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

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 (BmGDH_M0 ) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDH_M6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDH_M6 was also more stable than BmGDH_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, BmGDH_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.