Cofactor Regeneration Using Permeabilized Expressing NAD(P)-Dependent Glycerol-3-Phosphate Dehydrogenase.

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized cells in which the and genes were deleted and the gene, which encodes NAD(P)-dependent glycerol-3-phosphate dehydrogenase, was overexpressed. These manipulations were applied to block a side reaction (, the conversion of glycerol to dihydroxyacetone) and to switch the -encoding enzyme reaction to a -encoding enzyme reaction that generates both NADH and NADPH. We demonstrated the performance of the cofactor regeneration system using a lactate dehydrogenase reaction as a coupling reaction model. The developed biocatalyst involves an economical substrate, bifunctional regeneration of NAD(P)H, and simple reaction conditions as well as a stable environment for enzymes, and is thus applicable to a variety of oxidoreductase reactions requiring NAD(P)H regeneration.