Functional Analyses and Application of Microbial Lactonohydrolases

发现并鉴定了具有独特性质的微生物乳酸水解酶（分子内酯键水解酶）。从尖孢镰刀菌中提取的内酯水解酶可催化醛醛酸内酯和d -泛酰基内酯（D-PL）的对映选择性水解。该酶可用于外消旋PL的大规模光学分辨。在米曲霉中，尖孢镰刀菌内酰胺水解酶编码基因可以过表达。农杆菌可催化PL的不对称水解，但其立体特异性与镰刀菌酶相反。该酶基因也可在大肠杆菌转化细胞中表达。双氢香豆素水解酶（DCH）是一种双功能酶，它不仅能催化芳香族内酯的水解，还能在H_2O_2和双氢香豆素存在下催化一氯二烯酮的溴化反应。DCH还能水解几种线性酯，并可用于DL-β-乙酰硫代异丁酸甲酯的对映选择性水解和癸酸甲酯的区域选择性水解。DCH在体内与过氧化氢酶一起解毒过氧化物酸。

Microbial lactonohydrolases (intramolecular ester bond-hydrolyzing enzymes) with unique properties were found and characterized. The lactonohydrolase from Fusarium oxysporum catalyzes enantioselective hydrolysis of aldonate lactones and D-pantoyl lactone (D-PL). This enzyme is useful for the large-scale optical resolution of racemic PL.. The gene encoding latonohydrolase of F. oxysporum could be overexpressed in Aspergillus oryzae.The Agrobacterium tumefaciens enzyme catalyzes, asymmetric hydrolysis of PL, but the stereospecificity is opposite to that of the Fusarium enzyme. This enzyme gene also, could be expressed in E. coli transformant cells.Dihydrocoumarin hydrolase (DCH) from Acinetobacter calcoaceticus is a bifunctional enzyme, which catalyzes not only hydrolysis of aromatic lactones but also bromination of monochlorodirnedon in the presence of H_2O_2 and dihydrocoumarin. DCH also hydrolyzes several linear esters, and is useful for enantioselective hydrolysis of methyl DL-β-acetylthioisobutyrate and regioselective hydrolysis of methyl cetraxate. DCH in vivo detoxified peroxoacids in conjunction with the catalase.