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

• 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.