초록
<P>NADP<SUP>+</SUP>-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<SUP>+</SUP> offers significant benefits. NADP<SUP>+</SUP> 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 <I>E. col</I>i (EcGRX) and the glutathione reductase from <I>S. cerevisiae</I> (ScGR) for NADP<SUP>+</SUP> regeneration. This system employs inexpensive latent organic disulfides such as oxidized cysteine or 2-hydroxyethyl disulfide (HED) as oxidizing agents and allows NADP<SUP>+</SUP> recycling under both aerobic and anaerobic conditions with a TTN in excess of 5 × 10<SUP>5</SUP>, 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<SUP>+</SUP> recycling system has potential for biocatalytic applications in academic and industrial settings.</P><P><B>Graphic Abstract</B><BR><IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/accacs/2017/accacs.2017.7.issue-2/acscatal.6b03061/production/images/medium/cs-2016-03061z_0006.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/cs6b03061'>ACS Electronic Supporting Info</A></P>