Abstract
Objectives
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.
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Acknowledgments
This project is supported by the National Natural Science Foundation of China (31300661) and the High Technology Research and Development Program of China (863 Program) (2012AA06A203).
Supporting Information
Supplementary Table 1—Primers used in this study.
Supplementary Table 2—Characterization of wild-type and mutant ESHs.
Supplementary Fig. 1—Summary of the strategy for directed evolution of ESH.
Supplementary Fig. 2—SDS-PAGE of purified ESH and mutant ESHs.
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Zhang, C., Pan, H., Yao, L. et al. Single point mutations enhance activity of cis-epoxysuccinate hydrolase. Biotechnol Lett 38, 1301–1306 (2016). https://doi.org/10.1007/s10529-016-2078-3
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DOI: https://doi.org/10.1007/s10529-016-2078-3