabulated in Table X and they were essentially the same set forth.

The Effect of Temperature, pH, N and P on Dispersion Rates The effect of temperature on the rate of dispersion of No. 4 heating oil by EM-30 was determined by incubating at temperatures from +5 C to 40C with a 5C increment. Samples from each reaction flask were withdrawn at 10-minute intervals and percent dispersion of the oil was determined spectrophotometrically. The results are presented in Table XIV. At higher temperatures, the dispersion appears to be rapid. However, the optimum temperature range appears to be around 25-35C. It becomes difficult to define dispersion at temperatures lower than 10C because of the concomitant oil viscosity decrease and the slowing down of the metabolism of the microorganisms. However, there appears to be no significant change in the dispersion rates between 5 and 15C. These data suggest that the initial dispersion is dependent upon the particle size and amount of dispersant associated with the microbes. Apparently, the rate of initial dispersion by the microbial dispersant is independent of the temperature, however, further increase in the percent dispersion is closely associated with the optimum temperature for the biosynthesis of the dispersant. Additional experiments suggested that the pH of the reaction mixture is not critical. However, assimilable sources of nitrogen and phosphate and trace metals are necessary for the increase in biomass, and associated activities such as dispersion, degradation and transformation of the oil. Similarly, the rate of oil dispersion by microbes is dependent upon the strains which are capable of utilizing some fractions of crude oil as sources of carbon for the growth and for the biosynthesis of the dispersant, size,

52

Isolation of lipase organisms from soil sample


temperature, agitation, presence of assimilable sources of nitrogen and phosphate, and the chemical composition of the crude oil.

Effect of nitrogen source To increase lipase production by the fungus and considering that high nitrogen concentrations are typically used for the production of fungal lipases (21), medium A was supplemented with various organic and inorganic nitrogen sources (Table 1). Supplementation was most effective in the case of addition of inorganic salts, with the highest lipolytic activity (12 U/mL) obtained after 72 h with medium F (Fig. 1). However, with KNO3 as the sole source of nitrogen (medium G) the maximum lipolytic activity was relatively low (3.5 U/mL after 72 h). Since growth and lipase production did occur on this medium, KNO3 was included in the calculation of the C/N ratio for all media. Both of the media supplemented only with organic sources (C and D) performed similarly to medium A. In medium E, which contains yeast extract in addition to ammonium sulfate, the lipase production was high (9 U/mL). The specific activity was highest for the medium containing only inorganic nitrogen sources (297 U/mg, medium F), followed by the medium containing the combination of inorganic salts and yeast extract (130 U/mg, medium E). The specific activities attained with the other media were relatively low (59, 10 and 25 U/mg for media A, C and D, respectively). The lower specific activity values obtained for the fungus cultivated in media containing polypeptides could be due to the release of other proteins into the medium. Given that a higher lipolytic activity was obtained in a relatively short fermentation period (72 h) in a medium containing only ammonium sulfate and

53

Isolation of lipase organisms from soil sample


potassium nitrate as nitrogen sources (medium F), this medium was used as the base for the subsequent experiments.

Effect of carbon source Vegetable oils such as soybean, corn, sunflower, olive, palm and cottonseed oils, amongst others, are cited as inducers of lipase production, comprising, at times, the sole source of carbon in the medium (306-309). To evaluate the effect of carbon source on the production of lipase by P. aurantiogriseum, cultures were done with the addition of corn, soybean, sunflower and olive oils, each separately. For comparison, a culture was also grown with glucose as the sole carbon source. Considering 72 h of fermentation time, the lipolytic activities in the culture broth were approximately 5 U/mL when sunflower, corn and soybean oils were used, compared to an activity of 12.5 U/mL in the presence of olive oil (Fig. 2). Lipolytic activity was not detected when glucose was the sole carbon source, confirming that the presence of an inducer is necessary for P. aurantiogriseum to produce lipases (306). The maximum specific lipase activities obtained in these experiments were: 192 U/mg after 48 h with olive oil; 114 U/mg after 72 h with soybean oil; 220 U/mg after 112 h with sunflower oil and 138 U/mg after 112 h with corn oil. Therefore, although the volumetric activities were quite different in the presence of the various oils, the maximum specific activity did not vary so greatly. However, the time at which this maximum occurred did vary. Fatty acids present in the greatest proportions in these oils are oleic and linoleic acids. Triglycerides of olive oil and corn oil, which were the two best carbon sources for lipase production, contain 28 and 25 % oleic acid, respectively, while those of sunflower oil and soy oil contain 17 and 13 % oleic acid, respectively. On the other hand, sunflower, soy and corn oils have higher percentages of linoleic acid (51, 27 and 33 %, respectively), while olive oil contains only 3 % linoleic acid. Better lipase production appears therefore to be correlated with a higher content of oleic

54

Isolation of lipase organisms from soil sample


acid in the oil (309). Due to the higher volumetric activity obtained with olive oil, it was maintained in the fermentation medium for the remaining experiments, but it is important to note that sunflower oil, a less expensive substrate, could also be used for industrial scale production.

Effect of the nitrogen source concentration For fungi, relatively high nitrogen concentrations (and therefore lower C/N ratios) are typically required in order to favor the production of lipases over the production of other enzymes (306,311,313). To evaluate the effect of the concentration of the nitrogen source, cultures were done with increasing concentrations of ammonium sulfate while the concentration of olive oil was maintained constant, in such a manner as to give C/N ratios of 1, 2.5, 5 and 10. The maximum lipolytic activity obtained with a C/N ratio of 5 was 17 U/mL and with a C/N ratio of 2.5 it was 13 U/mL (Fig. 3). With an even higher C/N ratio of 10, lipase production was relatively poor. With a C/N ratio of 1 the amount of lipase produced was not significant and the mycelial growth in the flask was very poor. The peak volumetric activity was obtained between 72 and 96 h for all C/N ratios. The peak of the specific activity occurred after 48 h with initial C/N ratios of 2.5 and 5, and between 48 and 72 h with an initial C/N ratio of 10. The highest specific activity of 113 U/mg was obtained with a C/N ratio of 5. Reasonably high values of 103 and 83 U/mg were obtained for C/N ratios of 2.5 and 10, respectively.

Effect of the carbon source concentration The effect of the concentration of the carbon source on lipase production was studied with the addition of different concentrations (0.5, 1, 1.5 and 2.0 %) of olive oil. The concentrations of ammonium sulfate and potassium nitrate were maintained constant. The carbon source concentration has a strong influence on the production of lipase by P. aurantiogriseum, as

55

Isolation of lipase organisms from soil sample


shown in Fig.4. With an increase in olive oil concentration there was a decrease in the peak lipolytic activity attained. Fermentations done with 1.5 and 2 % olive oil had much lower peak activities, indicating an inhibitory effect on the production of lipase by P. aurantiogriseum.

Various cloning and recombinant studies for lipase studies We were able to use PCR to amplify small regions of lipase genes directly from chromosomal DNA. This was achieved by using highly degenerate consensus primers to the oxyanion hole (Jaeger et al., 1994) and active-site regions of lipase genes to amplify fragments of putative lipases. Using genomic-walking PCR (Morris et al., 1995, 1998), we were able to clone a complete lipase gene from amixed environmentalDNA sample and express it in a heterologous host. At the amino acid level, the lipase gene shares less than 20% similarity with any known lipase gene. Expression of the novel lipase gene was highly toxic to the Escherichia coli host, making it unlikely that the gene could have been cloned using an expression library strategy. We conclude that the PCR method described here is useful for lipase gene prospecting, since it allows cloning of genes from organisms that are unculturable (or yet-to-be-cultured) or possess lipase genes whose expression is highly toxic to the heterologous host.

56

Isolation of lipase organisms from soil sample


METHODS Bacterial strains, media and plasmids. Bacterial cloning experiments were carried out with either Escherichia coli DH5a or BL21-SI (Life Technologies) using standard techniques. E. coli DH5a was used in combination with general cloning vectors suitable for blue}white assays such as pUC18 (Yanisch-Perron et al., 1985) and pCR (Invitrogen). E. coli BL21-S1 was used in combination with the Novagen pET- 26b() vector containing the T7 promoter to clone and express the oli lipase gene. A Bacillus pumilis strain producing lipase was used to test the ability of the lipase-prospecting primers to selectively amplify lipase genes of known sequence directly from chromosomal DNA. DNA sequence determination. Automated sequencing was carried out according to the dideoxy chain-termination method of Sanger et al. (1977) using the ABI PRISM Ready Reaction Dye Terminator Sequencing Kit.

Biomass generation and DNA extraction procedure: Environmental biomass was obtained from an olive-oil enriched percolation conducted at 65 C from a natural New Zealand hot spring. High molecular mass DNA ("15 kb) was extracted from B. pumilis biomass and olive oil percolation biomass as described previously (Morris et al., 1998).

Database searching and computational analysis: Lipase gene sequences were obtained using the Entrez search and retrieval system at the National Center for Biotechnology Information (NCBI). Regions with homology to the lipase gene sequences were obtained using blastp at NCBI. Alignment of the olilipase gene with the Bacillus lipase

57

Isolation of lipase organisms from soil sample


genes was performed using clustalw at eBioinformatics

(http :}}www.bionavigator.com}).

Cloning of lipase gene fragments: DNA was extracted from either a pure bacterial strain or from environmental biomass.

Identification of conserved regions within lipase genes Lipases are serine hydrolases and their active site is composed of three residues : a serine, a histidine and a carboxylate residue (Jaeger et al., 1994). Lipase gene alignments show that conservation around the three active-site residues is low (Jaeger et al., 1994), making them dicult targets for genomic prospecting using PCR. However, such alignments reveal a fourth conserved site at a region corresponding to the lipase oxyanion hole (Jaeger et al., 1994). We examined the amino acid sequences of over 70 lipases from bacteria, archaea and eukaryotes containing the [GA] X-S-X-G sequence characteristic of the active site. Lipases possessing the active-site consensus sequence generally also possessed a region homologous to the oxyanion hole region of the Pseudomonas glumae lipase (Jaeger et al., 1994) located 60108 aa upstream of the active site. These results, combined with analysis of available lipase three-dimensional structures, indicated that the activesite consensus sequence and the oxyanion hole were the most promising sites for the design of PCR primers.

Characterization of conserved regions within lipase genes Although some homology was detected in most lipases at the oxyanion hole and the active-site consensus region, several factors make the

58

Isolation of lipase organisms from soil sample


design of PCR primers using these sites dicult. These factors include low homology, the presence of unfavourable amino acids such as serine within the conserved sequences, the absence of contiguous conserved amino acids and the shortness of the conserved sites (6 aa or less). The initial step in overcoming these problems was to identify and dene the sequence constraints on both the oxyanion hole region and the region surrounding the active-site serine residue. Alignment of sequences at the oxyanion site revealed the presence of a short hydrophobic region (6 aa) upstream of amoderately conserved His-Gly (HG) dipeptide. A related hydrophobic region could also be identied in lipases that did not possess the HGdipeptide, but rather possessed only a conserved Gly residue. A correlation was found between sequences at the oxyanion hole and sequences present at the active site, allowing division of the lipases into four groups as shown in Table 2. To demonstrate the capacity of PCR to amplify lipase gene fragments, we focused our eorts on designing primers to Group 1, the largest and most diverse of the lipase groups. This group includes lipases from archaeal, bacterial and eukaryotic sources. Using prosite (http :}}au.expasy.org}prosite}) nomenclature in which ambiguities are indicated by listing the acceptable amino acids for a given position between square parentheses ` [] ' and each element in a pattern is separated from its neighbour by a ` - ', the consensus sequence of the active-site region of group 1 lipases could be dened as [GA]-H-S-[MHQL] G-[GASTP]. Similarly, we could dene the oxyanion hole consensus as [PNTSVL]-[VIF]-[VIFL]-[VIFLM]-[VLCAISQ]-H-G. Examination of the two sites revealed that they were separated by between 60 and 110 aa (180330 bp).

59

Isolation of lipase organisms from soil sample


Design of lipase-prospecting primers Using CODEHOP primer design principles (Rose et al., 1998), we designed the primers to have a two-part structure, a consensus clamp and a degenerate core. However, for the design of the lipase-prospecting primers, lipases within the groups were not given any weighting, since it was considered that the most conserved' residues observed may be a consequence of the fact that most lipases have been derived from a limited phylogenetic background. Instead, the primers were designed to suit as many lipases as possible. Since the aim of our PCR strategy was to clone lipases from unknown genetic backgrounds, codon usage was not used to limit the degeneracy of primers. Bearing these criteria in mind, an initial set of primers were designed to be complementary to the active site and the oxyanion hole. In an iterative process, the sets of primers were examined and manually compared to the available lipase gene sequences to determine how many lipase gene sequences the primers would potentially bind to. The primer sequences were then modied to maximize the number of lipases that could be expected to be amplied. After repeating the process several times, a minimal set of three degenerate primers were designed that were complementary to the oxyanion hole (Table 1). This design was greatly facilitated by the observation that most of the hydrophobic residues upstream of the HG dipeptide (e.g. LVMIF) possessed a T nucleotide in the second position of the codon triplet. Using a similar process, a set of four degenerate primers were designed to be complementary to the active site of the lipases. In both sets (Table 1), the degeneracy of the primers went slightly beyond the generally accepted maximum levels of degeneracy for PCR (Rose et al., 1998). Identification of conserved lipase motifs in other members of the a/b hydrolases

60

Isolation of lipase organisms from soil sample


We searched for other proteins possessing sequences

homologous to those found in Group 1 to determine whether the consensus regions identied were unique for lipases. As shown in Table 3, both regions co-exist in several other a}b hydrolase proteins. Some of these proteins match exactly the consensus sequences found in Group 1 lipases. Therefore, it was concluded that the lipase-prospecting primers would also amplify gene fragments from a selected range of other a}b hydrolases, including dihydrolipoamide acetyltransferases, tropinesterases, lysophospholipases and halogen peroxidases. Testing of lipase-prospecting primers on B. pumilis chromosomal DNA PCR products were obtained for most combinations of the lipase-prospecting primers (Fig. 1a). Sequencing of several of these bands indicated that at least six unique sequences were amplied by the lipaseprospecting primers (Fig. 1a). Three of these amplication products showed homology to known or suspected a}b hydrolases (Table 4) and one of these was nearly identical to the equivalent region of the published B. pumilis lipase (accession no. A34992). To conrm that this gene fragment was derived from a functional lipase gene, primers BPUM1F and BPUM1R (Table 1) were designed to amplify the full-length B. pumilis lipase gene. Cloning of the amplied gene product into the pCR vector (Invitrogen) followed by plating onto lipase tester medium (Kouker&Jaeger, 1987) indicated the amplied lipase gene was functional and expressed under control of the lacZ promoter. Sequencing of the full-length gene indicated that it diered from the published B. pumilis gene at 9 aa positions and was identical to the sequence amplied by the lipase-prospecting primers (data not shown). As predicted, the lipase-prospecting primers also amplied fragments of other a}b hydrolase genes, including a gene with homology to a putative lysophospholipase from Bacillus halodurans (accession no.

61

Isolation of lipase organisms from soil sample


NCj002570) and a gene with homology to another conserved gene of B. halodurans (accession no. NPj242762) that shows some homology to known lipases. At least three other bands were amplied by the lipase-prospecting primers that did not correspond to structural gene fragments since no ORFs could be detected within the amplied sequences (Fig. 1). This is most likely a consequence of the highly degenerate nature of the primers and the nonstringent nature of the PCR conditions used. Overall, however, these results indicate that the lipase-prospecting primers are capable of selectively amplifying fragments of lipase genes from complex mixtures of chromosomal DNA. Cloning of a novel lipase gene from biomass To demonstrate that the lipase-prospecting primers can be used as a general method to clone unknown lipase genes, we used the primers to amplify DNA fragments from environmental biomass. DNA was extracted from an olive oil percolation performed using water from a New Zealand hot spring at Kairua Park, Rotorua (see Methods). Amplication of 16S rRNA sequences directly from the biomass DNA (see Methods), followed by subcloning and sequencing indicated that the bulk DNA contained DNA from a variety of dierent organisms, including some with greatest 16S homology to Pseudomonas sp., Leptospira sp., Cytophagales sp., Rhodothermus sp. and Desulfotomaculans sp. When the lipase primers were applied to the environmental biomass, many bands were obtained, including several of an appropriate size for a}b hydrolases (180320 bp) (Fig. 1b). Direct sequencing of a band generated by OXF1 and ACR1 primers (Table 1) demonstrated that it was composed of a single DNA species. Conceptual translation of the gene fragment revealed the presence of an ORF that displayed some homology to known lipase genes. Direct sequencing of the other bands failed to produce good quality sequence, presumably due to the presence of multiple sequences. This was not surprising given the complex nature of the PCR substrate. Since a candidate lipase gene fragment had been successfully

62

Isolation of lipase organisms from soil sample


amplied, these bands were not further investigated. Genomic-walking PCR (Morris et al., 1995) was used to obtain the full-length gene sequence for analysis. Conceptual translation of the gene revealed the gene had the highest (but still low) degree of similarity with the lipases from the mesophilic bacilli. Cloning lipases using PCR Chromosomal DNA was used as a template for amplication of lipase gene fragments using degenerate consensus primers OXF13 and ACR14 (Table 1) in all possible combinations. PCR reactions were performed according to the following conditions using a Perkin Elmer thermal cycler (model 480): template DNA was added to a nal concentration of 175 ng ll-" in a buer composed of 1AmpliTaq gold PCR buer, 5 mM MgCl#, 048 mM dNTPs, and 025 U AmpliTaq gold enzyme ll-". Forward and reverse primers were added at a nal concentration of 8 ng ll-". Five thermocycles were performed as follows: Seg1 (94C, 1 s), Seg2 (94 C, 1 min), Seg3 (37 C, 1 s), Seg4 (37 C 30 s), Seg5 (72 C, 2 min 20 s), Seg6 (72 C, 1 min). These ve cycles were followed by 35-step cycles as follows: Seg1 (95 C, 1min), Seg2 (50 C, 1 min), Seg3 (72 C, 1 min). Linker assembly, linker library construction and genomic-walking PCR were performed according to Morris et al. (1995, 1998). Forward and reverse genomic-walking primers are shown in Table 1. Amplification and cloning of full-length lipase genes using PCR. BPUM1F and BPUM1R primers were added to PCR reactions containing B. pumilis chromosomal DNA. After a 10 min 95 C activation of the AmpliTaq Gold polymerase (Perkin Elmer), 35 PCR cycles (95 C, 1 min; 45 C, 2 min; 72 C, 1 min) were performed. The sequence of the B. pumilis lipase gene was obtained by direct sequencing. OPL1F and OPL1R primers were added to PCR reactions containing DNA from the olive oil percolation. After a 10 min 95 C activation of the AmpliTaq Gold polymerase (Perkin

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Isolation of lipase organisms from soil sample


Elmer), 35 PCR cycles (95 C, 1 min; 45 C, 2 min; 72 C, 1 min) were performed. The sequence of the oli-lipase gene was conrmed by direct sequencing. Presumably due to toxicity to the E. coli host, the lipase gene could not be cloned into standard cloning vectors such as pUC (YanischPerron et al., 1985). The gene was cloned into a pET-26b() (Novagen) vector under control of the T7 promoter to allow the gene to be maintained and expressed in the E. coli host. To reduce basal expression levels, this synthetic T7lac promoter contains a 25 bplac repressor. The host strain BL21-SI contains a salt-inducible T7 RNA polymerase gene, allowing expression of high levels of recombinant proteins by the addition of salt to the induction medium. The primers OLIPFPET and OLIPRPET (see Table 1) incorporate the restriction sites BamHI and HindIII, respectively, allowing the directional in-frame ligation of the amplied fragment into pET-26b(). The expression vector pET-26b() carries an N terminal pelB signal sequence for periplasmic localization plus a C terminal tag of six histidine residues. Production and characterization of recombinant oli-lipase LuriaBertani medium (600 ml) without NaCl, supplemented with kanamycin (30 lg ml-"), was inoculated with 6ml of an overnight culture of E. coli strain BL21-SI (Life Technologies) harbouring the recombinant OlipET26b() plasmid. The culture was incubated at 22 C, with shaking, until the absorbance at 600nm reached 05. NaCl was added to a nal concentration of 300 mM to the culture broth to induce expression of the recombinant lipase and the growing culture was incubated for a further 4 h. To reduce fully induced expression levels, IPTG was not added to the broth, thus maintaining partial lacI repression of expression. Following induction, cells were harvested by centrifugation. Purication of the recombinant oli-lipase fusion protein was performed with Ni#+-nitrilotriacetic Magnetic Agarose Beads (Qiagen) following the manufacturer's instructions.

Characterization of the recombinant lipase

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Isolation of lipase organisms from soil sample


Lipase activity was detected by measuring the hydrolysis of pnitrophenyl esters (C%C")). Assays were performed in 120 mM universal buer (Britton & Robinson, 1931), pH 80, for 2 min under a variety of physiochemical conditions prior to measuring the increase in absorbance at 405 nm. The eect of temperature on the reaction rate was determined by incubating the puried recombinant lipase with the substrate at temperatures ranging from 30 to 70 C for 2 min. The enzyme activity was also assayed at pH values ranging from 3 to 10 (120 mM universal buer) at the optimal temperature for activity (50 C) for 2 min. Unit activity was dened as 1 lmol PNP liberated min-".

Expression and characterization of a novel lipase gene product from biomass DNA Although the entire putative lipase gene could be readily amplied using suitable PCR primers, it was found that the gene could not be readily cloned into the pCR cloning vector (Invitrogen) or other plasmids such as pUC18 (Yanisch-Perron et al., 1985) with lac-based promoter systems. In hindsight, this observation was presumably due to the high toxicity of the gene to the host since the lipase gene could be cloned into pET26b() (Novagen) which is characterized by low basal expression levels of genes cloned into the polylinker. The basal level of expression of genes cloned into pET26b() is very low due to a combination of repression by the lacI repressor and tight control of induction by the T7 promoter (Novagen). To minimize fully induced expression levels of the oli-lipase gene, only salt was added to induction cultures, rather than salt and IPTG required for full induction. Despite the tight control of lipase gene expression using this vector, yields of the puried protein using the T7 promoter system were low and microscopic examination of the cells revealed morphological abnormalities in the E. coli host expressing lipase. The puried recombinant oli-lipase was active between 30 and 70C, with an optimal temperature for activity at 4550

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C. At 60C, more than 50% of the initial activity of oli-lipase was detected. The optimal pH for activity of oli-lipase at 50 C was pH80, whereas no activity was detected at pH 30. Furthermore, at pH 10, the lipase displayed 65% of its initial activity. The substrate specicity of the puried oli-lipase demonstrated a preference for p-nitrophenyl esters with longer fatty acids ("C); data not shown). The lipase showed. P. J. L. Bell and others the highest activity (100%) towards pnitrophenyl caprate (C"!), whereas in the presence of p-nitrophenyl laurate (C"#) and p-nitrophenyl myristate (C"%), the enzyme displayed 82 and 57%of its maximal activity. In the presence of p-nitrophenyl butyrate (C%), only 20% of the initial activity of oli-lipase was detected. Lipases can be simply dened as hydrolysing long-chain acylglycerols ("10 carbon atoms) (Ferrato, 1997). Accordingly, based on its substrate specicity oli lipase is a true lipase. Current methods are not ideal for the direct cloning of lipases from environmental biomass for several reasons. In particular, environmental cultures will be composed of many organisms each secreting their own lipase making the purication of one specic lipase enzyme dicult. Since the design of oligonucleotide probes requires highly puried lipase enzyme, the presence of several lipase enzymes in mixed microbial populations complicates the use of this method for lipase gene prospecting. Uneven distribution of organisms within the biomass is also a problem, particularly if one or a few bacterial species are dominant in the population. In these cases, successful prospecting for novel lipases requires construction of large libraries to obtain complete coverage of the range of genomes present. Expressioncloning strategies can also fail due to diculties in achieving expression of a lipase in a heterologous host. For example, lipases such as those from both mesophilic and thermophilic bacilli are toxic to the E. coli host (Bell et al., 1999; Kim et al., 1998; Dartois et al., 1992). A consequence of this toxicity may be that bacteria containing lipase genes are poorly represented or absent

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Isolation of lipase organisms from soil sample


from an expression library based on environmental DNA. Functional expression of the lipase protein may also require a helper gene or foldase (Jaeger et al., 1994; Sullivan et al., 1999). Incompatibility of transcription and translation apparatus between the organism producing lipase and a heterologous host such as E. coli also limits the usefulness of expression libraries for lipase gene prospecting. For example, eukaryotic lipase genes may contain introns, and thus not be functionally expressed from genomic DNA libraries in bacterial hosts such as E. coli. In these cases, lipases must be isolated by constructing and screening cDNA libraries (e.g. Haas et al., 1991). Genes from bacterial species such as Thermus and unculturable organisms may also be dicult to express in E. coli due to both dierences in codon usage and fundamental dierences in the genetic information processing apparatus (Ishida & Oshima, 1996). Our study demonstrates that PCR methodology can be used to prospect for novel lipase genes directly from environment DNA despite the low homology observed between lipases. Since PCR methods allow the specic amplication of rare sequences within complex DNA mixtures, PCR-based technology, such as that described here, overcomes many of the problems associated with isolating lipase genes using library-based methods. Finally, the similarity between the conserved sites of the lipases and several other a}b hydrolases suggests that the PCR strategy used here to amplify lipase gene fragments can also be used to amplify regions of other a}b hydrolases of interest, such as halogen peroxidases and lysophospholipases.

Activation and inactivation of the enzyme Cofactors are not required for the expression of lipase activity. Divalent cations, such as calcium, generally stimulate the activity. It has been postulated that this is based on the formulation of calcium salts of long-chain fatty acids. The lipase activity is inhibited drastically by Co++, Ni++, Hg++, and

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Isolation of lipase organisms from soil sample


Sn++; and is slightly inhibited by Zn++, Mg++, EDTA, and SDS146. In H. lanuginosa S-38, sulphhydryl-reducing agents, like dithiothreitol, did not alter the enzyme activity, but did render it more susceptible to heat inactivation. Inactivation is accelerated by the addition of urea. Reducing compounds (cysteine, 2-mercaptoethanol), chelating agents, (EDTA, o-phenanthroline), and thiol group inhibitors (p-chloro mercuric benzoate, monoiodoacetate) did not show a detectable effect on lipase in M. pusillus, suggesting that lipase is not a metallo-enzyme and it does not require either free -SH group or an intact SS bridge for its activity. Spontaneous and cyclic AMP-induced lipase formation is greatly enhanced in Serratia marcescens SM-6 on exposure to glycogen, hyaluronate, pectin B, and gum arabic.

Objectives: Sample collection. Screening and cultivation of microbes on desired media.

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Subculturing and storage of cultures. Genomic DNA isolation from subcultured microbes. Qualitative and quantitative analysis of whole genomic DNA. PCR amplification of 16s rDNA gene. Molecular weight based confirmation of amplified product. Purification of amplified product. Sequencing of amplified product. NCBI BLAST analysis. Phylogenetic tree construction.

Materials and methods Sample collection:

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Samples were collected from the sites contaminated with lipids and oils from 5 different sites in Vijayawada. The soil samples are collected from 1 inch below the soil surface in sterile polythene bags.

Screening and cultivation of microbes and desired media Serial dilution:


Collected soil is to be mixed very well and dry it at 1100c.

1gm of soil was dissolve in 10ml of water by vortex.

Take 8 test tubes and add 9ml of water in each test tube and

label it as 10-1, 10-2 up to 10-8. Then take 1ml of water sample from the soil dissolved sample

and add that sample to 10-1 test tube.

Take 1ml of sample from the 10-1 test tube and mix it in 10-2 test

tube.

Take 1ml of sample from the 10-2 test tube and mix it in 10-3 test

tube.

Take 1ml of sample from the 10-3 test tube and mix it in 10-4 test

tube.

Take 1ml of sample from the 10-4 test tube and mix it in 10-5 test

tube.

Take 1ml of sample from the 10-5 test tube and mix it in 10-6 test

tube.

Take 1ml of sample from the 10-6 test tube and mix it in 10-7 test

tube.

Take 1ml of sample from the 10-7 test tube and mix it in 10-8 test

tube.

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Isolation of lipase organisms from soil sample

Preparation of nutrient agar medium: Composition: Luria Bertani agar (LB Agar) Agar agar Prepare the nutrient agar medium for 60ml. The diluted samples were plated on nutrient agar for total viable count. Then sterilize the medium, petriplates, L shaped loop in autoclave for 30 minutes. Pour the nutrient medium into 4 petriplates. After solidification 1ml of sample poured in petriplates spread it with L shaped loop.

Then incubate it for 24hrs at 340c. The dominant organisms were isolated and individually streaked on tributyrin agar plates.

The formation of halo zone around the colony on tributyrin agar was considered as the positive colony.

Sub culturing and storage of cultures Preparation of nutrient broth: LB Broth is dissolved in 25ml of distilled water. Take 5 test tubes and 5ml of nutrient broth in each test tube.
In one test tube inoculate the colony from the 10-6 culture plate. Then in another test tube inoculate the colony from 10-8 culture plate.

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In another test tube inoculate the colony from 10-8 with anti-biotic

culture plate.
Take another test tube and inoculate the colony which is similar in

morphology from the 10-6 and 10-8 culture plates.


Incubate the plates for 24 hrs a 360c by keeping the cotton plug for

each test tube.

Gram staining Take two sterilized slides.


In one slide take a loop of culture from nutrient growth in which similar

colonies from 10-6 and 10-8 are grown for 24hrs.


Take another slide and loop of culture from nutrient growth in which 10 8

with anti-biotic are grown.

Prepare the smear in these two slides and air dry it. Then add crystal violet and allow it to dry for 2 minutes and wash with distilled water. Then add grams iodine and allow it to dry for 30sec and wash it with distilled water. Then decolorized with acetone. Then counter stain with saffranin.

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Wash the strain with distilled water and see under microscope.

Observation: Gram +ve bacilli was observed 10-6 and 10-8 colony. Gram ve cocci was observed in 10-8 with anti-biotic inoculated culture.

Preparation of nutrient medium: Composition: 1. Nutrient broth 2. Cacl2 3. Agar agar. 4. SDS (sodium dodecyl sulphate) In 5ml of hot water SDS is dissolved. Then take nutrient broth, Cacl2, agar are dissolved in 45ml of water. Autoclave the medium for 30 minutes. After autoclave add SDS along the wall of flask and mix carefully with out bubbles. Pour the medium to the petriplates and grow the organism by streak plate method.
Incubate the petriplates for 24hrs at 360c. In one plate inoculate the colony with 10-6 and 10-8 from broth

culture.

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In another plate inoculate the colony with 10-8 from broth culture. In another plate inoculate the colony with 10-8 with anti-biotic and

broth culture.

Preparation of broth: LB agar is dissolved in 50ml of water. Take 5 test tubes; in each test tube 10ml of LB agar is poured. Then inoculate the colony from the culture plates.
Incubate it for 24hrs at 360c.

DNA isolation Steps: 1. Lysis of cells. 2. Precipitation of proteins. 3. Precipitation of nucleic acids. 4. Purification of nucleic acids.

Types of lysis: 1. Physical lysis like sonication, grinding, etc. 2. Chemical lysis i. SDS for denaturing the proteins ii. Placing cells in hypotonic solution. 3. Enzymatic lysis.

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Isolation of lipase organisms from soil sample


Genomic DNA isolation from subcultured microbes: The genomic DNA from isolated colonies were analysed by various protocols. The best method we optimized and used for isolation are follows.

DNA isolation by phenol choloroform method. DNA isolation by lysis buffer protocol. DNA isolation by rapid method. DNA isolation by helini ready template A & B method. DNA isolation by solution A,B,C method. Protocol 1: a. Take 1.5ml bacterial culture into sterilize appendab tube. b. Centrifuge at 10000 rpm.
c. Discard the supernatant and add 600 l of lysis buffer.

d. Dissolve the pellet by vortex.


e. Incubate at 650c for 15 minutes and allow it to cool at room

temperature.
f. Add 200 l of sodium acetate solution and mix the contents of the tube

by vortexing for 1 minute which precipitates the proteins. g. Centrifuge the tubes at 10000 rpm for 5 minutes. h. After centrifugation transfer the supernatent into the fresh tube.
i.

Add 600 l of isopropanol to the supernatant mix the solution well and incubate in -200c for 30 minutes and recover the precipitated DNA by centrifugating at 10000 rpm for 2 minutes.

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j. Discard the supernatant add 70% ethanol which precipitates remaining proteins. k. Mix the solution well to dissolve the pellet. l. Centrifuge the tubes at 10000 rpm for 1min. m. Discard the supernatant and air dry the pellet until the ethanol is completely evaporated.
n. Dissolve the pellet in 50 l of tris EDTA buffer which maintains the ph

and chelates the DNA from enzymatical degradation. Composition of lysis buffer: 1. Tris chloride 2. EDTA 3. SDS Composition of sodium acetate: 1. 5 molar of sodium acetate 2. Glacial acetic acid 3. Distilled water

Protocol 2:

a. Take 1.5ml of bacterial culture in sterilized appendab tube. b. Centrifuge at 10000 rpm for 10 minutes c. Discard the supernatant.
d. To the pellet add 320 l of solution-A (TRIS & EDTA) and 80ml of

solution-B (SDS) vortex the sample homogeneously.

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e. Incubate it at 650c for 15 minutes.

f. Cool the sample at room temperature for 5minutes.


g. Add 130 l of solution-C (sodium acetate) invert the sample for 4 or 5

times and incubate at room temperature for 5 minutes. h. Centrifuge at 10000 rpm for 10 minutes. i. Transfer the supernatant into a fresh tube. Now supernatant contains DNA & RNA. Add equal volumes of ice cold iso propanol. Gently invert and incubate it at room temperature for 5 minutes. j. Then centrifuge at 10000 rpm for 15 minutes. k. Discard the supernatant.
l.

To the pellet add 70% ethanol of 200 l without disturbing the pellet keep for 1 minute.

m. Centrifuge at 10000 rpm for 1 minute. n. Discard the supernatant and leave the pellet to an air dried the ethanol by inverting it on a paper for 10 minutes.
o. Suspend the pellet in 50 l of TE buffer and dissolve it.

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Isolation of lipase organisms from soil sample

Protocol 3: a. Take 1ml of bacterial culture in sterilized appendab tube. b. Centrifuged at 10000 rpm for 10 minutes. c. Discard the supernatant.
d. Dissolve the pellet in 100 l of ready template A.

e. Dissolve the pellet properly by vortex.


f. Add 100 l of ready template B.

g. Vortex the sample for 1 minute. h. Keep the solution 5 minutes for room temperature.
i.

Then incubate at 650c for 10 minutes.

j. Centrifuge at 10000 rpm for 5 minutes. k. Add equal volumes of P:C:I to the supernatant. l. Centrifuge at 10000 rpm for 5 minutes. m. Then transfer the aqueous layer into fresh tube. n. Then add equal volumes of C:I to the aqueous layer o. Then centrifuge at 10000 rpm for 5 minutes. p. Transfer the aqueous layer into fresh tube.
q. To that add 3 molar potassium acetate or 160 l ice cold iso propanol.

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r. Incubate at -200c for 5 minutes.

s. Centrifuge at 10000 rpm for 5 minutes. t. Discard the supernatant. u. Wash the pellet with 90% ethanol or 70% ethanol. v. Air dry the pellet w. Dissolve the pellet with TE buffer.

Protocol 4: a. Take 1ml of bacterial culture in sterilized appendab tube. b. Centrifuged at 10000 rpm for 10 minutes. c. Discard the supernatant.
d. Dissolve the pellet in 100 l of ready template A.

e. Dissolve the pellet properly by vortex.


f. Add 100 l of ready template B.

g. Vortex the sample for 1 minute. h. Keep the solution 5 minutes for room temperature.
i.

Then incubate at 650c for 10 minutes.

j. Centrifuge at 10000 rpm for 5 minutes. k. Take centrifugate for PCR.

Qualitative and Quantitative analysis of whole genomic DNA.

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Isolation of lipase organisms from soil sample


The isolated genomic DNA was subjected for spectrometric analysis methods. This analysis provides the information about the purity of whole genomic DNA. If the 260/280 ranges are above than 1.8 that indicates the good purity of DNA. If it is less than 1.8 we can suspect that the DNA was contaminated with proteins. Upto getting the values as 1.8, the sample has to be purified. After reaching the 1.8 as reading then we can go for molecular analysis.

PCR amplification of 16s rDNA gene: By general microbial base characterization we cant say the species and strain number. It is tie consuming and very laborious. 16s rDNA is unique for microbes. But by using the 16s rDNA gene we can say much information about the species and strain number also. Here we used to have idea about the Tm, Annealing temperature, forward primer sequence, reverse sequence etc.

Thermal profile as follows:

94

94c 72c 72c

2min 45 sec

45 sec 62c

3min

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Isolation of lipase organisms from soil sample

45 sec

Molecular weight based confirmation of amplified product: After successful amplification of 16s rDNA gene we can reveal the molecular weight and it will be confirmed with molecular weight marker.

Purification of amplified product: The amplified product should not be suitable for sequencing, because it have the chemicals like Mg+2, assay buffer, Taq DNA polymerase, dNTPs and other contaminants. So before subjecting this wehave to purify the DNA from all chemicals. In all the products Nal based purification is the best for removing the contaminants for effective sequence analysis.

Sequence of amplified product: The purified DNA was sent for sequencing by autoradiography.

NCBI BLAST analysis: The sequenced DNA was subjected to Bioinformatics Blast and this provides the information about the species and strain level information about our organism.

Phylogenetic tree construction: This provides the very close information abouhowfar organism relates with other organism available in the database.

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Isolation of lipase organisms from soil sample

RESULT

Agarose gel electrophoresis of 16S rDNA amplified Profiles of Isolates

For the molecular identification of isolated strains 16S-rDNA profiles were obtained with Taq I Polymerase. The product was then

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Isolation of lipase organisms from soil sample


electrophoresed in 2.5% agarose gel with along with a DNA size marker. (Figure)

Analysis of the Genotypic and Phenotypic Groups The biological significance, lipases hold tremendous potential for exploitation in biotechnology. They posses the unique feature of acting at the aqueous and non aqueous interface which distinguishes them from esterases (Verger, 1997; Schmidt and Verger, 1998). The concept of lipase interfacial activity evolved from restriction of their catalytic activity to interface between lipid and water. The catalytic activity of lipases depends largely on the aggregated state of substrates. Experimental evidences suggest that the activation involves unmasking and structuring of enzyme-active-site, through conformational changes, that require presence of oil-in water droplets. Recent studies on the structure of several lipases have provided some clues for understanding their hydrolytic activity, interfacial activation and stereo selectivity of lipases (Kazlauskas and Bornscheuer, 1998). Enzymes such as proteases and carbohydrases have been used industrially for a number of years and corner the largest share of the world wide enzyme market. 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. The lipases catalyze wide range of reactions, including hydrolysis, inter-esterification, alcholysis, acidolysis, esterification and aminolysis. They catalyse the hydrolysis of fatty acid ester bond in the

83

Isolation of lipase organisms from soil sample


triacylglycerol (TAG) and release free fatty acids (ffa) (Sheldon, 1993). Strains included are showed the same extracellular enzyme profiles (lipase, pectinase and amylase). Moreover their physiological properties were also similar. These strains were not able to grow at pH 3, and 5% or 6% NaCl concentrations. And also they could grow weakly at 4% NaCl concentration. Lotti and her group in the University of Milan, Italy have been studying various aspects of lipase production by Candida rugosa / cylindracea for about a decade. They (Lotti et al. 2001) undertook a flow cytometric study to evaluate growth-production process of Candida rugosa cells of different culture media. Neugnot et al. (2002) cloned and the over expressed the genecoding lipase from Candida parapsilosis CBS 604. Two ORFs (CpLIP1 and CpLIP2) wereisolated and the deduced 465-amino-acid protein sequences containedthe consensus motif (G-X-S-X-G) which is conserved among lipolyticenzymes.

Partial Sequence Analysis of 16S rRNA gene :

Sequencing results were obtained in a SEQ 44 personal sequencing system. The results were submitted to GenBank and the sequence was blasted to the NCBI Genbank. From the BLAST sequence Phylogenetic tree was constructed.

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Isolation of lipase organisms from soil sample

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CONCLUSIONS AND FUTURE PERSPECTIVE

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Isolation of lipase organisms from soil sample

In this study, soil contaminated with oils collected within different olive oil mills in Andhra Pradesh. The strains were screened for the presence of lipase extracellular enzyme activities. For enzyme screening SDS was used as substrate. In total 3 lipase (0.02% used as substrate), activities were detected. All of the isolates were Gram (+), endospore forming rods, thus they were identified as ..

Taq I was used for 16S- rDNA . By choosing the universal primers for 16S rRNA gene, partial sequence analysis was carried out. Sequencing results were submitted to GenBank and the sequence was blasted against the Genbank sequence and pylogenitic tree was constructed. Lipases produced by the isolated strains can be studied in respect of enzyme activity, purification and production. The genes coding for these enzymes can also be cloned to obtain recombinant thermoacidophilic enzymes.

LOTTI, Marina; BROCCA, Stefania and PORRO, D. High lipase production by Candida rugosa is associated with G1 cells. A flow cytometry study. Biotechnology Letters, 2001, vol. 23, no. 21, p. 1803-1808.

NEUGNOT, Virginie; MOULIN, Guy; DUBRENG, Eric and BIGEY, Frederic. The lipase /acyltransferase from Candida parapsilosis: Molecular cloning and characterization of purified recombinant enzymes. European Journal of Biochemistry, March 2002, vol. 269, no. 6, p. 1734-1745. VERGER, R. Interfacial activation of lipases: facts and artefacts. Trends in Biotechnology, January 1997, vol. 15, no. 1, p. 32-38.

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SHELDON, 0824791436. R.A. Chirotechnology-industrial synthesis of

optically-active compounds. Marcel Dekker Ltd., New York. 1993. 448 p. ISBN

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