Isolation, Purification and Characterization of Lipase from mutant Bacillus brevis.

Abstract

Lipases are glycerol ester hydrolases that catalyze the hydrolysis of triacylglycerol to free fatty acids and glycerol. Bacterial lipase producers were isolated from palm oil contaminated soil samples from palm oil processing sites. Four isolates showed a greater zone of clearance from the medium agar than the others indicating higher lipase activity. These four isolates were subjected to mutation using ethidium bromide and the bacterial isolates with higher zone of clearance was selected. The wild and mutantBacillus spp were further screened for lipase production by sub-merged fermentation and mutant B. brevis showed the highest lipase activity. The production of lipase from mutant Bacillusbrevis was carried out in a shake-flask at 30 ˚C in cultivation medium containing palm oil 10 mL (emulsified in Tween- 80 at a ratio of 10mL oil:1mL Tween); in amedium containing (NH4)2SO4(5g); Na2HPO4(6g); KH2PO4(2g); MgSO4(3g); CaCl2(3g)0.5M citrate-phosphate buffer pH 6.0 with agitation of 150 rpm for 7 days. The crude lipase was collected and followed by purification that involved 60 % (w/v) ammonium sulphate precipitation, ion-exchange chromatography on DEAE – Sephadex A-50 and gel-filtration chromatography on Sephadex G- 100 with a final yield of 5.6 % and purification fold of 19.05. The molecular weight of the purified enzyme was estimated to be 59 kDa after Sodium dodecyl Sulphatepolyacrylamide gel electrophoresis. The optimal temperature of purified lipase was 40 ˚C and it retained up to 55 % of its initial activity for 2 hr. The optimal pH was 9.0 and retained up to 54 % of its initial activity for 2 hours. Al3+ and Ca2+slightly increased the activity of the enzyme at a concentration of 10 mM while Cu2+ and Hg2+ decreased the activity of the enzyme at the same concentration. At 10 mM concentration, ethylene diamnine teteracetic acid (EDTA) and β-mercaptoethanol decreased the activity by 48 % and 50 % respectively while SDS and Urea has little effect on the enzyme.