一种来自荧光假单胞菌B68的具有对映体选择性的脂肪酶基因的克隆、表达及酶学研究
loning, Expression and Characterization of an Enantioselective Lipase LipB68 from Pseudomonas Fluorescens Strain B68
本研究对一株筛选到的荧光假单孢菌Pseudomonas fluorescens B68产的脂肪酶LipB68进行了克隆表达与酶学性质研究。从B68的基因组中克隆得到了脂肪酶LipB68基因的完整序列。基因lipB68 由1431个碱基组成其开放阅读框（ORF），编译476个氨基酸，理论分子量为50,193 Da。同源性分析表明，脂肪酶LipB68属于脂肪酶家族I.3。将基因lipB68克隆于表达载体pET-28a中，实现了在大肠杆菌BL21（DE3）中以包涵体形式的高量表达。纯化后的脂肪酶LipB68以对硝基苯酚癸酸酯为水解底物时，具有族中已知最低的最适反应温度——20℃。同时，该脂肪酶也显示了对DMF和乙腈有良好的耐受性。固定化在布条上后，该脂肪酶对外消旋的苯乙醇和苯丙醇有较高的对映体选择性（E值分别为220和190）。
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 a-phenylethanol and a-phenylpropanol (E=220, and 190 respectively).
郑一涛、江正兵、魏东芝、罗宇、马昱澍
生物工程学分子生物学微生物学
克隆，酶学性质，表达，脂肪酶，荧光假单胞菌
clone enzyme property expression lipase Pseudomonas fluorescens
郑一涛,江正兵,魏东芝,罗宇,马昱澍.一种来自荧光假单胞菌B68的具有对映体选择性的脂肪酶基因的克隆、表达及酶学研究[EB/OL].(2006-03-02)[2025-08-16].http://www.paper.edu.cn/releasepaper/content/200603-14.点此复制
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