Overexpression and characterization of a lipase from Bacillus subtilis.
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 degrees C and pH 8.5 for hydrolysis of p-nitrophenyl caprylate. The values of Km and Vm were found to be 0.37 mM and 303 micromol mg-1 min-1, respectively. The substrate specificity study showed that p-nitrophenyl caprylate is a preference of the enzyme. The metal ions Ca2+, K+, and Mg2+ can activate the lipase, whereas Fe2+, Cu2+, and Co2+ 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.
Ma
Jisheng
J
Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, 2519 Jiefang Road, Changchun 130023, PR China.
Zhang
Zuoming
Z
Wang
Baijing
B
Kong
Xiangju
X
Wang
Yuguo
Y
Cao
Shugui
S
Feng
Yan
Y
D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D013379:Substrate Specificity; D013656:Taurocholic Acid / Q000494:pharmacology; D013696:Temperature
D001426:Bacterial Proteins; D008670:Metals; D011994:Recombinant Proteins; D013656:Taurocholic Acid; D008049:Lipase