L Bora, M Kalita
lipase, rice bran, solid substrate fermentation, sp lbn 4
L Bora, M Kalita. Production of extra cellular lipase from Bacillus sp LBN 4 by solid state fermentation. The Internet Journal of Bioengineering. 2008 Volume 3 Number 2.
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 industries 1 . 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
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 121 ° C for 20min, cooled to room temperature and inoculated with 10% of 12h grown seed culture of
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 100 ° C 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 µ 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 pH8.0 and glycine NaOH 9-10.0) at 50mM concentration. Temperature effect on lipase production was determined by carrying out the incubation at different temperatures in the range of 30-80 ° C at pH 7.0. The effects of metal ions were also studied.
The maximum lipase yield from
Table 1: Effect of different substrates on lipase production by Bacillus sp in solid state fermentation
The increase in lipase production may be due to the presence of additional factors like nitrogen and oil compounds which acts as stimulating factors in the medium. The addition of wheat bran increased the yield upto 70% whereas marginal increase was obtained with coconut oil (20%). Sekhon
In most of the earlier reports the lipase production was reported after prolonged fermentation period (72-192h) whereas in our study the maximum lipase yield was observed after 36h, (Sekhon
(Sharma et al 2002) . Similar results were also observed in
The optimum lipase activity was observed at pH 7.0 (Fig 2). The activity was found to be decreasing on increasing the pH values. The initial pH of the growth medium was important for lipase production. The pH in and around 7.0 was found to be preferable for bacteria for better growth and lipase production. Similar results have been reported for other
Divalent cation plays an important role in enzyme production. Earlier reports suggested that K + and Mg ++ are essential for lipase production. To study the effect of metal ions KCl, NaCl and MgCl2 at a concentration of 0.5, 1 and 1.5% (w/v) were added to the medium and incubated. Optimum concentration was found to be 1% in all three cases (Fig 3) .The best lipase production was achieved with Na+ followed by K + and Mg ++ . Similar results of lipase stimulation have been has been reported (Van ort et al 1989).
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