一种具有立体选择性新酯酶的菌种筛选和基因克隆

Screening of strain producing an novel esterase with high enantioselectivity and molecular cloning of the enzyme gene

摘要:

从土样分离到一株产生具有立体选择性酯水解酶的恶臭假单孢菌(Pseudomonas putida NH33)。构建P.putida NH33的基因组文库,并在E.coli中进行酯酶活性筛选,得到一个含有4.7kb插入片段的阳性克隆。对这个克隆的DNA片段进行序列分析,表明它含有一个1142个碱基的开放阅读框,为编码381个氨基酸的酯酶。推测的酯酶氨基酸序列与其它丝氨酸酯水解酶具有共同的保守基序GXSXG。把该蛋白在E.coli BL21(DE3)中进行表达,并用金属亲和层析纯化至单一条带。利用纯化的酶水解2-芳基丙酸乙酯制备2-芳基丙酸的S型异构体,产物的光学纯度ee_p>99%,说明此酶可8用于手性药物的合成。该酯酶是一个新酶,其基因序列已递交GenBank,登记号为AY896293。

Abstract:

A novel strain producing an enantioselective lipolytic enzyme was isolated from soil samples, and identified as Pseudomonas putida NH33. A genomic library of P. putida NH33 was constructed and screened for esterase activity in E. coli. One positive clone was isolated, and subsequent analyses of the plasmid by restriction mapping revealed a 4.7kb DNA fragment carrying esterase gene. The nucleotide sequence of the DNA was found to contain an open reading frame of 1142 nucleotides encoding esterase of 381 amino acid residues and designated PPEst. The primary structure of the esterase exhibited 35%～40% homology to those of related enzymes from various sources and 80%～90% homology to esterases from the genus Pseudomonas. Amino acid sequence deduced from the nucleotide sequence contains of the consensus active site sequence, GXSXG, of serine esterase. The PPEst fragments were cloned into the expression vector pET-22b(+) and transformed into E. coli BL21 (DE3), and the recombinant protein fused with 6×His at its C-terminus was purified to homogeneity by a single immobilized metal ion affinity chromatographic step. The molecular mass of the esterase was determined to be approximately 42kDa by SDS_PAGE. The purified enzyme could convert ethyl esters of 2-arylpropanoic acid to S-isomer of 2-arylpropanoic acids with an optical purity of >99%.The result suggests that this esterase is excellent biocatalyst for synthesis of chiral pharmaceuticals. Ｔhe enzyme is an novel esterase, and its nucleotide sequence has been submitted to GenBank under accession number AY896293.