Improving catalytic performance of an arylacetonitrilase by semirational engineering
Ya-Ping Xue1,2, Biao Jiao1,2, Deng-En Hua1,2
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China.
Abstract:
Arylacetonitrilases have been widely acknowledged as important alternatives to chemical catalysts for synthesizing optically pure 2-hydroxyphenylacetic acids from nitriles. In this work, two residues (Thr132 and Ser190) located at the catalytic tunnel in the active site of an arylacetonitrilase nitA from uncultured organisms were mutated separately by site-directed mutagenesis. Ser190 was demonstrated to be the critical position which has a greater influence on arylacetonitrilase nitA activity than Thr132. The replacement of serine at position 190 with glycine increases its activity toward mandelonitrile and (o, m, p)-chloromandelonitrile, whereas replacing it with leucine abolished its activity. The best mutant S190G exhibited threefold higher specific activity toward mandelonitrile compared with that of wild-type nitA, which rendered it promising for industrial application. Homology modeling and molecular docking experiments were in agreement with the kinetic assays and support the improved catalytic performance.
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