Title of Invention	AN IMPROVED PROCESS FOR THE PRODUCTION OF STABLE LIPASE
Abstract	The invention relates to a process for production of stable lipase in which a novel solid substrate medium containing wheat bran and gingelly cake oil in the ratio ranges between 1:0.11 to 1:9.0, preferably in the range of 1:0.14 to 1:3.0 is used for growing a strain of Aspergillus niger, deposited at CLRI and having accession no. CLRI BTFL by which more active and stable lipase is extracted.

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The present invention relates to an improved process for the production of stable lipase. The lipase of the present invention exhibits wide thermal stabilty between 4°-60°C and it is also stable in the wide pH range of 2.0-11.0. More particularly, it is produced from a fungal source using solid state fermentation technique. It has wide ranging industrial applications. The enzyme of the present invention may be used as a degreasing agent in leather tanning industry. It has enormous potential applications as an additive to laundry detergent formulations in detergent industry. It may also be used for the production of industrially important fatty acids from fats and oils in oleochemical industry. Chemical industry can use this enzyme for synthesis of esters and acylation of glucose. It has as well an important application in food and pharmaceutical industries for interesterification of butterfat, resolution of racemic mixtures and synthesis of peptides. It may even be used in the generation of dairy flavors for the dairy industry. The major advantage of using lipase is that unlike conventional enzymes, it does not require any cofactor to catalyse any reaction. Lipases, chemically known as triacylglycerol acylhydroiases, belong to the class of serine hydrolases. 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. As reported by Macrae and Hammond (Biotechnology Genetic Engineering Reviews, 3, 193-219, 1985), they are capable of reversing the reaction in the absence of water. The reverse reaction leads to esterification and formation of glycerides from fatty acid and glycerol. 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 acids as reported by Iwai and Meiko (Yushi, 32, 84-91, 1989) and Bjorkling at al (Trends in Biotechnology, 9, 360-363, 1991). Lipases occur in animals, plants and microorganisms. Based on their occurrence, they are classified as plant lipases, animal lipases and microbial iipases. As reported by Brockerhoff and Jensen (Lipolytic enzymes, Academic Press, New York, pp. 35, 1974), pancreatic lipases are generally purified by adsorption to calcium phosphate and aluminium hydroxide followed by zone electrophoresis to yield pure enzyme, although this method yields only a milligram of enzyme per run. Plant lipases are usually obtained from castor bean, wheat germ, oats and rice bran. As reported by Brockerhoff and Jensen (Lipolytic enzymes, Academic Press, New York, pp. 130-139, 1974), the dehulled beans are ground and centrifuged to remove the precipitate debris, followed by chromatography to yield the pure enzyme. Moreoever, sufficient care has to be taken to avoid contact with potent allergenic proteins and dry seed powders during the early stage of purification. Thus, to summarise, the following major constraints are involved in the production of plant or animal lipase on commercial scale: i) Individual plant/animal source would not contain abundant lipase to make the extraction commercially viable, thereby requiring the availability of a large number of these sources on regular basis, although the supply of the said sources, which are essentially biological in nature, is inelastic, ii) Tedious extraction procedures are involved in the purification of plant and animal lipases. The above limitations associated with the plant and animal sources have prompted the researchers to explore the microbial source for the production of lipase enzyme for commercial purpose. Most microbial Upases are extracellular in nature as reported by Lotrakul and Dharmsthiti (World Journal of Microbiology and Biotechnology, 13, 163 - 166, 1997) and this property rules out the possibility of laborious extraction procedures and unnecessary precautions in handling operations. Moreover, maintaining a single microbial strain ensures generation of any desired amount of enzymes simply by subculturing the strain as and when required, thereby assuring an abundant supply of the enzyme. As reported by Macrae and Hammond (Biotechnology Genetic Engineering Reviews, 3, 193-219, 1985), microbial Upases are preferred for commercial applications due to their multifold properties, easy extraction procedures, unlimited supply and increased stablility compared to plant and animal Upases. The microbial lipase may again be either from bacterial or fungal origin. As reported by Sidhu et al (Indian Journal of Microbiology, 38, 9-14, 1998), bacterial Upases exhibit thermoresistance of upto 55° C and pH stability in the range of 7.0-9.0 respectively. Fungal Upases have in general been found to be stable over a wider range of pH 4.0-10.5 and temperature upto 70°C, as reported by Rapp (Enzyme and Microbial Technology, 17, 832-838, 1995). Moreover, unlike bacterial Upases, most of the fungal Upases such as those of Aspergillus niger, Rliizopus delemar, Mucor miehei exhibit 1,3-regiospecificity, making them suitable for various interesterification and transesterification reactions. Molds are thus considered to be the better microbial source for lipase production on commercial basis. The general process for producing microbial lipase may be either submerged fermentation technique (SmF) or solid state fermentation (SSF) technique. As reported by Solis-Pereira et al (Applied Microbiology and Biotechnology, 39, 36-41, 1993), submerged fermentation (SmF) systems have been extensively used for the production of industrially important enzymes like lipases, proteases, tannases, amylases, cellulases and pectinases. SmF essentially involves culturing of a strain by inoculating the strain in a liquid medium under dynamic aerated condition, followed by the separation of the en2yme produced. Production of lipase using submerged fermentation system necessitates the separation of the resulting biomass from the culture broth followed by concentrating the resulting liquid, thereby increasing the cost of production. Another major limitation of the SmF system is the requirement of more investment in terms of cost intensive chemicals and also the laborious methods required for the complete sterilization of the constituents forming the production medium, which may otherwise eventually lead to precipitation problem hindering the growth of the organism. On the other hand, the SSF system uses solid substrates as the medium for the gro\vth of a microorganism, which produces the enzyme and the whole substrate is used as the source of the enzyme, thereby avoiding sterilisation and subsequent downstream processing, eventually ensuring that the cost of production does not rise. As reported by Lonsane et al (Enzyme and Microbial Technology, 7, 258-265, 1985) and Lonsane and Ramesh (Advances in Applied Microbiology, 75, 1-48, 1992), SSF possesses many economical advantages, including superior volumetric productivity, use of simpler machinery and an inexpensive substrate, simpler downstream processing and lower energy requirements. In addition, there is low waste water output and consequently, there are fewer problems with waste water treatment than are experienced with submerged fermentation. Furthermore, in SSF systems, the metabolites obtained are more concentrated and purification procedures are less expensive. Moreover, as reported by Ramesh and Lonsane (Applied Microbiology and Biotechnology, 35, 591-593, 1991) and Solis-Pereira et al (Applied Microbiology and Biotechnology, 39, 36-41, 1993), SSF systems are capable of minimizing catabolite repression which is one of the major drawbacks of SmF systems. Work on fungal lipases started as early as 1950's. As reported by Godtfredsen (Microbial enzymes and Biotechnology, edited by Fogarty and Kelly, Elsevier, Amsterdam, pp. 255-273, 1990), and according to a comprehensive review presented by Brokerhoff and Jensen (Lipolytic enzymes, Academic Press, New York, pp. 25-175, 1974), fungal sources like Aspergillus niger, Candida cylindracea, Humicola lanuginosa, Mucor miehei, Rhizopus delemar, Rhizopus japonicus, Rhizopus niveus and Rltizopus oryzae are the chief producers of commercial lipases and the molds belonging to the genus Aspergillus are the most prominent among them, as reported by Lazar and Schroder (Microbial degradation of natural products, edited by Winkelmann, VCH, Weinhein, pp. 267-291, 1992). Pal et al ( Journal of Fermentation Technology, 56, 593-598), Raman (Ph.D Thesis, University of Delhi. 1994) and Ohnishi et al (Journal of Fermentation and Bioengineering, 77, 490-495, 1994) have reported production of lipase from several species of Aspergilli such as Aspergillus niger, Aspergillus terreus and Aspergillus oryzae using SmF system with lipase activity as low as 0.46-5.0 U/ml. This limitation of low lipase activity, which eventually renders the fermentation process commercially non-viable, has been overcome by Kamini et al (Indian Journal of Microbiology, 37, 85-89, 1997), who have isolated a new strain of Aspergillus niger to produce lipase using submerged fermentation technique, in which an isolated strain ofAspergillus niger has been cultured in a medium the composition of which isgiven hi Table 1. Table 1 (Table Removed) The resulting lipase, which has been found to have an activity of 35.4 U/ml in 48h of fermentation, is stable at a pH in the range of 7.0- 9.0 and temperature in the range of 4°- 45°C. As stated by Hesseltine (Biotechnology and Bioengineering, 14, 517-532, 1972), Solid State Fermentation (SSF) with fungal strains result in much greater productivity than submerged fermentation. Moreover, liquid media are far from the natural environment of fungi and they decrease the importance of specific phenomena such as osmotic resistance or cytoplasmic transfer. A number of microorganisms are capable of growing on solid substrates such as oil cakes, oil seeds, wheat bran, rice bran, coffee husk, spent barley, sugarcane bagasse etc., which can be used either individually or in different combinations for the production of fungal enzymes. The fungus grows rapidly on moist solid substrates. Earlier, strains of Aspergillus niger have been cultured on solid substrates such as sugarcane bagasse, cassava meal, wheat bran and corn corbs, computer cards and saw dust as reported by Oriol et al (Journal of Fermentation Technology, 66, 57-62, 1988), Raimbault and Alazard (European Journal of Applied Microbiology and Biotechnology, 9, 199-210, 1980), Fukumoto et al (Journal of General and Applied Microbiology, 9, 353- 361, 1963) and Madamwar et al (Journal of Fermentation Bioengineering, 67, 424-426, 1989) respectively. Mixtures of substrates such as wheat bran and sugarbeet head have been used as substrates for the production of pectinase, hemicellulase and amylase from Aspergillus a\\>amori, as reported by Mushikova et al (Microbial Synthesis, /, 25-28, 1978), while mixtures of saw dust with rice bran and powdered rice straw with wheat germ have been used for the production of cellulase from Trichoderma viride as reported by Toyama (Biotechnology and Bioengineering Symposium, No.6, 207-219, 1976). The following table shows the activities of different types of Upases produced by Aspergillus niger, Penicillium candidum, Mucor javanicus, Candida rugosa, Candida sp., Aspergillus oryzae and Rhizopus delemar, as reported by Fukumoto et al (Journal of General and Applied Microbiology, 9, 353-361, 1963), Rivera-Munoz et al (Biotechnology Letters, 13, 271-280, 1991), Saiki et a! (Agricultural Biological Chemistry, 32, 1458-1463, 1968), Venkata Rao et al (Process Biochemistry, 28, 385-389, 1993), Bharat Bhushan et al (Biotechnology Letters, 16, 841 - 842, 1994) , Ohnishi et al (Journal of Fermentation and Bioengineering, 77, 490-495, 1994) and Christen et al (Biotechnology Techniques, 9 , 597-600, 1995) respectively using SSF system. Table 2 (Table Removed) WS -Wet substrate; DS^-Dry substrate; IDM -Initial dry matter The above table (Table 2) also indicates that the selection of the solid substrate plays a very important role in the SSF system for producing enzymes exhibiting wide ranging activities. As reported by Umesh Kumar (Master of Engineering Thesis submitted to the Birla Institute of Technology, Pilani, 1995), lipase produced by a fungal strain of Aspergillus niger using wheat bran alone in the SSF system exhibited pH stability in the range of 6.0-8.0 and thermal stability in the range of 4°-40°C, while that produced by the same" system using gingelly oil cake alone as substrate exhibited pH stability in the range of 4.0-10.0 and temperature stability in the range of 4°-50°C as reported by Kamini et al (Process Biochemistry, 33, 505-511, 1998). Moreover, the Km values of the enzymes produced by wheat bran and gingelly oil cake alone have been found to be as high as 6.89xlO"2 and 4.5 5xlO"2 respectively implying that the reactivity of the resulting enzymes are low. Rao et al (Process Biochemistry, 28, 385-389, 1993) could increase the activity of the resulting lipase in rice bran based SSF system by adding urea and maltose as nitrogen and carbon sources respectively, but these additions not only increased cost of production, but also the possibility for bacterial contamination necessitating for control mechanisms to arrest the contamination, which would otherwise have reduced the efficiency of the system. As reported by Bhushan et al (Biotechnology letters, 16, 841-842, 1994), in SSF system, the duration of fermentation for lipase production has been found to be as high as 96-144h. No prior art is available on the production of lipase using a combination of the substrates The main objective of the present invention is to provide an improved process for the production of lipase for industrial applications, which obviates the drawbacks such as, (i) low yield (1.0-7.0 U/g wet substrate; 303.0 U/g dry substrate) (ii) narrow pH tolerance (4.0.-9.0) (iii) narrow temperature stability (upto 45°C) These have been removed by adopting the following measures: (a) by the use of a combination of substrates (b) by mutating the organism suitably (c) this has resulted in increased yield (d) with high pH tolerance (2.O.-11.0) (e) with high thermal stability (4°-60°C) Another objective of the present invention is to use Aspergillus niger, deposited at Central Leather Research Institute (CLRI) and having accession no. CLRI BTFL for producing lipase. The organism has inter alia the following characteristics and can be made available to the public as per normal official procedures: 1. This organism belongs to the class Ascomycetes and family Eitroliaceae. 2. The colonies growing on Czapek-Dox agar at 25°C attain a diameter of 4- 5cm within 7 days. 3. The mycelium is well developed, profusely branched, septate and hyaline. 4. The hyphal cells are multinucleate. 5. The colonies grow fast bearing spherical black conidia. 6. The sporulation is dense and the conidial heads radiate tending to split into loose columns with age. 7. The Aspergillns niger has been mutated by growing over a specifically recombination of substrates to enable to get a lipase having stability over a wide pH range 2.0-11.0 and temperature of 4°-60°C making it useful and adaptable for industrial purposes. Furthermore, it is capable of producing lipase upto 370.0 U/g dry substrate when compared to the previously reported activities such as 1.0-7.0 U/g wet substrate and 303.0 U/g dry substrate. The organism has been isolated in the following way: In order to isolate a potent strain which produces lipase, a sample of curd was enriched with sunflower oil and incubated for 3-4 days. This sample was then serially diluted and dilutions of 106, 107 and 10s were plated on Czapek-Dox agar plates supplemented with tributyrin, the composition of which is given in Table 3. Table 3 (Table Removed) The plates were incubated at 30°C for 3-4 days. The colony of the fungal strain used in this study produced the maximum halozone among other strains, measuring about 9mm in diameter which indicates extracellular lipase activity. To confirm the lipolytic activity of this strain, the culture which was maintained on Czapek-Dox agar slants, was then inoculated in Czapek-Dox liquid medium supplemented with olive oil, ammonium sulphate, potassium dihydrogen orthophosphate, disodium hydrogen orthophosphate, zinc sulphate and manganese sulphate, the percentage commposition of which is given in Table 4. Table 4 (Table Removed) The culture was grown for a maximum of 72h. At 24h intervals, the samples were filtered and the filtrate was assayed for lipase activity. The enzyme activity was found to be 8.0 U/ml at 72h of fermentation. This culture identified as AspergiUus niger was then used for subsequent studies on lipase production. Yet another objective of the present invention is to provide an improved process to produce lipase within a short time using a combination of solid substrates. Still another objective of the present invention is to provide lipase enzyme having pH stability in the range of 2.0-11.0. Yet another objective of the present invention is to provide lipase enzyme exhibiting thermal stability in the range of 4°-60°C. Still another objective of the present invention is to provide lipase enzyme having broad stability in the presence of different commercial laundry detergents. Yet another objective of the present invention is to provide lipase enzyme having remarkable stability in the presence of organic solvents. Still another objective of the present invention is to provide lipase enzyme as an additive in detergent formulations for ecofriendly applications. Yet another objective of the present invention is to provide lipase enzyme as a biocatalyst in the hydrolysis of oils and fats thereby minimising thermal energy consumption and resulting in the production of sufficiently pure reaction products. Accordingly the present invention provides an improved process for the production of stable lipase, wherein the said process comprising the steps of: a) growing the spores of the strain of Aspergillus niger on Czapek - Dox agar medium by known method; b) inoculating the spore suspension obtained from step (a) in pre sterilized solid substrate medium containing wheat bran and gingelly oil cake in a ratio ranging from 1:0.11-1:9.0 to obtain a moldy solid mass. c) extracting the lipase enzyme from the moldy solid mass obtained from step (b) by known method; d) separating the lipase enzyme from enzyme extract obtained from step (c). In an embodiment of the present invention, the propotion of wheat bran and gingelly oil cake may be preferably in the range of 1:0.14 - 1:3.0. In another embodiment of the present invenrtion, the moisture content of the solid substrates may be in the range of 30-85%. In yet another embodiment of the present invention, the concentration of spore suspension is in the range of 104-1010 spores/g of substrate. In still another embodiment of the present invention, the depth of the solid substrate medium may be in the range of 3-30mm. In yet another embodiment of the present invention, the inoculation temperature of the solid substrate medium is in the range of 20-45 degree C. In still another embodiment of the present inevtion, the extraction of thr moldy mass may be effected by such as grinding, simple percolation, counter current extraction, plug flow extraction. In yet another embodiment of the present invention, the separation may be carried out by such as centrifugation, filtration, gravity separation. A solid substrate medium is prepared by mixing wheat bran and gingelly oil cake at a ratio in the range of 1:0.11 to 1:9.0, preferably ranging between 1:0.14 to 1:3.0 and the resulting mixture is moistened with water, while maintaining the moisture content of the substrate in the range of 30-85%. This medium is then sterilised by conventional method. The isolate of the strain of Aspergillus niger CLRI BTFL is grown on Czapek-Dox agar slants for at least 72h at a temperature in the range of 28-37°C by known method. The spore suspension, prepared thereby, is then inoculated in the sterilized solid substrate medium, while maintaining the concentration of the inoculum in the range of 104 to 1010 spores per gm of the substrate. The depth of the inoculated medium is adjusted in the range of 3-30 mm and then incubated at a temperature in the range of 20° - 45°C under static conditions for a period in the range of 12h-150h. The resulting moldy mass, formed thereby, is subjected to enzyme extraction by conventional method and the extracted enzyme is finally separated by known method to obtain lipase enzyme. The novelty and non-obviousness of the present invention lies not only in the selection of the constituents of the substrates to form the solid substrate medium, but also in providing their proportions, thereby providing, compared to the hitherto known processes, an improved, cost-effective solid state fermentation process for producing lipase enzyme with, unlike the conventional available enzymes, wider stability to pH, temperatures, detergents and solvents for industrial applications , by using a fungal strain in an environmentally friendly way. 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 9.0g of wheat bran and l.Og of gingelly oil cake were taken in a 250ml Erlenmeyer flask and then distilled water was added to it with continuous shaking to ensure that the initial moisture content of the substrate mixture was 67%, M'/V. The flask was then autoclaved at 151bs/in2 at 121°C and after a period of 20 minutes, it was allowed to cool down to a temperature of 30°C. The spores of the isolate of the strain of Aspergillus m'ger, designated as CLRI BTFL, were streaked on a Czapek-Dox agar slant with the help of an inoculating needle and the slant was incubated at 28°C to facilitate the growth of the organism. After a period of 72h, 2ml of sterile distilled water was added to the slant and the mycelia were separated using a sterile glass rod. The spore concentration of the resulting spore suspension was found to be 4.3x 1010 spores/ml of the suspension. 1.0ml of the above spore suspension was inoculated into the solid substrate medium using a pipetteman and the resulting inoculated substrate was mixed well using a sterile glass rod. The depth of the substrate mixture in the flask was found to be lOmm.The flask was then incubated at 45°C under static condition. After 12h of incubation, Ig of the resulting mouldy substrate was taken in a mortar and 10ml of distilled water was added to it. This suspension was then subjected to grinding with the help of a pestle to obtain the lipase enzyme extract, which was centrifuged at 10,000rpm for 5min. at 30°C to get the lipase enzyme. This lipase was assayed for enzyme activity by the titrimetric method and the activity was found to be 32.6 U/g jiry substrate. 50U of this crude enzyme was used to remove olive oil stain efficiently from cotton fabric at a temperature of 30°C with a washing time of 20 minutes in the presence of 0.1%, w/v of the commercial detergent, Surf Excel. EXAMPLE 2 l.Og of wheat bran and 9.0g of gingelly oil cake were taken in a 1000ml Erlenmeyer flask and then distilled water was added to it with continuous shaking to ensure that the initial moisture content of the substrate mixture was 75%, ti'/v. The flask was then autoclaved at 151bs/in2 at 121°C and after a period of 20 minutes, it was allowed to cool down to a temperature of 30°C. The spores of the isolate of the strain of Aspergillus niger, designated as CLRI BTFL, were streaked on a Czapek-Dox agar slant with the help of an inoculating needle and the slant was incubated at 32°C to facilitate the growth of the organism. After a period of 96h, 2ml of sterile distilled water was added to the slant and the mycelia were separated using a sterile glass rod. The spore concentration of the resulting spore suspension was found to be 3.23x 109 spores/ml of the suspension. 0.75ml of the above spore suspension was inoculated into the solid substrate medium using a pipetteman and the resulting inoculated substrate was mixed well using a sterile glass rod. The depth of the substrate mixture in the flask was found to be 3mm.The flask was then incubated at 37°C under static condition. After 150h of incubation, Ig of the resulting mouldy substrate was taken in a mortar and 10ml of distilled water was added to it. This suspension was then subjected to grinding with the help of a pestle to obtain the lipase enzyme extract, which was centrifuged at 10,000rpm for 5min. at 30°C to get the lipase enzyme. This lipase was assayed for enzyme activity by the titrimetric method and the activity was found to be 8.4 U/g dry substrate. This lipase enzyme was used for the enzymatic hydrolysis by incubating 5rnl of this enzyme of effective activity of SOU, Ig of coconut oil and 15ml of O.IM phosphate buffer of pH 7.0, at 30°C in a shaker rotating at 200rpm over a period of 72h to yield free fatty acids. EXAMPLE 3 2.5g of wheat bran and 7.5g of gingelly oil cake were taken in a 500ml Erlenmeyer flask and then distilled water was added to it with continuous shaking to ensure that the initial moisture content of the substrate mixture was 33%, w/v. The flask was then autoclaved at 151bs/in2 at 121°C and after a period of 20 minutes, it was allowed to cool down to a temperature of 30°C. The spores of the isolate of the strain of Aspergillus niger, designated as CLRI BTFL, were streaked on a Czapek-Dox agar slant with the help of an inoculating needle and the slant was incubated at 34°C to facilitate the growth of the organism. After a period of 84h, 2ml of sterile distilled water was added to the slant and the mycelia were separated using a sterile glass rod. The spore o concentration of the resulting spore suspension was found to be 1.08 x 10 spores/ml of the suspension. 1.0ml of the above spore suspension was inoculated into the solid substrate medium using a pipetteman and the resulting inoculated substrate was mixed well using a sterile glass rod. The depth of the substrate mixture in the flask was found to be lOmm.The flask was then incubated at 30°C under static condition. After 96h of incubation, Ig of the resulting mouldy substrate was taken in a mortar and 10ml of distilled water was added to it. This suspension was then subjected to grinding with the help of a pestle to obtain the lipase enzyme extract, which was centrifuged at 10,000rpm for 5min. at 30°C to get the lipase enzyme. This lipase was assayed for enzyme activity by the titrimetric method and the activity was found to be 95.3 U/g dry substrate. 50U of this crude enzyme was used to remove olive oil stain efficiently from cotton fabric at a temperature of 37°C with a washing time of 40min. in the presence of 0. l%,\v/v of the commercial detergent, Ariel. EXAMPLE 4 50.Og of wheat bran and 50.Og of gingelly oil cake were taken in a 1000ml beaker and then distilled water was added to it with continuous stirring to ensure that the initial moisture content of the substrate mixture was 30%, w/v. The beaker was then autoclaved at 151bs/in2 at 121°C and after a period of 20 minutes, it was allowed to cool down to a temperature of 30°C. The spores of the isolate of the strain of Aspergillus niger, designated as CLRI BTFL, were streaked on a Czapek-Dox agar slant with the help of an inoculating needle and the slant was incubated at 36°C to facilitate the growth of the organism. After a period of 72h, 2ml of sterile distilled water was added to the slant and the mycelia were separated using a sterile glass rod. The spore concentration of the resulting spore suspension was found to be 4.3 x 105 spores/ml of the suspension. 10ml of the above spore suspension was inoculated into the solid substrate medium using a pipetteman. The resulting inoculated substrate was mixed well using a sterile glass rod and was then transferred to a sterilised aluminium tray of 18x20cm dimension, while adjusting the depth of the substrate mixture to 30mm height. The tray was then incubated at 25°C under static condition. After 144h of incubation, Ig of the resulting mouldy substrate was taken in a mortar and 10ml of distilled water was added to it. This suspension was then subjected to grinding with the help of a pestle to obtain the lipase enzyme extract, which was centrifuged at 10,000rpm for 5min. at 30°C to get the lipase enzyme. This lipase was assayed for enzyme activity by the titrimetric method and the activity was found to be 50.2 U/g dry substrate. This lipase enzyme was used for the enzymatic hydrolysis by incubating 5ml of this enzyme of effective activity of 50U, Ig of olive oil and 15ml of 0.1M phosphate buffer of pH 7.0, at 30°C in a shaker rotating at 200rpm over a period of 72h to yield free fatty acids. EXAMPLE 5 8.75g of wheat bran and 1.25g of gingelly oil cake were taken in a 250ml Erlenmeyer flask and then distilled water was added to it with continuous stirring to ensure that the initial moisture content of the substrate mixture was 67%, w/v. The flask was then autoclaved at 151bs/in2 at 121°C and after a period of 20 minutes, it was allowed to cool down to a temperature of 30°C. The spores of the isolate of the strain of Aspergilhis niger, designated as CLRI BTFL, were streaked on a Czapek-Dox agar slant with the help of an inoculating needle and the slant was incubated at 30°C to facilitate the growth of the organism. After a period of 84h, 2ml of sterile distilled water was added to the slant and the mycelia were separated using a sterile glass rod. The spore concentration of the resulting spore suspension was found to be 1.08 x 108 spores/ml of the suspension. 1.0ml of the above spore suspension was inoculated into the solid substrate medium using a pipetteman and the resulting inoculated substrate was mixed well using a sterile glass rod. The depth of the substrate mixture in the flask was found to be 15mm. The flask was then incubated at 30°C under static condition. After 48h of incubation, Ig of the resulting mouldy substrate was taken in a mortar and 10ml of distilled water was added to it. This suspension was then subjected to grinding with the help of a pestle to obtain the lipase enzyme extract, which was centrifuged at 10,000rpm for 5min. at 30°C to get the lipase enzyme. This lipase was assayed for enzyme activity by the titrimetric method and the activity was found to be 202.3 U/g dry substrate. The enzyme exhibited wide stability between pH 2.0 - 11.0 at 37°C for 30min. Also, the enzyme was thermotolerant exhibiting broad stability between 4 - 60 C for 3Qmin. at pH 7.0. EXAMPLE 6 40.Og of wheat bran and 60.Og of gingelly oil cake were taken in a 1000ml beaker and then distilled water was added to it with continuous stirring to ensure that the initial moisture content of the substrate mixture was 85%, w/v. The beaker was then autoclaved at 151bs/in2 at 121°C and after a period of 20 minutes, it was allowed to cool down to a temperature of 30°C. The spores of the isolate of the strain of Aspergillus niger, designated as CLRI BTFL, were streaked on a Czapek-Dox agar slant with the help of an inoculating needle and the slant was incubated at 37°C to facilitate the growth of the organism. After a period of 96h, 2ml of sterile distilled water was added to the slant and the mycelia were separated using a sterile glass rod. The spore concentration of the resulting spore suspension was found to be 4.3 x 106 spores/ml of the suspension. 10ml of the above spore suspension was inoculated into the solid substrate medium using a pipetteman. The resulting inoculated substrate was mixed well using a sterile glass rod and was then transferred to a sterilised aluminium tray of 16x18cm dimension, while adjusting the depth of the substrate mixture to 25mm height. The tray was then incubated at 20°C under static condition. After 60h of incubation, the enzyme in the resulting mouldy substrate was extracted by the conventional counter-current technique with a contact time of 30min. The extracts, thus obtained, were filtered using Whatman filter paper No.4 to get the lipase enzyme in the filtrate. This lipase was assayed for enzyme activity by the titrimetric method and the activity was estimated to be 308.4 U/g dry substrate. The enzyme exhibited good stability in presence of the commercial detergents like Ariel, Henko, Rin, Surf ultra and Surf excel. Iml/mg of this enzyme was used in combination with these commercial detergents at 30°C to remove fat based stains. EXAMPLE 7 7.5g of wheat bran and 2.5g of gingelly oil cake were taken in a 250ml Erlenmeyer flask and then distilled water was added to it with continuous stirring to ensure that the initial moisture content of the substrate mixture was 62%, w/v. The flask was then autoclaved at 151bs/in2 at 121°C and after a period of 20 minutes, it was allowed to cool down to a temperature of 30°C. The spores of the isolate of the strain of Aspergillus niger, designated as CLRJ BTFL, were streaked on a Czapek-Dox agar slant with the help of an inoculating needle and the slant was incubated at 30°C to facilitate the growth of the organism. After a period of 72h, 2ml of sterile distilled water was added to the slant and the mycelia were separated using a sterile glass rod. The spore concentration of the resulting spore suspension was found to be 4.3 x 108 spores/ml of the suspension. 0.5ml of the above spore suspension was inoculated into the solid substrate medium using a pipetteman and the resulting inoculated substrate was mixed well using a sterile glass rod. The depth of the substrate mixture in the flask was found to be 15mm.The flask was then incubated at 30°C under static condition. After 72h of incubation, the enzyme in the resulting mouldy substrate was extracted by the conventional plugflow technique. The extracts, thus obtained were filtered using Whatman filter paper No.4 to get the lipase enzyme in the filtrate. This lipase was assayed for enzyme activity by the titrimetric method and the activity was estimated to be 369.7 U/g dry substrate. The enzyme showed remarkable stability in hexane, heptane, cyclohexane, isooctane and benzene at 30°C for Ih. The main advantages of the present invention are the following: i) This Aspergilhis niger CLRI BTFL strain shows enhanced lipase production within short time compared to other strains. ii) The cost of the substrates used in the production of lipase makes this SSF process economically viable, iii) The enzyme exhibited broad pH and temperature stabilities in the range of pH 2.0-11.0 and temperature 4°-60°C. iv) Stability of the enzyme is good in presence of different commercial detergents and organic solvents, v) The lipase is effective as an additive in laundry formulations for the removal of fat based stains in laundries, vi) The enzyme is found to be effective hi the hydrolysis of oils and fats to yield optimum amount of free fatty acids. We Claim: 1. An improved process for the production of stable lipase, wherein the said process comprising the steps of: a) growing the spores of the strain of Aspergillus niger on Czapek - Dox agar medium by known method; b) inoculating the spore suspension obtained from step (a) in pre sterilized solid substrate medium containing wheat bran and gingelly oil cake in a ratio ranging from 1:0.11-1:9.0 to obtain a moldy solid mass. c) extracting the lipase enzyme from the moldy solid mass obtained from step (b) by known method; d) separating the lipase enzyme from enzyme extract obtained from step (c). 2. A process as claimed in claim 1, wherein the said Czapek - Dox medium contains sodium nitrate, di potassium hydrogen orthophosphate, potassium chloride, magnesium sulphate, sucrose, agar and tributin. 3. A process as claimed in claim 1, wherein the mycelia are separated with the help of a sterile glass rod by adding sterile distilled water to the culture of Aspergillus niger to obtain the spore suspension. 4. A process as claimed in claim 1, wherein the concentration of spore suspension is in the range of 104-1010 spores/g of substrate. 5. A process as claimed in claim 1, wherein wheat bran and gingelly oil cake preferably used in a ratio 1:0.14-1:3.0. 6. A process as claimed in claim 1, wherein incubation temperature of the inculated solid substrate medium is in the range of 20-45 degree C. 7. A process as claimed in claim 1, wherein the separation of lipase is carried out by centrifugation, filtration or gravity separation. 8. An improved process for the production of stable lipase substantially as herein described with reference to the examples.

Documents:

1556-del-1999-abstract.pdf

1556-del-1999-claims.pdf

1556-del-1999-correspondence-others.pdf

1556-del-1999-correspondence-po.pdf

1556-del-1999-description (complete).pdf

1556-del-1999-form-1.pdf

1556-del-1999-form-19.pdf

1556-del-1999-form-2.pdf