来源于Bacillus sp.AR9 D-海因酶半理性设计的研究  

The Semi-rational Designing of D-hydantoinase from Bacillus sp.AR9

作　　者：樊帅[1] 刘坤 金媛媛[1] 杨兆勇[1] FAN Shuai;LIU Kun;JIN Yuanyuan;YANG Zhaoyong(Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050,China)

机构地区：[1]中国医学科学院医药生物技术研究所,北京100050

出　　处：《生物技术进展》2021年第3期386-392,共7页Current Biotechnology

基　　金：国家自然科学基金项目(81761128016,8187131584)

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K_(m)值基本与野生型HYD相似,而k_(cat)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k _(cat)/K_(m)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。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 K_(m) 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 k cat compared with HYD.Specifically,enhanced catalytic efficiencies(k_(cat)/K_(m))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.

关 键 词：D-海因酶 半理性设计 饱和突变 酶动力学 

分 类 号：Q814[生物学—生物工程]