Title of Invention

A PROCESS FOR THE PRODUCTION OF NOVEL LIPASE

Abstract

The present invention relates to a process for the production of a novel lipase. The lipase of the present invention is produced by using a new bacterial strain for industrial applications. This enzyme so obtained has higher stability over a wide range of pH of 2 to 10, temperature of 4 to 80°C. It has shelf life up to 90 days having potential use in leather industry as a degreasing agent for beam house processing,. It may also be used as an additive to laundry detergent formulations. The enzyme is a potential hydrolyzing agent for industrially important fats and oils while producing fatty acids. It may find use in esterificaiton, transesterification and interesterification reactions. Moreover, resolution of racemic mixtures is possible by using this enzyme. It has also a potential use in pharmaceutical industry as a digestive aid.

Full Text

The present invention relates to a process for the production of a novel lipase. The lipase of the present invention is produced by using a new bacterial strain for industrial applications. This enzyme so obtained has higher stability over a wide range of pH of 2 to 10, temperature of 4 to 80°C. It has shelf life upto 90 days having potential use in leather industry as a degreasing agent for beamhouse processing. It may also be used as an additive to laundry detergent formulations. The enzyme is a potential hydrolysing agent for industrially important fats and oils while producing fatty acids. It may find use in esterification, transesterification and interesterification reactions. Moreover, resolution of racemic mixtures is possible by using this enzyme. It has also a potential use in pharmaceutical industry as a digestive aid. Lipase is widely distributed in vertebrates, plants and microorganisms. The natural substrates for lipase, as stated by Tombs (Biotechnology in food industry, Edited by MP Tombs, Prentice Hall, New Jersey, pp 127-146, 1991), are triacylglycerols having very low solubility in water. Under natural conditions, lipase catalyses the hydrolysis of ester bonds at the interface between an insoluble substrate phase and the aqueous phase in which the enzyme is dissolved. As reported by Macrae and Hammond (Biotechnology Genetic Engineering Reviews, 3, 193-219, 1985), under certain experimental conditions such as in the absence of water, lipase is capable of reversing the reaction. The reverse reaction leads to esterification and formation of glycerides from fatty acids and glycerol. As reported by Mukherjee and Hills (Lipase, their Structure, Biochemistry and Application, Edited by P Wooley and SB Petersen, Cambridge University Press, Cambridge, pp 49-75, 1994) lipase can be obtained from plants and most of the efforts in this area have been devoted to seed lipases from castor bean (Ricimis comrmmis), Vernonia anthelmintic, wheat germ, oats and rice bran. The enzyme may be produced either by grinding the plants followed by enzyme extraction or by solvent extraction method. Different categories of animal lipases, viz. pancreatic, blood, stomach, hepatic, lingual, brain, muscle, placenta, butter, egg, fish and invertebrates have been studied by Barrowman and Darnton (Gastroenterology 59, 13-21, 1970), Hamosh (Lipases , Edited by B Borgstrom and HL Brockman, Elsevier Amsterdam, 49-81, 1984) and Carriere et al. (Federation of European Biochemists Society Letters, 338, 63-68, 1994).The enzyme is produced from the respective animal organs by grinding the animal source followed by enzyme extraction using solvent extraction method. Though lipases from different plants and animals have been studied extensively over the years for various end uses, the main limitation of getting lipase lies in the availability and collection of the respective animal or plant sources for commercial production. Moreover, the availability of the amount of enzyme in the above sources is limited. For example, 40 gms of delipidated powder of pancreas yields only lOOO units of lipase enzyme. Again, as reported by Brockerhoff and Jensen (Lipolytic enzymes, Academic press, New York, p- 36, 1974), since the enzyme available in the above sources is intracellular in nature, additional downstream processing is necessary to extract the enzyme. General biological significance for lipases (triacyl glycerol acyl hydrolase, Enzyme Code 3.1.1.3) has attained importance with the advent of biotechnology. As reported by Macrae and Hammond (Biotechnology Genetic Engineering Reviews, 3, 193-219, 1985); Iwai and Mieko (Yushi, 32, 84-91, 1989), microbial lipases have high stability and specificity in both aqueous and non-aqueous media. Apart from their biological significance, microbial lipases have multifarious applications in many areas viz. leather processing industries, detergent formulations, oleochemistry, organic synthesis, nutrition, pharmaceuticals, cosmetics, textile, food processing etc. According to Bjorkling et al. (Trends in Biotechnology, 9, 360-363, 1991), the study of lipases is very important for mankind as development of new lipase enzyme technologies will result in their expansion into new areas and will have tremendous impact on an array of industries As reported by Thirstrup et al (Biochemistry, 33, 2748-2756, 1994), it is now well established that microbial lipases are preferred to those from other sources for commercial applications due to their multifaceted applications, easy manipulation, optimal production strategies and easy down stream processing. The advent of recombinant DNA technology for the improvement of microorganisms is one of the major reasons for choosing microbes as a first choice. The major advantage of using microbes is that unlike plant and animal sources, which have to be grown every time for harvesting the particular source of enzyme for processing, thereby lengthening the production time, maintenance of one microbial strain ensures production of desired quantity of an enzyme by subculturing the strain in the production media as and when required. As reported by Rao and Lakshmanan (Indian Chemical Engineer, 33(4), 9-10, 1994), activity of microbial lipase can be as high as 27500 Units/ml of enzyme. As mentioned by Priest (Encyclopaedia of Microbiology, 2, 81-93, 1992), microorganisms are characterised by the fact that they can synthesize extracellular enzymes, whose activity is many times greater than the intracellular level of activity and most of these enzymes are inducible in nature.The similar opinion has been expressed by Okeke and Okoto (Biotechnology Letters, 72, 747-750, 1990), who have reported that microbial Upases are inducible in some cases by the inclusion of lipid substrates in the growth media. The effect of culture conditions and media components has been extensively studied by Cordenons et al. (Biotechnology Letters, 75, 633-638, 1996); Kambourova et al. (Folia Microbiologia, 41, 146-148, 1996) and Montesinos et al (Applied Biochemistry Biotechnology 59, 25-37, 1996). Mostly, addition of lipids in the culture broth is found to increase the exo-lipase production as studied by Khan et al (Biochimica Biophysica Acta, 732, 68-77, 1967). According to Rao and Lakshmanan (Indian Chemical Engineer, 33(4), 9-10, 1994), common nitrogen sources for lipase production are soya bean meal, peptone, yeast extract, casein hydrolysate and corn steep liquor. As reported by Brune and Gotz (Microbial degradation of natural products, edited by Winkelmann, VCH, Weinheim, pp 243-263, 1992) and Peterson and Drablos (Lipases, their structure, biochemistry and application edited by Wolley and Petersen Cambridge University Press, Cambridge, pp 23-48, 1994) a relatively smaller number of bacterial lipases have been studied as compared to plant and fungal Upases. Microbial lipases are most active within the temperature range of 30°C-40°C. The lipase of Talaromyces emersonii has been reported by Oso (Canadian Journal of Botany, 56, 1840-1843, 1978) to have an optimum temperature of 40°C . Fukumoto et al (Journal of General and Applied Microbiology, 9, 353-361, 1963) have reported a lipase from Aspergilhis niger to be stable at 50°C. Aisaka and Terada (Agricultural Biological Chemistry, 89, 817-822, 1981) have reported a lipase from Rhizopus japonicus with good stability at 50°C. Yamaguchi et al (Agricultural Biological Chemistry, 37, 999-1005, 1973) have also reported a lipase from Chromobacterium viscosum exhibiting stability at 50°C and Humicolo lanuginosa lipase has been observed by Liu et al (Agricultural Biological Chemistry, 37, 157-163, 1973) to be stable at 60°C. However, Watanabe et al (Agricultural Biological Chemistry, 41, 1353-1358, 1977) have reported that a lipase from the bacterium Pseitdomonas nitroreducens variety thermotolerance has been found to be stable even at 70°C. Soberon-Chavez and Palmeros (Critical Reviews of Microbiology,20, 95-105,1994) have reviewed the literature extensively and have observed that Pseitdomonas Upases display special biochemical characteristics like thermoresistance upto 70°C and pH stability in the range of 6.5-10, which are not common among the Upases produced by other microorganisms. According to the review report of Soberon-Chavez and Palmeros (Critical Reviews of Microbiology, 20, 95-105, 1994), Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas cepacia and Pseudomonas glumae are the prominent lipase producers among the Pseudomonas group and they have been extensively studied for their biochemical properties. No lipase has however been reported to be stable over pH range as wide as 2-10. There has also been no report of a lipase exhibiting wider thermal stability in the range of 4-80°C. Therefore, new isolates producing highly active and stable enzyme systems are always preferred. No prior art is available on the production of lipase from Ralstonia pickettii The main objective of the present invention is to provide a process for the production of a novel lipase, which obviates the drawbacks stated above. Another objective of the present invention is to use Ralstonia pickettii, deposited at Central Leather Research Institute (CLRI) and having accession no. CLRI BTL1 for producing lipase. The organism has inter alia the following characteristics and can be made available to the public as per normal official procedures. l.The microorganism of the present invention belongs to class Pseudomonadales. 2. It is slimy, white in appearance. 3.It appears pinkish with fluorescence when exposed to light in nutrient agar medium. 4.1t appears darker in Luria Berteni medium. 5.In potato dextrose agar medium, it appears slimy white with no fluorescence. 6.1t is a gram negative organism. 7. It is a short rod shaped organism. 8. Sporulation of the organism is negative. 9.It has got motility. 10. It produces catalase, oxidase. 11.It can utilise the carbohydrates like glucose,xylose, mannitol, maltose,trehalose, lactose,cellobiose and fructose. The organism has been isolated in the following way. Soil sample was collected from the rhizosphere of rice field and was diluted with sterile distilled water to 106 times. An aliquot of this was spread on a screening medium having the composition, as mentioned in Table I below. Table 1 (Table Removed) The plates with the screening medium were incubated at 37°C. After 96hrs. colonies were observed for zone of lipolysis. The most potent colony was isolated and was identified as Ralstonia picketti. Yet another objective of the present invention is to prepare a medium for Ralstonia pickettii CLRIBTL1 for maximum lipase production. Still another objective of the present invention is to provide a thermotolerant lipase from Ralstonia pickettii CLRI BTL1 having stability over temperature range of 4- 80°C. Yet another objective of the present invention is to provide an ecofriendly option for degreasing in leather manufacture. Still another objective of the present invention is to provide lipase which has wider stability in the pH range 2-10. Yet another objective of the present invention is to provide an enzyme having stability in the presence of different commercial laundry detergents. Still another objective of the present invention is to provide an eco-friendly additive for detergent formulations for the removal of olive oil from cotton fabric. Yet another objective of the present invention is to provide an option to reduce the consumption of energy for fat splitting by incorporating the use of lipase and running the process at around 30°C. Accordingly, the present invention provides a process for the production of a novel lipase which comprises i. Culturing the isolate of the strain of Ralstonia picketiii, having the characteristics as herein described, deposited in Central Leather Research Institute, Chennai and designated as CLRI BTL1, grown in a liquid nutrient medium over a period of at least 12hrs in a known manner, in a conventional production medium such as herein described under agitation for a period of at least 24hrs. ii. separating the supernatant as a source of lipase from the culture broth, produced in step (i), by known centrifugation method optionally followed by purifying the said lipase by conventional methods such as salt precipitation, chromatography. In an embodiment of the present invention, the pH of the nutrient medium may be in the range of 6-8. In another embodiment of the present invention, the agitation of medium may be carried out at maximum 200 rpm. In yet another embodiment of the present invention, the nutrient medium may be prepared by mixing natural infusions and sodium chloride in different combinations. In still another embodiment of the present invention, the natural infusions used may be such as beef extract, yeast extract, malt extract, soyabeen meal, peptone, tryptone, casamino acids in different combinations. In yet another embodiment of the present invention, the proportion of the natural infusion and sodium chloride may be in the range of 1:0.4 to 1:0.6 In still another embodiment of the present invention, salts used for preparing the production medium may be such as magnesium sulphate, calcium chloride, manganese sulphate, cobalt chloride, zinc sulphate, copper sulphate in different combinations. In yet another embodiment of the present invention, the secondary carbon source used for preparing the production medium may be such as vegetable oils, mineral oils. In still another embodiment of the present invention, the primary carbon source used for preparing the production medium may be such as glucose, starch, sucrose, galactose, lactose, fructose, xylose, mannitol. In yet another embodiment of the present invention, the nitrogen source used for preparing the production medium may be such as urea, ammonium sulphate, ammonium chloride, ammonium sodium hydrogen phosphate, diammonium hydrogen phosphate, ammonium di hydrogen phosphate. In still another embodiment of the present invention the pH of the production medium may be adjusted in the range of 5-9. In yet another embodiment of the present invention, the agitation of the production medium may be in the range of 25 to 200 rpm. In still another embodiment of the present invention, the centrifugation may be effected at 8000 to 12000 rpm for 10 to 20 minutes. In yet another embodiment of the present invention, the salt precipitation may be carried out preferably by ammonium sulphate method of 20% to 80% w/v. In still another embodiment of the present invention, the chromatographic method used may be preferably hydrophobic interaction chromatography. Any other similar conventional methods known to a person in the art, though not outlined, can be adopted. The isolate of Ralstonia pickettii is precultured for 12h-24h at 30°C-40°C in liquid nutrient medium, maintained at a pH in the range of 6-8. The preculture is agitated upto a maximum of 200 rpm. 0.5%v/v, of the precultured cells having a turbidity in the range of 0.4-0.8 O.D.( Optical Density) are inoculated into the production medium, maintained at a pH in the range of 5-9 at a temperature of 30°-50° C. The cells are allowed to grow over a period of 24-120 h under shaking in the range of 25 to 200rpm. Finally the cells are harvested by centrifugation and the culture filtrate is used as the crude lipase enzyme. The novelty and non-obviousness of the present invention lies in the use of a new strain of Ralstonia and formulating its production medium for producing lipase enzyme with wider stability to heat, pH and detergents with broad substrate specificity, thereby providing an eco-friendly option for industrial applications. The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention. EXAMPLE 1 One loop of the Ralstonia pickettii CLRI BTL1 strain was picked up from the nutrient agar slant and was precultured in 50 ml nutrient broth taken in 100 ml Erlenmeyer flask. The composition of the nutrient broth is given in the following table ( Table-2). Table 2 (Table Removed) Preculture was done at 35°C for 18 h in a rotary incubator shaker at 200 rpm. Then the production medium was prepared as per the following composition (Table-3) Table 3 (Table Removed) 0.5 ml of the precultre was inoculated in the production medium. Cultures were grown at 30°C ± 2°C and pH 6.5 on a rotary incubator shaker at 150 rpm. After 96 h incubation, the cells were centrifuged at 8000 rpm for 10 min at 4°C and the supernatant liquid containing the crude lipase was decanted. 2.5 ml of this crude enzyme solution was mixed with 600 mg of commercial laundry detergent 'ariel', dissolved in 50 ml of 0.05M, sodium phosphate buffer of pH 7, made upto 100 ml with distilled water and was used to remove oil stain on cotton fabric. EXAMPLE 2 One loop of the Ralstoniapickettii CLRI BTLl strain was picked up from the nutrient agar slant and was precultured in 50 ml nutrient broth taken in 100 ml Erlenmeyer flask. The composition of the nutrient broth is given in the following table ( Table-4). Table-4 (Table Removed) Preculture was done following the conditions stated in the above table. Then the production medium was prepared as per the following composition (Table-5) Table 5 (Table Removed) 0.5 ml of the preculture was inoculated in the production medium. Cultures were grown at 30°C ± 2°C, pH 6.0 on a rotary incubator shaker at 150 rpm. After 96 h incubation, the cells were centrifuged at 8000 rpm for 10 min at 4°C and the supernatant liquid containing the crude lipase is decanted. Enzymatic hydrolysis of oil was carried out by weighing Ig of coconut oil in a 100 ml conical flask. 15ml of phosphate buffer pH 6.5 was added followed by 75U of enzyme solution. The mixture was incubated for 72h at 37°C with constant agitation of 150 rpm. and was used to produce free fatty acids from coconut oil EXAMPLE 3 One loop of the Ralstonia pickettii CLRI BTLl strain was picked up from the nutrient agar slant and was precultured in 50 ml nutrient broth taken in 100 ml Erlenmeyer flask. The composition of the nutrient broth is given in the following table (Table-6). Table 6 (Table Removed) Preculture was done following the conditions stated in the above table. Then the production medium was prepared as per the following composition (Table-7) Table 7 (Table Removed) 0.5 ml of the precultre was inoculated in the production medium. Cultures were grown at 30°C ± 2°C, pH 5.5 on a rotary incubator shaker at 150 rpm. After 96 h incubation, the cells were centrifuged at 8000 rpm for 10 min at 4°C and the supernatant liquid containing the crude lipase was decanted. Degreasing of pickled greasy woolly sheep skins was done using the above enzyme at a concentration of 250 Units/ml/min at pH 8.0 and at 32°C ± 2°C. Degreasing was of the order of 70.5% when 250 Units of lipase was used. EXAMPLE 4 One loop of the Ralstoniapickettii CLRI BTLl strain was picked up from the nutrient agar slant and was precultured in 50 ml nutrient broth taken in 100 ml Erlenmeyer flask. The composition of the nutrient broth is given in the following table (Table-8). Table 8 (Table Removed) Preculture was done following the conditions stated in the above table. Then the production medium was prepared as per the following composition (Table-9) Table 9 (Table Removed) 0.5 ml of the precultre was inoculated in the production medium. Cultures were grown at 34°C ± 2°C, pH 7.0 on a rotary incubator shaker at 150 rpm. After 120 h incubation, the cells were centrifuged at 8000 rpm for 10 min at 4°C and the supernatant liquid containing the crude lipase was decanted. 2.5 ml of this crude enzyme solution was mixed with 600 mg of commercial laundry detergent 'ariel', dissolved in 50 ml of 0.05M, sodium phosphate buffer of pH 7, made upto 100 ml with distilled water and was used to remove oil stain on cotton fabric. EXAMPLE -5 One loop of the Ralstonia pickettii CLRJ BTLl strain was picked up from the nutrient agar slant and was precultured in 50 ml nutrient broth taken in 100 ml Erlenmeyer flask. The composition of the nutrient broth is given in the following table ( Table-10). Table 10 (Table Removed) Preculture was done following the conditions stated in the above table. Then the production medium was prepared as per the following composition (Table-11) Table 11 (Table Removed) 0.5 ml of the precultre was inoculated in the production medium. Cultures were grown at 37°C ± 2°C, pH 7.5 on a rotary incubator shaker at 125 rpm. After 72 h incubation, the cells were centrifuged at 8000 rpm for 10 min at 4°C and the supernatant liquid containing the crude lipase was decanted. This crude lipase was purified by ammonium sulphate precipitation method at 40 % w/v saturation. EXAMPLE 6 One loop of the Ralstonia pickettii CLRIBTL1 strain picked up from the nutrient agar slant and was precultured in 50 ml nutrient broth taken in 100 ml Erlenmeyer flask. The composition of the nutrient broth is given in the following table ( Table-12). Table 12 (Table Removed) Preculture was done following the conditions stated in the above table. Then the production medium was prepared as per the following composition (Table-13) Table 13 (Table Removed) 0.5 ml of the precultre was inoculated in the production medium. Cultures were grown at 50°C ± 2°C, pH 8.0 on a rotary incubator shaker at 150 rpm. After 48 h incubation, the cells were centrifuged at 8000 rpm for 10 min at 4°C and the supernatant liquid containing the crude lipase is decanted. Purification of lipase was done by hydrophobic interaction chromatography using octyl Sepharose CL-4b columns. Binding of the enzyme was done with 2% ammonium sulphate in 0.05M, sodium phosphate buffer of pH 6.5 and elution was carried out using 20% isopropanol in 0.05M, sodium phosphate buffer of pH 6.5. EXAMPLE 7 One loop of the Ralstonia pickettii CLRIBTL1 strain picked up from the nutrient agar slant and was precultured in 50 ml nutrient broth taken in 100 ml Erlenmeyer flask. The composition of the nutrient broth is given in the following table ( Table-14). Table 14 (Table Removed) Preculture was done following the conditions stated in the above table. Then the production medium was prepared as per the following composition (Table-15) Table 15 (Table Removed) 0.5 ml of the precultre was inoculated in the production medium. Cultures were grown at 45°C ± 2°C, pH 8.5 on a rotary incubator shaker at 175 rpm. After 24 h incubation, the cells were centrifuged at 8000 rpm for 10 min at 4°C and the rsupernatant liquid containing the crude lipase is decanted. Purification was done by ammonium sulphate precipitation at 40 % saturation w/v, followed by hydrophobic interaction chromatography using octyl Sepharose CL-4b columns. Binding of the enzyme was done with 2% ammonium sulphate in 0.05M, sodium phosphate buffer of pH 6.5 and elution was carried out using 20% isopropanol in 0.05M, sodium phosphate buffer of pH 6.5. EXAMPLE 8 One loop of the Ralsionia pickettii CLRIBTL1 strain picked up from the nutrient agar slant and was precultured in 50 ml nutrient broth taken in 100 ml Erlenmeyer flask. The composition of the nutrient broth is given in the following table (Table-16). Table 16 (Table Removed) Preculture was done following the conditions stated in the above table. Then the production medium was prepared as per the following composition (Table-17) Table 17 (Table Removed) 0.5 ml of the precultre was inoculated in the production medium. Cultures were grown at 34°C ± 2°C, pH 9.0 on a rotary incubator shaker at 150 rpm. After 72 h incubation, the cells were centrifuged at 8000 rpm for 10 min at 4°C and the supernatant liquid containing the crude lipase is decanted. Degreasing of pickled greasy woolly sheep skins was done using the above enzyme at a concentration of 250 Units/ml/min at pH 8.0 and at 32°C ± 2°C. Degreasing was of the order of 70.5% when 250 Units of lipase was used. EXAMPLE 9 One loop of the Ralstoniapicketlii CLRI BTLl strain picked up from the nutrient agar slant and was precultured in 50 ml nutrient broth taken in 100 nil Erlenmeyer flask. The composition of the nutrient broth is given in the following table ( Table-18). Table 18 (Table Removed) Preculture was done following the conditions stated in the above table. Then the production medium was prepared as per the following composition (Table-19) Table 19 (Table Removed) 0.5 ml of the precultre was inoculated in the production medium. Cultures were grown at 30°C ± 2°C, pH 6.5 on a rotary incubator shaker at 150 rpm. After 96 h incubation, the cells were centrifuged at 8000 rpm for 10 min at 4°C and the supernatant liquid containing the crude lipase is decanted. Enzymatic hydrolysis of oil was carried out by weighing Ig of coconut oil in a 100 nil conical flask. 15ml of phosphate buffer pH 6.5 was added followed by 75U of enzyme solution. The mixture was incubated for 72h at 37°C with constant agitation of 150 rpm. and was used to produce free fatty acids from coconut oil. EXAMPLE 10 One loop of the Ralstoniapickettii CLRI BTLl strain picked up from the nutrient agar slant and was precultured in 50 ml nutrient broth taken in 100 ml Erlenmeyer flask. The composition of the nutrient broth is given in the following table ( Table-20). Table 20 (Table Removed) Preculture was done following the conditions stated in the above table. Then the production medium was prepared as per the following composition (Table-21). Table 21 (Table Removed) 0.5 ml of the precultre was inoculated in the production medium. Cultures were grown at 30°C ± 2°C, pH 6.5 on a rotary incubator shaker at 150 rpm. After 96 h incubation, the cells were centrifuged at 8000 rpm for 10 min at 4°C and the supernatant liquid containing the crude lipase is decanted. Degreasing of pickled greasy woolly sheep skins was done using the above enzyme at a concentration of 250 Units/ml/min at pH 8.0 and at 32°C ± 2°C. Degreasing was of the order of 70.5% when 250 Units of lipase was used. The main advantages of the present invention are the following: i) Unlike the conventional lipase, the enzyme of the present invention has wider stability in the pH range 2 to 10 and temperature in the range of 4°C to 80°C. ii) This enzyme has been found to be stable in the presence of different commercial detergents like aerial, surf-ultra at a concentration of 7mg/ml. iii) The lipase of the present invention has been found to be able to remove olive oil from cotton fabrics with better efficiency in the presence of available detergents. iv) The enzyme of the present invention can be used for degreasing greasy pelt either individually or as an additive to an available commercial detergent based degreasing agent. It provides degreasing of about 70% at a concentration of 250Units per 30g of the pickled skin, v) Ralstonia pickettii CLRI BTL1 lipase is observed to be effective in hydrolysis of industrially important oils for fatty acid production at ambient temperatures with very little product charring or discolouration, vi) The cost of production is low as a simple salt medium is used, vii) Ralstonia picksttii CLRI BTL1 lipase is effective in the removal of greasy soil when used as an additive in laundry formulations. We claim 1. A process for the production of a novel lipase which comprises 1. Culturing the isolate of the strain of Ralstonia picketlii, having the characteristics as herein described, deposited in Central Leather Research Institute, Chennai and designated as CLRI BTL1, grown in a liquid nutrient medium over a period of at least 12hrs in a known manner, in a conventional production medium such as herein described under agitation for a period of at least 24hrs, ii. separating the supernatant as a source of lipase from the culture broth, produced in step (i), by known centrifugation method optionally followed by purifying the said lipase by conventional methods such as salt precipitation, chromatography. 2. A process, as claimed in claim 1, wherein the pH of the nutrient medium is in the range of 6-8. 3. A process, as claimed in claims 1& 2, wherein the agitation of medium is carried out at maximum 200 rpm. 4. A process, as claimed in claims 1 to 3, wherein the nutrient medium is prepared by mixing natural infusions and sodium chloride in different combinations. 5. A process, as claimed in claims 1 to 4, wherein the natural infusions used are such as beef extract, yeast extract, malt extract, soyabeen meal, peptone, tryptone, casamino acids in different combinations. 6. A process, as claimed in claims 1 to 5, wherein the proportion of the natural infusion and sodium chloride is in the range of 1:0.4 to 1:0.6 7. A process, as claimed in claims 1 to 6, wherein salts used for preparing the production medium are such as magnesium sulphate, calcium chloride, manganese sulphate, cobalt chloride, zinc sulphate, copper sulphate in different combinations. 8. A process, as claimed in claims 1 to 7, wherein the secondary carbon source used for preparing the production medium are such as vegetable oils, mineral oils. 9. A process, as claimed in claims 1 to 8, wherein the primary carbon source used for preparing the production medium is such as glucose, starch, sucrose, galactose, lactose, fructose, xylose, mannitol. 10. A process, as claimed in claims 1 to 9, wherein the nitrogen source used for preparing the production medium is such as urea, ammonium sulphate, ammonium chloride, ammonium sodium hydrogen phosphate, diammonium hydrogen phosphate, ammonium di hydrogen phosphate. 11. A process, as claimed in claims 1 to 10, wherein the pH of the production medium is adjusted in the range of 5-9. 12. A process, as claimed in claims 1 to 11, wherein the agitation of the production medium is in the range of 25 to 200 rpm. 13. A process, as claimed in claims 1 to 12, wherein the centrifugation is effected at 8000 to 12000 rpm for 10 to 20 minutes. 14. A process, as claimed in claims 1 to 13 , wherein the salt precipitation is carried out preferably by ammonium sulphate method of 20% to 80% w/v. 15. A process, as claimed in claims 1 to 14, wherein the chromatographic method used is preferably hydrophobic interaction chromatography. 16. A process for the production of a novel lipase as explained with reference to the examples.

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