Dataset Information

Enzymatic characterization of a recombinant carbonyl reductase from Acetobacter sp. CCTCC M209061.

ABSTRACT:

Background

Acetobacter sp. CCTCC M209061 could catalyze carbonyl compounds to chiral alcohols following anti-Prelog rule with excellent enantioselectivity. Therefore, the enzymatic characterization of carbonyl reductase (CR) from Acetobacter sp. CCTCC M209061 needs to be investigated.

Results

A CR from Acetobacter sp. CCTCC M209061 (AcCR) was cloned and expressed in E. coli. AcCR was purified and characterized, finding that AcCR as a dual coenzyme-dependent short-chain dehydrogenase/reductase (SDR) was more preferred to NADH for biocatalytic reactions. The AcCR was activated and stable when the temperature was under 35 °C and the pH range was from 6.0 to 8.0 for the reduction of 4'-chloroacetophenone with NADH as coenzyme, and the optimal temperature and pH were 45 °C and 8.5, respectively, for the oxidation reaction of isopropanol with NAD⁺. The enzyme showed moderate thermostability with half-lives of 25.75 h at 35 °C and 13.93 h at 45 °C, respectively. Moreover, the AcCR has broad substrate specificity to a range of ketones and ketoesters, and could catalyze to produce chiral alcohol with e.e. >99% for the majority of tested substrates following the anti-Prelog rule.

Conclusions

The recombinant AcCR exhibited excellent enantioselectivity, broad substrate spectrum, and highly stereoselective anti-Prelog reduction of prochiral ketones. These results suggest that AcCR is a powerful catalyst for the production of anti-Prelog alcohols.Graphical abstractThe biocatalytic reactions conducted with the recombinant AcCR.

SUBMITTER: Wei P

PROVIDER: S-EPMC5573764 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Enzymatic characterization of a recombinant carbonyl reductase from <i>Acetobacter</i> sp. CCTCC M209061.

Wei Ping P Cui Yu-Han YH Zong Min-Hua MH Xu Pei P Zhou Jian J Lou Wen-Yong WY

Bioresources and bioprocessing 20170828 1

<h4>Background</h4><i>Acetobacter</i> sp. CCTCC M209061 could catalyze carbonyl compounds to chiral alcohols following anti-Prelog rule with excellent enantioselectivity. Therefore, the enzymatic characterization of carbonyl reductase (CR) from <i>Acetobacter</i> sp. CCTCC M209061 needs to be investigated.<h4>Results</h4>A CR from <i>Acetobacter</i> sp. CCTCC M209061 (AcCR) was cloned and expressed in <i>E. coli</i>. AcCR was purified and characterized, finding that AcCR as a dual coenzyme-depen ...[more]

PMID: 28913159

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Project description:BackgroundCandida parapsilosis (R)-carbonyl reductase (RCR) and (S)-carbonyl reductase (SCR) are involved in the stereoconversion of racemic (R,S)-1-phenyl-1,2-ethanediol (PED) to its (S)-isomer. RCR catalyzes (R)-PED to 2-hydroxyacetophenone (2-HAP), and SCR catalyzes 2-HAP to (S)-PED. However, the stereoconversion efficiency of racemic mixture to (S)-PED is not high because of an activity imbalance between RCR and SCR, with RCR performing at a lower rate than SCR. To realize the efficient preparation of racemic mixture to (S)-PED, an in situ expression of RCR and a two-stage control strategy were introduced to rebalance the RCR- and SCR-mediated pathways.ResultsAn in situ expression plasmid pCP was designed and RCR was successfully expressed in C. parapsilosis. With respect to wild-type, recombinant C. parapsilosis/pCP-RCR exhibited over four-fold higher activity for catalyzing racemic (R,S)-PED to 2-HAP, while maintained the activity for catalyzing 2-HAP to (S)-PED. The ratio of k cat /K M for SCR catalyzing (R)-PED and RCR catalyzing 2-HAP was about 1.0, showing the good balance between the functions of SCR and RCR. Based on pH and temperature preferences of RCR and SCR, a two-stage control strategy was devised, where pH and temperature were initially set at 5.0 and 30 °C for RCR rapidly catalyzing racemic PED to 2-HAP, and then adjusted to 4.5 and 35 °C for SCR transforming 2-HAP to (S)-PED. Using these strategies, the recombinant C. parapsilosis/pCP-RCR catalyzed racemic PED to its (S)-isomer with an optical purity of 98.8 % and a yield of 48.4 %. Most notably, the biotransformation duration was reduced from 48 to 13 h.ConclusionsWe established an in situ expression system for RCR in C. parapsilosis to rebalance the functions between RCR and SCR. Then we designed a two-stage control strategy based on pH and temperature preferences of RCR and SCR, better rebalancing RCR and SCR-mediated chiral biosynthesis pathways. This work demonstrates a method to improve chiral biosyntheses via in situ expression of rate-limiting enzyme and a multi-stage control strategy to rebalance asymmetric pathways.