Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents

Abstract Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH ( Bm GDH M0 ) in the presence of large amounts of 1‐phenylethanol by directed evolution. Among the resulting mutants, Bm GDH M6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2‐fold increase in tolerance against 10 % ( v / v ) 1‐phenylethanol. Moreover, Bm GDH M6 was also more stable than Bm GDH M0 when exposed to hydrophobic and enzyme‐inactivating compounds such as acetophenone, ethyl 2‐oxo‐4‐phenylbutyrate, and ethyl ( R )‐2‐hydroxy‐4‐phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, Bm GDH M6 was successfully used for the asymmetric reduction of deactivating ethyl 2‐oxo‐4‐phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.