Cloning, Overexpression, and Characterization of a High Enantioselective Nitrilase from Sphingomonas wittichii RW1 for Asymmetric Synthesis of (R)-Phenylglycine
Cloning, Overexpression, and Characterization of a High Enantioselective Nitrilase from Sphingomonas wittichii RW1 for Asymmetric Synthesis of (R)-Phenylglycine
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DOI：
10.1007/s12010-014-0845-y
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发表时间：
2014-05-01
影响因子：
3
通讯作者：
Wei, Dong-Zhi
中科院分区：
文献类型：
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作者：
Qiu, Jian;Su, Er-Zheng;Wei, Dong-Zhi
本研究从鞘氨醇单胞菌（Sphingomonaswittichii）RW 1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BL 21（DE 3）中进行了高效表达。重组腈水解酶经纯化至均一，分子量为40 kDa。pH和温度的最佳值分别为pH 8.0和40 A ℃。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E.以大肠杆菌BL 21/ReSWRW 1全细胞为催化剂，在pH6.0的条件下，对（R）-苯甘氨酸的不对称合成进行了动力学拆分。产率为46%，对映体过量（ee）为95%，这使其成为合成（R）-苯基甘氨酸的有前途的生物催化剂。
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 A degrees 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.