一种来自荧光假单胞菌B68具有对映体选择性脂肪酶基因克隆、表达及酶学研究.pdfVIP

Cloning, Expression and Characterization of an Enantioselective Lipase LipB68 from Pseudomonas * Fluorescens Strain B68 ** ** Yu Luo, Yitao Zheng, Zhengbing Jiang, Yushu Ma and Dongzhi Wei State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, PRC 200237 Abstract A lipase-producing bacterium strain B68 was screened and identified as Pseudomonas fluorescens sp. Novel lipase gene lipB68 was cloned and sequenced by the method of GenomeWalker. Sequence analysis revealed an open reading frame of 1,431 nucleotides, encoding a protein with 476 amino acids and a molecular mass of 50,193 Da, being a member of lipase subfamily I.3. The lipB68 gene was expressed in E. coli BL21 (DE3) in the form of inclusion body and the purification and protein refolding were conducted. Lipase lipB68 had a favorable reaction temperature of 20°C with p -Nitrophenyl caprate as substrate, lower than all the known lipases from subfamily I.3. And it also showed good stability to organic solvents such as DMF and acetonitrile. After being immobilized on fabric of cellulose, lipase lipB68 showed high enantioselectivity at the resolution of racemic α-phenylethanol and α-phenylpropanol (E=220, and 190 respectively).