A Versatile Disulfide-Driven Recycling System for NADP+ with High Cofactor Turnover Number

NADP(+)-dependent enzymes are important in many biocatalytic processes to generate high-value chemicals for the pharmaceutical and food industry; hence, a cost-effective, efficient, and environmentally friendly recycling system for the relatively expensive and only marginally stable enzyme cofactor NADP(+) offers significant benefits. NADP(+) regeneration schemes have previously been described, but their application is severely limited by the low total turnover numbers (TTN) for the cofactor. Here, we report a glutathione-based recycling system that combines glutaredoxin from E. coli (EcGRX) and the glutathione reductase from S. cerevisiae (ScGR) for NADP(+) regeneration. This system employs inexpensive latent organic disulfides such as oxidized cysteine or 2-hydroxyethyl disulfide (HED) as oxidizing agents and allows NADP(+) recycling under both aerobic and anaerobic conditions with a TTN in excess of 5 x 10(5), indicating that each regeneration cycle is 99.9998% selective toward forming the cofactor. Accordingly, for each 1 mol of product generated, less than $0.05 of cofactor is needed. Finally, the EcGRX/ScGR pair is compatible with eight enzymes in the guanosine monophosphate (GMP) biosynthetic pathway, giving the corresponding isotopically labeled nucleotide in high yield. The glutathione-based NADP(+) recycling system has potential for biocatalytic applications in academic and industrial settings.