文献类型：期刊文献

中文题名：半理性设计提高短链醇脱氢酶在(S)-1-(4-氟苯基)乙醇合成中的应用

英文题名：Semi-rational design to enhance short-chain alcohol dehydrogenases in the synthesis of(S)-1-(4-fluorophenyl)ethanol

作者：谢静雯[1];孟仪方[1];叶文杰[1];王华磊[1];魏东芝[1]

机构：[1]华东理工大学生物工程学院,上海200237

年份：2025

卷号：44

期号：5

起止页码：2515

中文期刊名：化工进展

外文期刊名：Chemical Industry and Engineering Progress

收录：;EI(收录号：20252118474287);北大核心:【北大核心2023】；

基金：国家自然科学基金(22078097)。

语种：中文

中文关键词：(S)-1-(4-氟苯基)乙醇;半理性设计;醇脱氢酶;不对称还原;共表达

外文关键词：(S)-1-(4-fluorophenyl)ethanol;semi-rational design;alcohol dehydrogenase;asymmetric reduction;co-expression

摘要：(S)-1-(4-氟苯基)乙醇是一种重要的手性药物中间体,被广泛用于阿尔兹海默症治疗等多种药物的合成当中。目前,利用生物不对称还原法合成该手性醇时普遍存在制备效率低下的问题。为建立(S)-1-(4-氟苯基)乙醇酶法高效合成工艺,本研究对实验室包含323种醇脱氢酶的酶库进行筛选,得到了具备高立体选择性(e.e.=99.9%)和相对活性的短链醇脱氢酶Dp ADH,随后通过半理性设计经过多轮迭代突变成功获得了催化效率提高30.8倍的优良变体M3(N164C/S195W/F157A)。为进一步提高生物催化效率,构建了醇脱氢酶突变体M3与葡萄糖脱氢酶Bs GDH的共表达菌株,在100g/L底物投料下可于7h内实现99.6%的转化率。分子动力学模拟结果显示,M3变体增加了与底物间有利的π-π相互作用并稳定了底物结合构象。本研究为(S)-1-(4-氟苯基)乙醇的制备提供了经济高效的合成途径,并为醇脱氢酶Dp ADH的分子改造和机制解析提供了指导,提高了该酶的工业应用潜力。
(S)-1-(4-fluorophenyl)ethanol is an important chiral drug intermediate,which is widely used in the synthesis of various drugs such as Alzheimer’s disease therapeutics.Currently,preparation inefficiencies are prevalent in the synthesis of this chiral alcohol using bioasymmetric reduction.In order to establish an enzymatic and efficient synthesis process of(S)-1-(4-fluorophenyl)ethanol,this study screened a laboratory enzyme library containing 323 alcohol dehydrogenases,obtained DpADH,a short-chain alcohol dehydrogenase possessing high stereoselectivity(e.e.value of 99.9%)and relative activity,and further improved the catalytic performance of the enzyme by semi-rational design.The excellent variant M3(N164C/S195W/F157A)with a 30.8-fold increase in catalytic efficiency was successfully obtained using multiple rounds of iterative mutagenesis.To improve the efficiency of coenzyme delivery,a co-expression strain of alcohol dehydrogenase variant M3 with glucose dehydrogenase BsGDH was constructed,which showed 98.5%conversion of 4-fluoroacetophenone(100g/L)within 7h of reaction.Molecular dynamics simulations showed that the variant M3 increased the favourableπ-πinteractions with the substrate and stabilised the substrate-bound conformation.This study provides an economical and efficient synthetic route for(S)-1-(4-fluorophenyl)ethanol,and provides guidance for the molecular modification and mechanistic elucidation of DpADH,which enhances the potential of the enzyme for industrial applications.