Production purification and characterization of solvent Tolerant lipase from Bacillus sphaericus MTCC 7542

Abstract

Lipases, triacylglycerol hydrolases, are an important group of biotechnologically relevant enzymes and find immense applications in food, dairy, detergent and pharmaceutical industries. Catalytic activity can be enhanced greatly by using enzyme in organic solvent rather than in aqueous media. In organic solvents enzymes can catalyze reactions that are impossible in water, become more stable and exhibit new behavior. In the present study, solvent tolerant Bacillus sphaericus MTCC 7542 and Bacillus pumilis MTCC 2296 were identified from various Bacillus species via the direct plating method using 30% (v/v) organic solvents. Optimization studies were undertaken to improve lipase production in basal media. The effect of inducer was studied and the highest yield of 0.148 ± 0.003 IU/mL on 48 h was obtained at 1% of olive oil. The effect of inorganic nitrogen source studied and the maximum yield of 0.191 ± 0.004 IU/mL on 48 h was obtained with the media supplemented with 0.2% ammonium chloride. The optimizations of the media components on lipase production have successfully carried out by the Central Composite Design (CCD). The optimized media composition for lipase production was determined to be 1.0% olive oil, 0.5% yeast extract, 0.2% glucose, 0.2% ammonium chloride. Statistical method of media optimization led to a 3.5-fold increase in lipase production (0.30 ± 0.003 IU/mL) over the unoptimized media. The purified lipase protects 90% of its activity after incubating for 60 min at 40°C in the presence of solvents. Metal ions such as Mg2+, Ca2+, Cu2+ and K+ were found to enhance the lipase activity, while Fe3+, Zn2+, Na+ and Al3+ions were inhibited. The presence of Tween 80 was enhanced the lipase activity, while SDS, 2-mercaptoethanol EDTA, Triton X 100, Sodium perchlorate show the inhibited the lipase activity. newline newline newline