Qiu Jian, Su Er-Zheng, Wang Hua-Lei, Cai Wen-Wen, Wang Wei, Wei Dong-Zhi
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
Appl Biochem Biotechnol. 2014 May;173(2):365-77. doi: 10.1007/s12010-014-0845-y. Epub 2014 Mar 25.
In this study, a high (R)-enantioselective nitrilase gene from Sphingomonas wittichii RW1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The recombinant nitrilase was purified to homogeneity with a molecular weight of 40 kDa. The pH and temperature optima were shown to be pH 8.0 and 40 °C, respectively. The purified nitrilase was most active toward succinonitrile, approximately 30-fold higher than that for phenylglycinonitrile. Using the E. coli BL21/ReSWRW1 whole cells as biocatalysts, the kinetic resolution for asymmetric synthesis of (R)-phenylglycine was investigated at pH 6.0. A yield of 46 % was obtained with 95 % enantiomeric excess (ee), which made it a promising biocatalyst for synthesis of (R)-phenylglycine.
在本研究中，从维氏鞘氨醇单胞菌RW1中克隆了一个高（R）-对映选择性腈水解酶基因，并在大肠杆菌BL21（DE3）中进行了过表达。重组腈水解酶被纯化至同质，分子量为40 kDa。结果表明，最适pH和温度分别为pH 8.0和40℃。纯化的腈水解酶对丁二腈的活性最高，比对苯基甘氨腈的活性高约30倍。使用大肠杆菌BL21/ReSWRW1全细胞作为生物催化剂，在pH 6.0下研究了不对称合成（R）-苯基甘氨酸的动力学拆分。获得了46%的产率和95%的对映体过量（ee），这使其成为合成（R）-苯基甘氨酸的一种有前景的生物催化剂。
