来源于Bacillus sp. AR9 D-海因酶半理性设计的研究 |
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作者姓名: | 樊帅 刘坤 金媛媛 杨兆勇 |
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作者单位: | 中国医学科学院医药生物技术研究所, 北京 100050 |
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基金项目: | 国家自然科学基金项目(81761128016;8187131584)。 |
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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。
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关 键 词: | D-海因酶 半理性设计 饱和突变 酶动力学 |
收稿时间: | 2021-04-03 |
The Semi-rational Designing of D-hydantoinase from Bacillus sp. AR9 |
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Authors: | FAN Shuai LIU Kun JIN Yuanyuan YANG Zhaoyong |
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Institution: | Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China |
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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. |
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Keywords: | D-hydantoinase semi-rational design site-directed saturation mutagenesis enzyme kinetics |
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