Production and Optimization of Thermostable lipase from a Thermophilic Bacillus sp LBN 4

摘要

The Bacillus sp isolated from the hot spring of Tawang district of Arunachal Pradesh, India was studied. Maximum enzyme production was achieved after 20 Hrs of cultivation. The olive oil and yeast extract was found to be the most suitable substrate for enzyme production. The enzyme had temperature optima of 65°C. Over 80% of its peak activity was at the pH range of 7-8, with an optimum of 8.0. The enzyme was found to be most stable at 80°C for 10 minutes. Almost 80% of the residual activity was retained at 80°C. CaCl2 was found to stimulate the lipase activity by 40%. Introduction To satisfy the global requirement of the industrial process we should have enzymes showing optimal activities at maximum degree of extremity of pH, temperature and salt concentration. Thermophilic microorganisms are found to be potential and good alternative source of thermostable enzymes (Brock 1985). The extremophilic, especially thermophilic bacteria can be isolated from the natural high temperature environments distributed throughout the world and found in association of tectonically active sites (Brock 1985). Lipases (EC 3.1.1.3) are comprised a group of enzymes which catalyse the hydrolysis of triacylglycerols. In the recent years, the interest on lipase has grown significantly. The development of technologies using lipases for the synthesis of novel compounds will result in their expansion into new areas and increase in number of industrial applications (Bjorkling et al. 1991). Lipases are extremely versatile enzymes, showing many interesting properties of industrial applications. Microbial lipases are high in demand due to their specificity of reaction, stereo specificity and less energy consumption than conventional methods. Microbial lipases have wide application in the processing of food, leather, domestic, industrial wastes, cosmetic, detergents and pharmaceutical industries (Ghosh et al. 1996, Saxena et al. 1999). In this paper isolation of lipolytic Bacillus sp from a hot spring of Arunachal Pradesh its production and characterization of the enzyme are reported. Material and Methods Isolation The samples were collected from the thermal spring of kitpi area of Tawang district of Arunachal Pradesh, India. This thermal spring is situated adjacent to the river Jung. Water and sediments showed a characteristics hydrogen sulfide odour. The samples were enriched at 50°Cin the nutrient broth supplemented with olive oil. Lipolytic isolates were selected on tributyrate agar medium (Collins and Lyne 1989) using glycerol tributyrate as substrate. Isolates having a higher ratio of clearing zone to the colony size were grown in the liquid culture and level of lipase production was determined from the cell free culture supernatant fluid. Characterization and identification of the isolate was made following Bergeys Manual of Systematic Bacteriology (Sneath 1986).Enzyme productionThe medium of the enzyme production was composed of peptone 2%, starch 2%, KH2PO4 0.5%, (NH4)2SO4 0.1%, (NH4)2CO4 0.1%, MgSO4. 7H2O 0.1%, pH 7.5. The medium was inoculated with 2 ml of overnight culture and incubated at 50°C. After 24 hours of incubation the culture was centrifuged and the cell free culture supernatant fluid was used as the enzyme source.Effect of culture variable on lipase productionFor the study of lipase production from the crude enzyme, starch was replaced by other carbon sources such as glucose, fructose, sucrose, and lactose and olive oil. Each of the substrates (1%w/v) was used as sole carbon source while tested for their ability to induce lipase production. The crude substrates were autoclaved for 30 minutes at 121°Cin the autoclave before use. The effect of nitrogen sources on the lipase production was analyzed by supplementing production medium with different nitrogen sources (1%w/v) like yeast extract, gelatin, tryptone, potassium nitrate and ammonium sulphate and enzyme activity was assayed.Enzyme assayAll the experiments were run in triplicate. The