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

摘要： D-对羟基苯甘氨酸是一种重要的精细化工品，在制药行业具有广泛的应用前景。酶法是生产D-对羟基苯甘氨酸的主要手段，但由于缺乏高催化效率的酶而限制了D-对羟基苯甘氨酸的生产。为了提高来自Bacillus sp. AR9的D-海因酶（HYD）的催化效率，进而提高D-对羟基苯甘氨酸的产量，对HYD的底物结合通道进行分析，选取底物通道瓶颈处的氨基酸进行饱和突变和筛选，以提高HYD的催化效率。结果显示，突变体F159S、F159A和F65V的活性相较于野生型HYD分别提高了51%、40%和17%，通过对突变体F65V、F159S和双位点突变F65V/F159S的酶动力学研究发现，突变体的Km值基本与野生型HYD相似，而kcat是野生型HYD的1.3、1.9和2.0倍，最终双位点突变F65V/F159S的催化效率kcat/Km是野生型HYD的2.4倍。高催化效率突变体的获得，以及对突变体动力学的分析，对酶法制备D-对羟基苯甘氨酸具有重要的研究意义和应用价值。

关键词: D-海因酶, 半理性设计, 饱和突变, 酶动力学