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A NADH-accepting imine reductase variant: Immobilization and cofactor regeneration by oxidative deamination

Paper ID Volume ID Publish Year Pages File Format Full-Text
22646 43368 2016 8 PDF Available
Title
A NADH-accepting imine reductase variant: Immobilization and cofactor regeneration by oxidative deamination
Abstract

•The Streptomyces sp. GF3587 imine reductase variant K40A accepts NADH as cofactor.•Alternative cofactors (BNAH, F420) are not accepted by investigated IREDs.•Immobilization on EziG™ carriers facilitates reusability of the catalyst.•The K40A variant enabled 88% conversion of 4% (w/v) 2-methylpyrroline to 2-methylpyrolidine.•NADPH regeneration by oxidative deamination of N-methyl-3-aminopentane could be demonstrated.

Engineering cofactor specificity of enzymes is a promising approach that can expand the application of enzymes for biocatalytic production of industrially relevant chemicals. Until now, only NADPH-dependent imine reductases (IREDs) are known. This limits their applications to reactions employing whole cells as a cost-efficient cofactor regeneration system. For applications of IREDs as cell-free catalysts, (i) we created an IRED variant showing an improved activity for NADH. With rational design we were able to identify four residues in the (R)-selective IRED from Streptomyces GF3587 (IR-Sgf3587), which coordinate the 2′-phosphate moiety of the NADPH cofactor. From a set of 15 variants, the highest NADH activity was caused by the single amino acid exchange K40A resulting in a 3-fold increased acceptance of NADH. (ii) We showed its applicability using an immobilisate obtained either from purified enzyme or from lysate using the EziG™ carriers. Applying the variant and NADH, we reached 88% conversion in a preparative scale biotransformation when employing 4% (w/v) 2-methylpyrroline. (iii) We demonstrated a one-enzyme cofactor regeneration approach using the achiral amine N-methyl-3-aminopentanone as a hydrogen donor co-substrate.

Keywords
Imine reductase; Cofactor specificity; Rational design; Immobilization; Preparative scale biocatalysis; Coupled oxidative deamination
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A NADH-accepting imine reductase variant: Immobilization and cofactor regeneration by oxidative deamination
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 230, 20 July 2016, Pages 11–18
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us