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Issue 8, 2019
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Engineering of a keto acid reductase through reconstructing the substrate binding pocket to improve its activity

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Abstract

(R)-2-Hydroxy-4-phenylbutyric acid ((R)-HPBA) is a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. Asymmetric reduction of 2-oxo-4-phenylbutyric acid (OPBA) by keto acid reductases represents a promising approach for (R)-HPBA production due to its high atom economy and theoretical yield of up to 100%. However, the low activity of keto acid reductases restricts their industrial application. In this study, it is the first time that a keto acid reductase from Leuconostoc lactis (LlKAR) with superior activity was designed for the reduction of OPBA to (R)-HPBA through reconstruction of the substrate binding pocket. Several residues (Leu127, Thr129, Leu244, Leu245, and Ala250) were selected for mutagenesis. Through three-round mutagenesis, multiple positive mutants were picked out. Compared with wild-type LlKAR, the mutant L244G/A250G/L245R exhibited 3.5-fold improvement in specific activity and 6.80-fold improvement in catalytic efficiency (Kcat/Km). Using L244G/A250G/L245R coupled with Exiguobacterium sibiricum glucose dehydrogenase (GDH) for cofactor regeneration, the productivity of (R)-HPBA was up to 85.7 mM h−1 with >99% enantiomeric excess in the reaction system. The engineered L244G/A250G/L245R represents a potential and competitive biocatalyst for practical application in the synthesis of (R)-HPBA.

Graphical abstract: Engineering of a keto acid reductase through reconstructing the substrate binding pocket to improve its activity

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Article information


Submitted
23 Dec 2018
Accepted
18 Mar 2019
First published
19 Mar 2019

Catal. Sci. Technol., 2019,9, 1961-1969
Article type
Paper

Engineering of a keto acid reductase through reconstructing the substrate binding pocket to improve its activity

D. Wang, H. Li, S. Xia, Y. Xue and Y. Zheng, Catal. Sci. Technol., 2019, 9, 1961
DOI: 10.1039/C8CY02586J

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