红球菌AJ270对映选择性腈水合酶的纯化及特性研究

Purification and characterization of the enantioselective nitrile hydratase from Rhodococcus sp. AJ270.

作者信息

Song Liya, Wang Meixiang, Yang Xiuqing, Qian Shijun

机构信息

State Key Laboratories of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

出版信息

Biotechnol J. 2007 Jun;2(6):717-24. doi: 10.1002/biot.200600215.

DOI:10.1002/biot.200600215

PMID:17330219

Abstract

Nitrile hydratase (NHase, EC 4.2.1.84) from Rhodococcus sp. AJ270 was purified with 23.96% yield after sonication, ammonium sulfate fractionation, ion exchange, hydrophobic and gel-filtration column chromatography. The enzyme showed intriguing characteristics: it hydrated not only aliphatic and heterocyclic nitriles but also aromatic ones. Some substrates were also hydrated enantioselectively to the corresponding amides. The enantiomeric excess (ee) value of the enzyme hydrating trans-2,2-dimethyl-3-phenylcyclopropanecarbonitrile was 84.7. The enzyme is composed of two subunits: an alpha subunit and beta subunit of 22 975 Da and 23 493 Da, respectively. The optimal temperature and pH for the catalytic reaction of the enzyme was 25 degrees C and pH 7.6. The enzyme activity of the purified NHase was strongly inhibited by some oxidizing agents and heavy metals.

摘要

来自红球菌属AJ270的腈水合酶（NHase，EC 4.2.1.84）经超声处理、硫酸铵分级分离、离子交换、疏水和凝胶过滤柱色谱法纯化后，产率为23.96%。该酶表现出有趣的特性：它不仅能使脂肪族和杂环腈水合，还能使芳香族腈水合。一些底物也能对映选择性地水合生成相应的酰胺。该酶使反式-2,2-二甲基-3-苯基环丙烷甲腈水合的对映体过量（ee）值为84.7。该酶由两个亚基组成：分别为22 975 Da和23 493 Da的α亚基和β亚基。该酶催化反应的最适温度和pH分别为25℃和pH 7.6。纯化的NHase的酶活性受到一些氧化剂和重金属的强烈抑制。