Gene mining-based identification of aldo–keto reductases for highly stereoselective reduction of bulky ketones

Abstract

Background: Aldo-keto reductase (AKR) or alcohol dehydrogenases (ADH)-mediated stereoselective reduction of prochiral carbonyl compounds is an efficient way of preparing single enantiomers of chiral alcohols. However, steric hindrance of substrate affects the catalytic performance of enzymes. The present study aims to discover and identify AKRs/ADHs capable of catalyzing highly stereoselective reduction of sterically hindered ketones. Results:Five AKRs from different microorganisms (CaCR, ScCR, KmCR, CPR-C1, and CPR-C2) were identified through gene mining, and overexpressed in recombinant Escherichia coli BL21(DE3). The specific activity and stereoselectivity of the AKRs were further evaluated towards various ketoesters and heterocyclic ketones, which are sterically bulky and are valuable in industrial applications. Each purified enzyme exhibited catalytic activity to one or more of the tested substrates. Among the enzymes, ScCR showed a broader substrate spectrum compared to the others. Regarding K m values related to substrate association, we also provided insights into the specificity and preference of certain enzymes. Consequently, enantiopure (R)-methyl mandelate, ethyl (R)-mandelate, ethyl (R)-2-hydroxy-4-phenylbutyrate, and (S)-N-benzyl-3-pyrrolidinol (> 99%e.e.) were obtained through the identified AKRs. Conclusion:The stereospecific AKRs were obtained through gene mining, which possesses the potential for application in the preparation of important optically active alcohols.

Stereoselective carbonyl reductases and their roles in asymmetric synthesis of bulky chiral hydroxyl compounds

“…向进化技术是又一种提高酶催化活性、拓宽底物专一 性、强化立体选择性的有效途径和手段 [54] . Gong等 [56] ; 基于底物广谱性表征, 从序列-结构-功 能等角度系统比较和分析底物分子结构与选择性之间 的关系, 解析羰基还原酶选择性识别大基团底物及其 催化不对称还原的分子机制 [22] ; 基于序列-结构-功能 三者之间的内在关系和底物结构对酶催化性能的影响 规律及酶结构功能的共进化关系, 结合酶蛋白质分子 从头设计方法(de novo design), 建立调控选择性识别 和催化不同基团结构底物的精准分子设计策略 [57] , 从 而进一步拓展和强化立体选择性羰基还原酶的催化性 能和工业应用属性. Chiral hydroxyl compounds have been widely used in pharmaceutical, agrochemical, fine chemicals, and functional materials industries, due to their unique physical and chemical properties.…”

Multi-Enzymatic Cascade for Efficient Deracemization of dl-Pantolactone into d-Pantolactone

“…Specifically, known l- pantolactone dehydrogenases are membrane-bound enzymes, and ascertaining their heterologous expression remains challenging [ 13 , 18 ]. Mining novel enzymes or engineering known enzymes is essential to meet the requirements of activity and enantioselectivity for the efficient deracemization of dl -pantolactone [ 19 , 20 ].…”

Chemo-catalytic synthesis of biomass-derived furanyl diethers: green and renewable bio-diesel components

“…49 The used alcohol solvents have a huge impact on transfer hydrogenation and etherication reactions owing to their different reduction potentials and steric hindrance. [50][51][52] BAMFs produced by ether-ication with different alcohols have different combustion performances and blending ratios from ordinary commercial diesels, although all of them were regarded as biofuels or additives. Therefore, it is necessary to discuss the physicochemical properties of various BAMFs and their synthesized systems in detail, respectively.…”