Woo Jung-Hee, Kang Ji-Hyun, Kang Sung Gyun, Hwang Young-Ok, Kim Sang-Jin
Marine Biotechnology Research Centre, Korea Ocean Research & Development Institute, P.O. Box 29, Ansan, 425-600, South Korea.
Appl Microbiol Biotechnol. 2009 Apr;82(5):873-81. doi: 10.1007/s00253-008-1791-9. Epub 2008 Dec 16.
A gene encoding a putative epoxide hydrolase (EHase) was identified by analyzing an open reading frame of the genome sequence of Novosphingobium aromaticivorans, retaining the conserved catalytic residues such as the catalytic triad (Asp177, Glu328, and His355) and the oxyanion hole. The enantioselective EHase gene (neh) was cloned, and the recombinant EHase could be purified to apparent homogeneity by one step of metal affinity chromatography and further characterized. The purified N. aromaticivorans enantioselective epoxide hydrolase (NEH) showed enantioselective hydrolysis toward styrene oxide, glycidyl phenyl ether, epoxybutane, and epichlorohydrin. The optimal EHase activity toward styrene oxide occurred at pH 6.5 and 45 degrees C. The purified NEH could preferentially hydrolyze (R)-styrene oxide with enantiomeric excess of more than 99% and 11.7% yield after 20-min incubation at an optimal condition. The enantioselective hydrolysis of styrene oxide was also confirmed by the analysis of the vicinal diol, 1-phenyl-1,2-ethanediol. The hydrolyzing rates of the purified NEH toward epoxide substrates were not affected by as high as 100 mM racemic styrene oxide.
通过分析食芳烃新鞘氨醇菌（Novosphingobium aromaticivorans）基因组序列的一个开放阅读框，鉴定出一个编码假定环氧化物水解酶（EHase）的基因，该基因保留了保守的催化残基，如催化三联体（Asp177、Glu328和His355）和氧阴离子洞。对映选择性环氧化物水解酶基因（neh）被克隆出来，重组环氧化物水解酶可通过一步金属亲和层析纯化至表观均一，并进一步进行表征。纯化后的食芳烃新鞘氨醇菌对映选择性环氧化物水解酶（NEH）对氧化苯乙烯、缩水甘油基苯基醚、环氧丁烷和环氧氯丙烷表现出对映选择性水解作用。对氧化苯乙烯的环氧化物水解酶最佳活性出现在pH值6.5和45℃条件下。纯化后的NEH在最佳条件下孵育20分钟后，可优先水解（R）-氧化苯乙烯，对映体过量率超过99%且产率达到11.7%。通过对邻二醇1-苯基-1,2-乙二醇的分析也证实了氧化苯乙烯的对映选择性水解。纯化后的NEH对环氧化物底物的水解速率不受高达100 mM外消旋氧化苯乙烯的影响。
