Production of extra cellular lipase from Bacillus sp LBN 4 by solid state fermentation

摘要

Bacillus sp LBN4 produced lipase during solid substrate fermentation. Maximum lipase production was 14.0IUg-1 with rice bran as sold substrate. 50% more lipase production was achieved when rice bran was supplemented with soyabean mixture. Supplementation of the medium with wheat bran led to 70% increase in lipase production. Optimum temperature and pH were 500C and 7.0 respectively. Na+ induced more lipase than K+ and Mg++ Introduction Lipases are hydrolytic enzymes that catalyses the cleavage of ester bonds in triglycerides and producing glycerol and free fatty acids. These are biotechnologically relevant enzymes and find potential applications in detergent, food, pharmaceutical, leather, paper and pulp industries1. However for commercial applications development of low cost processes and production of lipases using simple and inexpensive substrates such as agro industrial residues are more favorable. Solid state fermentation is an low cost alternative and involves the growth and metabolism of microorganisms on moist solids without free flowing water. Solid state fermentation (SSF) has many advantages over submerged fermentation (SmF) including simplification of the fermentation media, low capital investment, absence of complex machinery, reduced energy requirement and improved product recovery (Losane et al 1983; Satyanarayana 1994). Major group of microorganisms used in Solid state fermentation are bacteria, actinomycetes, fungi and yeast. The vast majority of literature on Solid state fermentation refers to the fungi and yeast using different solid substrates ( Pandey et al 1999; Rao et al 1993; Falony et al 2006; Kamini et al 1997) The exploitation of new microorganisms capable to produce lipase in Solid state fermentation conditions would be useful for industrial applications in the near future. A strain of Bacillus Sp LBN 4 was taken from stock repository which was originally isolated from the alkaline soil of hot spring of Arunachal Pradesh was investigated for production of extra cellular lipase by solid state fermentation using rice bran as a solid substrate.Solid state fermentation was carried out in 250ml conical flasks. 10 g of rice bran was mixed with mineral salt medium and final moisture ratio of 1:2 w/v was achieved. The pH of the mixture was adjusted to 7.0 in sodium phosphate buffer. The above mixture was autoclaved at 1210C for 20min, cooled to room temperature and inoculated with 10% of 12h grown seed culture of Bacillus sp and incubated at 400C for different time intervals (12, 24, 36,48h). Enzyme extraction was carried out by adding 10 ml of 0.2M phosphate buffer having pH 7.0 to each flask and agitating in the orbital shaker at 200rpm for 1h. The particulate matter was filtered through a muslin cloth and centrifuged at 10,000rpm for 30 minutes. The clear supernatant was used for lipase assay.In another experiment, effects of various cheap substrates like soyabean oil, soybean meal, Wheat bran and coconut oil was studied by adding each of the substrate in the rice bran mixture and the lipase activity was determined accordingly.Lipase activity was determined by (Watnabe et al method 1977). The reaction mixture containing 5ml of olive oil emulsion composed of 25 ml olive oil and 75 ml 2% polyvinyl alcohol solution, 4ml of 0.2M tris buffer, 1ml of 110mM CaCl2 and 1ml enzyme solution. The control containing boiled inactivated enzyme (at 1000C for 5 minutes) was treated similarly. After the incubation, the enzyme activity was blocked by 20 ml of acetone ethanol (1: 1) mixture and liberated free fatty acid was titrated against 0.02 M NaoH using phenolphthalein as indicator. One unit of lipase was defined as the amount of enzyme, which liberates 1 m mol of fatty acid/ min under standard assay conditions. The enzyme activity was expressed as Ug-1 dry substrate.The effect of pH on lipase production was studied in a pH range of 5- 10.0 using different buffers ( citrate buffer 3-6, sodium phosphate, 6-7, Tris HCL p