Gene mining-based identification of aldo\u2013keto reductases for highly stereoselective reduction of bulky ketones

Yan Xu,Yao Nie,Chen Liang,Xiaoqing Mu

doi:10.1186/s40643-018-0220-x

Yan Xu, Yao Nie + Show 2 more

Open Access

https://doi.org/10.1186/s40643-018-0220-x

Copy DOI

Abstract

BackgroundAldo–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.ResultsFive 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 Km 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.ConclusionThe stereospecific AKRs were obtained through gene mining, which possesses the potential for application in the preparation of important optically active alcohols.

Highlights

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
Aldo–keto reductase (AKR) or alcohol dehydrogenase (ADH)-mediated stereoselective reduction of prochiral carbonyl compounds is an efficient way of preparing single enantiomers of chiral alcohols due to the high chemo, enantio, and regioselectivity of the enzymes (Ma et al 2013)
The gene mining approach was adopted to find potential enzymes that can catalyze the reduction of bulky ketones using the amino acid sequence of CaAKR as high-performance liquid chromatography (HPLC) condition (R,S)-1b (R,S)-2b (R,S)-3b (R,S)-4b (R,S)-5b

Summary

Introduction

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. 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. Aldo–keto reductase (AKR) or alcohol dehydrogenase (ADH)-mediated stereoselective reduction of prochiral carbonyl compounds is an efficient way of preparing single enantiomers of chiral alcohols due to the high chemo-, enantio-, and regioselectivity of the enzymes (Ma et al 2013). The majority of prochiral ketones used in industrial applications are structurally complex.

Methods

Results

Conclusion