Single point mutations enhance activity of cis-epoxysuccinate hydrolase.
OBJECTIVE
To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.


RESULTS
By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg(-1) (k cat /K m , 9.8 ± 0.1 mM(-1) s(-1)), which was 230 % higher than that of wild-type enzyme 355 U mg(-1) (k cat /K m , 5.3 ± 0.1 mM(-1) s(-1)). However, the thermostability of the mutant F10Q slightly decreased.


CONCLUSIONS
The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.
Institute of Biochemistry, Zhejiang University, Hangzhou, 310058, Zhejiang, China.
Changling
C
Zhang
Institute of Biochemistry, Zhejiang University, Hangzhou, 310058, Zhejiang, China.|Hangzhou Bioking Biochemical Engineering Co., Ltd., Hangzhou, 311106, Zhejiang, China.
Haifeng
H
Pan
Institute of Biochemistry, Zhejiang University, Hangzhou, 310058, Zhejiang, China.
Lingli
L
Yao
Hangzhou Bioking Biochemical Engineering Co., Ltd., Hangzhou, 311106, Zhejiang, China.
Wenna
W
Bao
Hangzhou Bioking Biochemical Engineering Co., Ltd., Hangzhou, 311106, Zhejiang, China.
Jinxin
J
Wang
Institute of Biochemistry, Zhejiang University, Hangzhou, 310058, Zhejiang, China. xier1975@163.com.|Hangzhou Bioking Biochemical Engineering Co., Ltd., Hangzhou, 311106, Zhejiang, China. xier1975@163.com.
Zhipeng
Z
Xie
Institute of Biochemistry, Zhejiang University, Hangzhou, 310058, Zhejiang, China.|Hangzhou Bioking Biochemical Engineering Co., Ltd., Hangzhou, 311106, Zhejiang, China.
Jianguo
J
Zhang
D002384:Catalysis; D019020:Directed Molecular Evolution; D004789:Enzyme Activation / Q000235:genetics; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation / Q000235:genetics*
D006867:Hydrolases
Cis-epoxysuccinate hydrolase; Directed evolution; Error-prone PCR; L(+)-Tartrate; Site-directed mutagenesis