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Cloning, Expression and Characterization of a Highly Active Alcohol Dehydrogenase for Production of Ethyl (S)-4-Chloro-3-Hydroxybutyrate.
Indian Journal of Microbiology ( IF 2.1 ) Pub Date : 2019-03-18 , DOI: 10.1007/s12088-019-00795-0 Yi-Hao Zhu 1 , Cai-Yun Liu 1 , Sha Cai 1 , Li-Bin Guo 1 , In-Won Kim 2 , Vipin C Kalia 2 , Jung-Kul Lee 2 , Ye-Wang Zhang 1
Indian Journal of Microbiology ( IF 2.1 ) Pub Date : 2019-03-18 , DOI: 10.1007/s12088-019-00795-0 Yi-Hao Zhu 1 , Cai-Yun Liu 1 , Sha Cai 1 , Li-Bin Guo 1 , In-Won Kim 2 , Vipin C Kalia 2 , Jung-Kul Lee 2 , Ye-Wang Zhang 1
Affiliation
A novel alcohol dehydrogenase from Bartonella apis (BaADH) was heterologous expressed in Escherichia coli. Its biochemical properties were investigated and used to catalyze the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is a chiral intermediate of the cholesterol-lowering drug atorvastatin. The purified recombinant BaADH displayed 182.4 U/mg of the specific activity using ethyl 4-chloroacetoacetate as substrate under the conditions of 50 °C in pH 7.0 Tris–HCl buffer. It was stable in storage buffers of pH 7 to 9 and retains up to 96.7% of the initial activity after 24 h. The Km and Vmax values of BaADH were 0.11 mM and 190.4 μmol min−1 mg−1, respectively. Synthesis of (S)-CHBE catalyzed by BaADH was performed with a cofactor regeneration system using a glucose dehydrogenase, and a conversion of 94.9% can be achieved after 1 h reaction. Homology modeling and substrate docking revealed that a typical catalytic triad is in contact with local water molecules to form a catalytic system. The results indicated this ADH could contribute to the further enzymatic synthesis of (S)-CHBE.
中文翻译:
用于生产(S)-4-氯-3-羟丁酸乙酯的高活性酒精脱氢酶的克隆,表达和表征。
一种来自巴氏杆菌的新型醇脱氢酶(BaADH)在大肠杆菌中异源表达。研究了其生物化学性质,并将其用于催化(S)-4-氯-3-羟基丁酸乙酯((S)-CHBE)的合成,该乙酯是降胆固醇药物阿托伐他汀的手性中间体。以4-氯乙酰乙酸乙酯为底物,在pH 7.0 Tris-HCl缓冲液中于50°C条件下,纯化的重组BaADH表现出182.4 U / mg的比活。它在pH 7至9的存储缓冲液中稳定,并在24小时后保留高达96.7%的初始活性。BaADH的K m和V max值为0.11 mM和190.4μmolmin -1 mg -1, 分别。BaADH催化的(S)-CHBE的合成是通过使用葡萄糖脱氢酶的辅因子再生系统进行的,反应1小时后的转化率为94.9%。同源性建模和底物对接显示典型的催化三联体与局部水分子接触形成催化系统。结果表明该ADH可能有助于(S)-CHBE的进一步酶促合成。
更新日期:2019-03-18
中文翻译:
![](https://scdn.x-mol.com/jcss/images/paperTranslation.png)
用于生产(S)-4-氯-3-羟丁酸乙酯的高活性酒精脱氢酶的克隆,表达和表征。
一种来自巴氏杆菌的新型醇脱氢酶(BaADH)在大肠杆菌中异源表达。研究了其生物化学性质,并将其用于催化(S)-4-氯-3-羟基丁酸乙酯((S)-CHBE)的合成,该乙酯是降胆固醇药物阿托伐他汀的手性中间体。以4-氯乙酰乙酸乙酯为底物,在pH 7.0 Tris-HCl缓冲液中于50°C条件下,纯化的重组BaADH表现出182.4 U / mg的比活。它在pH 7至9的存储缓冲液中稳定,并在24小时后保留高达96.7%的初始活性。BaADH的K m和V max值为0.11 mM和190.4μmolmin -1 mg -1, 分别。BaADH催化的(S)-CHBE的合成是通过使用葡萄糖脱氢酶的辅因子再生系统进行的,反应1小时后的转化率为94.9%。同源性建模和底物对接显示典型的催化三联体与局部水分子接触形成催化系统。结果表明该ADH可能有助于(S)-CHBE的进一步酶促合成。