Intl. J. of Appl. Biol. (2011) 2(2): 54-57 ISSN No: 2231-3370 Optimization Studies on a Thermophilous Fungus, Malbranchea pulchella var. sulfurea on Lipase Synthesis S. Bhuvaneswari Udaya Prakash1., S. Geetha2 and N.K. Udaya Prakash2 1 Research and Development, MARINA LABS, 40, Anna Nedum Pathai, Choolaimedu, Chennai 600 094 s_bhuvaneswari8@yahoo.co.in 2 Research and Development, Veltech Dr. RR Dr. SR Technical University, Avadi, Chennai 600 062 ABSTRACT Thermophilous fungus sp. Malbranchea pulchella var. sulfurea was isolated from the soil samples of edible oil extracting unit present near Chennai. The fungus produced a large clear zone on lipolytic agar medium suggesting its lipase activity. The lipase enzyme secreted by Malbranchea pulchella var. sulfurea is extracellular, which is widely used in various applications in industries. The lipase synthesis by this fungus, is optimized using various factors like concentration of Soybean meal, Carbon source, Nitrogen source, pH, different oil substrate and different temperature. Maximum activity was observed when Soybean meal of 10 g concentration, Glucose as Carbon source, Ammonium Nitrite as Nitrogen source, 6 pH value, Castor oil as oil source and 45°C as the temperature is used. Keywords: Thermophilous fungi, Malbranchea, Lipase, Soybean meal, Castor oil INTRODUCTION Lipase are enzymes that catalyses, the hydrolysis and the synthesis of esters formed from glycerol (Immanuel et. al., 2008) and long chain fatty acids. They are ubiquitous in nature, produced by animals, plants and fungi and other organisms. Lipases are currently attracting an enormous attention because of their biotechnological application. Fungi are broadly recognized as one of the best lipase producers and used in food industry. Lipases from fungi (Essamri et. al., 1998) have been widely used for biotechnological application in dairy industry, oil processing, and production of surfactants and preparation of pure pharmaceuticals (Viniegera et. al., 2003). The thermophilc fungi have advantages of less cross contamination and more enzyme stability as compared to mesophilic fungi. The thermophilic fungi are promising sources of thermostable enzymes for biotechnological application. Most thermophilic fungi grow in simple media containing carbon and nitrogen sources and necessary mineral salts; they are mostly autotrophic to vitamins. Arima et al., (1972) purified an extracellular lipase from Humicola lanuginosa strain Y-38, isolated from compost in Japan. The enzyme was produced in a medium containing soybean oil, starch, corn steep liquor, and antifoaming agent. Omar et. al., (1987a and (b) reported that the productivity and thermostability of lipase differed with different strains of H. lanuginosa. A lipase gene from H. lanuginosawas cloned and expressed in Aspergillus oryzae (Huge-Jensen et al., 1989). The structure of H. lanuginosa lipase (Derewenda et al., 1994) was similar to that of R. miehei lipase. Rhizomucor miehei, formerly called Mucor miehei, also produces active extracellular lipase. The isolation and purification methods for M. miehei lipase have been described by Huge-Jensen et. al., (1987). A review on the physiology and enzyme synthesis by thermophilous fungi was carried by Maheshwari et al., (2000). However, there was no report available on the synthesis of Lipase by the fungus, Malbranchea pulchella var. sulfurea and hence the study is conducted. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show different not only among strains of the same species. Several factors can affect extracellular lipase production from thermophilic fungi such as pH, temperature, media composition and substrate. The objective of this study is to optimize the synthesis of lipase by the thermophilous fungus, Malbranchea pulchella var. sulfurea by Solid State Fermentation for the extracellular lipase production using soybean meal as a substrate and optimization of various parameters controlling synthesis of lipase. April-June (2011) Optimization Studies on a Thermophilous ... MATERIALS AND METHODS The fungus: The fungus, Malbranchea pulchella var. sulfurea was isolated from the soil sample of an edible oil extracting unit situated in a suburban (Guduvanchery) area of Chennai. 55 RESULTS AND DISCUSSION The fungi isolate under investigation Malbranchea pulchella var. sulfurea showed lipolytic potential of the diameter of hydrolytic zone of 10mm after 3 days of incubation at 45°C on YpSs agar plate. Optimization parameters Substrate: Soyabean were used as substrates. 10 gm, 15gm and 20gm of Soyabean meal was suspended in 25 ml of YpSs (Yeast Phosphate Soluble Starch) media in a 250 ml flask and sterilized. It was cooled before using. The fungus was then inoculated into the flask for 5 days. After a stipulated time period the fermented samples were drawn and homogenized and a small amount of sample was taken from each flask for extraction and subsequent analysis. Enzyme Extraction: The crude enzyme from the fermented material was extracted by simple extraction method. The fermented substrate was mixed thoroughly with 100 ml of 0.05 M of phosphate buffer (pH 7.0) and then shaking the mixture in a rotary shaker (200 rpm) at 30° C for 2 hours. The crude enzyme obtained from centrifugation and was used to determine enzyme activity. Lipase Assay (Titrametric assay method): The enzyme was assayed by titrimetric method using olive oil. A unit lipase is defined as the amount of enzyme which releases one micromole of fatty acids per unit under specified assay conditions. One ml of the culture supernatant was added to the 5 ml of reaction mixture. The liberated fatty acid was titrated against 0.1N NAOH using phenolphthalein as indicator. The end point was indicated by appearance of pink colour. The enzyme unit was calculated as follows and expressed in u/g of Lipase activity = Fresh weight of biomass plate Fresh weight of biomass Effect of substrate concentration The variable amount of soyabean meal (10 g, 15 g and 20 g)was added along with the YPSs medium and was incubated at 45°C for 5 days and 10 g showed maximum lipase production where as least production was found in 20 g Effect of carbon source Glucose, lactose and starch were carbon source. Glucose was found to be source, as compared to others because maximum lipase production on Glucose, the lipase production was found in starch. used as the best it gives minimum Effect of Nitrogen source The effect of nitrogen source such as Ammonium Sulphate, Ammonium Nitrate and Sodium Nitrite was studied for the production of lipase. Ammonium Nitrate was the best nitrogen source among the 3 nitrogen source studied for extracellular lipase production by Malbranchea pulchella var. sulfurea. The minimum lipase production was found in Sodium Nitrite. Effect of pH on enzyme production As pH is the important parameter required for the growth of fungal culture in respective media so lipase activity got affected with basic pH, this indicates that suitable pH is responsible for fungal growth in the media. The data obtained clearly indicates that there is a strong influence of pH on lipase enzyme production. Thus the maximum activity was reported at pH 6. Optimization of Medium Parameters: The lipase synthesis by this fungus, is optimized using various factors like concentration of Soybean meal (10 g, 15 g and 20 g), Carbon source (Glucose, Lactose and Starch), Nitrogen source (Ammonium nitrate, Ammonium Nitrite and Sodium Nitrate), pH (5, 5.5 and 6), different oil (Olive oil, Coconut oil and Castor oil) substrate and different temperature (45°C, 50°C and 55°C). Effect of Oil source The effect of oil source such as olive oil, coconut oil, castor oil was studied for extracellular lipase production by Malbranchea pulchella var. sulfurea proved to be the best oil source stimulating extracellular lipase production. The minimum lipase production was found in Cocount oil. The Maximum lipase production was found in Castor oil. Intl. J. of Appl. Biol. 56 Table 1: Effect of different parameters on lipase activity. Lipase activity (U g ds-1) Parameters Substrate concentration Carbon Source Nitrogen Source pH Oil Source Temperature 10g 15 g 20 g Glucose Lactose Starch Ammonium Sulphate Ammonium Nitrate Sodium Nitrite 5 5.5 6 Olive oil Coconut oil Castor oil 400 315 250 156 150 117 416 666 195 45°C 250 315 400 278 264 658 300 50°C 291 55°C 230 Vol. 2 No. 2 recovery method for the production of industrial enzymes. Lipase production by various microorganisms has been found to be induced by fatty acids, lipids especially by vegetative oil, such as soybean, sunflower, olive oil, corn oil and others also by a variety of surfactants (Corzo Revah, 1999). The fungus, Malbranchea pulchella var. sulfurea is found to produce different enzymes, i.e., Xylanase (Matsou and Yasui, 1985), b-xylosidase (Matsuo et al.,, 1977a and b), Lipoamide dehydrogenase (Stevensen and Gaucher, 1975; Voordouw et al., 1974; Voordouw and Roche, 1975), thermomycolase, the extracellular serine protease (Ong and Gaucher, 1976), Two fungal sources of thermostable alkaline proteases were identified based on the zone of clearing of casein agar by culture filtrates as a semiquantitative assay of proteolytic activity of Malbranchea pulchella var. sulfurea (Ong and Gaucher, 1973). Lipoamide dehydrogenase (LD) also called dihydrolipoyl dehydrogenase, is a component of the multienzyme pyruvate dehydrogenase complex. It catalyzes the reduction of NAD1 with dihydrolipoamide. LD from the thermophilic fungus Malbranchea pulchella was purified to homogeneity by a simple three-step procedure consisting of salt precipitation, affinity chromatography, and ion-exchange chromatography (McKay and Stevensen, 1979). Effect of Temperature CONCLUSION The effect of temperature for the cultivation of lipase was studied by incubating the production media in different temperature such as 45°C, 50°C, and 55°C. Forty five degrees was found to be optimum temperature for extracellular lipase production by Malbranchea pulchella var. sulfurea. Malbranchea pulchella var. sulfurea is the proved to be the best lipase producer. Various physic chemical parameters were studied to determine the optimum conditions for its lipase production. The obtained results showed that the medium composition for lipase production was (soy bean meal − 10 g, Ammo nium nitrate – 1 g, Glucose – 1 g, castor oil – 1 ml) at pH 6 which has to be maintained at 45°C . 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