Abstract Bioreduction catalyzed by alcohol dehydrogenase/reductase is one of the most valuable biotransformation processes widely used in industry. The (S)-2-Chloro-1-(3, 4-difluorophenyl) ethanol is a key chiral synthon for synthesizing the antithrombotic agent ticagrelor. Herein, a new alcohol dehydrogenase (named Rhky-ADH) identified from Rhodococcus kyotonensis by an enzyme promiscuity-based genome mining method was successfully cloned and functionally expressed in Escherichia coli. The whole cell biocatalyst harboring Rhky-ADH was biochemically characterized and was shown to be able to convert 2-Chloro-1-(3, 4-difluorophenyl) ethanone to (S)-2-Chloro-1-(3, 4-difluorophenyl) ethanol with more than 99 % enantiomeric excess (ee) and 99 % conversion. Our data showed that the optimum temperature and pH for Rhky-ADH were 25 °C and pH 8.0, respectively. The addition of NADH and an appropriate concentration of isopropanol enhanced the activity of Rhky-ADH, and 1 mM Mn2+ increased the enzyme activity by about 8 %. Substrate specificity experiments showed that Rhky-ADH had notable enzyme promiscuity and could reduce several ketones with high stereoselectivity. Our investigation on this novel enzyme adds another rare biocatalyst to the toolbox for producing chiral alcohols, which are widely used in the pharmaceutical industry.

中文翻译：

---

使用来自京都红球菌的新型乙醇脱氢酶立体选择性合成替格瑞洛及其多种类似物的关键中间体

摘要 乙醇脱氢酶/还原酶催化的生物还原是工业上广泛应用的最有价值的生物转化过程之一。(S)-2-Chloro-1-(3, 4-difluorophenyl) 乙醇是合成抗血栓剂替格瑞洛的关键手性合成子。在此，通过基于酶混杂的基因组挖掘方法从京都红球菌中鉴定出的一种新的乙醇脱氢酶（命名为 Rhky-ADH）被成功克隆并在大肠杆菌中进行了功能表达。含有 Rhky-ADH 的全细胞生物催化剂的生化特征表明能够将 2-Chloro-1-(3, 4-difluorophenyl) 乙酮转化为 (S)-2-Chloro-1-(3, 4-difluorophenyl) ) 乙醇对映体过量 (ee) 超过 99%，转化率为 99%。我们的数据表明 Rhky-ADH 的最适温度和 pH 值为 25 °C，pH 值为 8.0，分别。添加NADH和适当浓度的异丙醇可增强Rhky-ADH的活性，1mM Mn2+使酶活性提高约8%。底物特异性实验表明，Rhky-ADH 具有显着的酶混杂性，可以以高立体选择性还原几种酮。我们对这种新型酶的研究为生产广泛用于制药行业的手性醇的工具箱增加了另一种罕见的生物催化剂。