Single point mutations enhance activity of cis-epoxysuccinate hydrolase.

Abstract

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate. 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 Umg(-1) (k cat /K m , 9.8±0.1mM(-1)s(-1)), which was 230% higher than that of wild-type enzyme 355Umg(-1) (k cat /K m , 5.3±0.1mM(-1)s(-1)). However, the thermostability of the mutant F10Q slightly decreased. 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.