Overexpression and characterization of a lipase from Bacillus subtilis

• Published in: Protein expression and purification Vol. 45; no. 1; pp. 22 - 29
• Main Authors: Ma, Jisheng, Zhang, Zuoming, Wang, Baijing, Kong, Xiangju, Wang, Yuguo, Cao, Shugui, Feng, Yan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 2006
• Subjects: Bacillus subtilis; Bacillus subtilis - drug effects; Bacillus subtilis - enzymology; Bacterial Proteins - chemistry; Bacterial Proteins - drug effects; Bacterial Proteins - genetics; Base Sequence; Characterization; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genetic Vectors - genetics; Hydrogen-Ion Concentration; Kinetics; Lipase; Lipase - chemistry; Lipase - drug effects; Lipase - genetics; Metals - pharmacology; Molecular Sequence Data; Molecular Weight; Overexpression; Recombinant Proteins - chemistry; Recombinant Proteins - drug effects; Recombinant Proteins - genetics; Substrate Specificity; Taurocholic Acid - pharmacology; Temperature; Characterization; Overexpression; Lipase; Bacillus subtilis
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2005.06.004

A novel plasmid, pBSR2, was constructed by incorporating a strong lipase promoter and a terminator into the original pBD64. A mature lipase gene from Bacillus subtilis strain IFFI10210, an existing strain for lipase expression, was cloned into the plasmid pBSR2 and transformed into B. subtilis A.S.1.1655. Thus, an overexpression strain, BSL2, was obtained. The yield of lipase is about 8.6 mg protein/g of wet weight of cell mass and 100-fold higher than that in B. subtilis strain IFFI10210. The recombinant lipase was purified in a three-step procedure involving ammonium sulfate fractionation, ion exchange, and gel filtration chromatography. Characterizations of the purified enzyme revealed a molecular mass of 24 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, maximum activity at 43 °C and pH 8.5 for hydrolysis of p-nitrophenyl caprylate. The values of K m and V m were found to be 0.37 mM and 303 μmol mg −1 min −1, respectively. The substrate specificity study showed that p-nitrophenyl caprylate is a preference of the enzyme. The metal ions Ca 2+, K +, and Mg 2+ can activate the lipase, whereas Fe 2+, Cu 2+, and Co 2+ inhibited it. The activity of the lipase can be increased about 48% by sodium taurocholate at the concentration of 7 mM and inhibited at concentrations over 10 mM.