关键词: D-海因酶, 半理性设计, 饱和突变, 酶动力学

Abstract: D-4-hydroxyphenylglycine is a high-value chemical intermediate and has a wide range of applications in the pharmaceutical industries. The enzymatic method is the main way for the production of D-4-hydroxyphenylglycine, but the production of D-4-hydroxyphenylglycine is limited due to the lack of enzymes with high catalytic efficiency. In order to improve the catalytic efficiency of D-hydantoinase (HYD) from Bacillus sp. AR9 and the production of D-4-hydroxyphenylglycine, the analysis of substrate tunnel and site-directed saturation mutagenesis were performed. The activity of variants F159S, F159A and F65V were increased by 51%, 40% and 17% compared to the HYD, respectively. The enzyme kinetics of HYD and mutants illustrated that the Km of mutants (F65V, F159S and F65V/F159S) were similar for HYD. However, F65V, F159S and F65V/F159S showed the 1.3-, 1.9- and 2.0-fold increase in kcat compared with HYD. Specifically, enhanced catalytic efficiencies (kcat/Km) was achieved by the F65V/F159S, representing 2.4 times higher catalytic efficiency than that of HYD. The obtained high-catalytic efficiency mutants and the analysis of mutant kinetics were of important research significance and application value for the biotransformation of D-4-hydroxyphenylglycine.

Key words: D-hydantoinase, semi-rational design, site-directed saturation mutagenesis, enzyme kinetics