[Directed evolution of lipase of Bacillus pumilus YZ02 by error-prone PCR].
Random mutagenesis on Bacillus pumilus lipase YZ02 gene was conducted by using error-prone PCR strategy. Through two cycles of directed evolution, two optimum mutants BpL1-7 and BpL2-1369 with lipase activity improved 2 folds and 6 folds respectively were screened. The sequence of BpL2-1369 lipase gene showed that four nucleotides substitution, T61C, C147T, A334G and T371A have occurred, and three of them caused amino acid changes. Thus, amine acid Ser21 was changed into Pro21, Arg112 to Gly112, and Leu124 to His124. According to the 3D structure of Bacillus pumilus lipase mimicked by SWISS-MODEL Repository, three mutated amino acids were located at the third amino acid of the first alpha-helix, the turn between the fourth and fifth beta fold, and the first amino acid of the fifth beta fold, respectively. The BpL and BpL2-1369 genes were ligated into pET28a vector, and transferred into E. coli BL21 (DE3). After induced by IPTG the lipases were purified and characterized. The results showed that the specific activity of the evolved lipase was 1.31-fold than that of the wild lipase, and the Km decreased from 8.24 mmol/L to 7.17 mmol/L. The pH stability of the evolved lipase was better than wild lipase when pH>8.0.
Huang
Ying
Y
Key Laboratory of Molecular Bio-physics, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. huangyin588@sina.com.cn
Cai
Yong
Y
Yang
Jiangke
J
Yan
Yunjun
Y
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering; D020539:Sequence Analysis, Protein
D008049:Lipase