The chromosomal DNA of Bacillus subtilis SK320 was amplified using sfp0 gene (642bp) specific primers and the gene was cloned into E. coli DH5a using plasmid vector pGEM-T (3kb). Expression of the gene showed higher biosurfactant production (2.20 gm/lit) in the clone E. coli pSKP0, as compared to the parent Bacillus subtilis SK320 (1.2 gm/lit) when grown on olive oil as the sole substrate. Biosurfactant of Clone E. coli pSKP0 was found to be a powerful lipopeptide and reduced the surface tension of water from 72 to 35 dynes/cm as compared to 40.1 by Bacillus subtilis SK320. Expression studies during growth reveled the linkage of the biosurfactant to an esterase enzyme. Maximum biosurfactant activity was observed during the mid log phase of growth during which esterase activity was also maximum. Ion-exchange chromatography using Q Sepharose purified the extracellular esterase from pSKP0 and resolved it into three components; designated as P01, P02 and P03. All the three esterases were found to be heterogeneous in nature. Gel-filtration chromatography (Sephadex G-75) further resolved the esterases into their sub-components. The sub-components were further purified to homogeneity by poly-acrylamide gel electrophoresis as observed by activity and silver staining. The esterase enzyme from clone E.coli pSKP0 showed mol wt ranging from 14 to 120 kda and an isozyme pattern similar to Bacillus subtilis SK320.
Biosurfactants, olive oil, Bacillus subtilis SK320, esterase, isozyme, cloning, genetic regulation
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