Enzymatic vs. Fermentative Synthesis: Thermostable Glucose Dehydrogenase Catalyzed Regeneration of NAD(P)H for use in Enzymatic Synthesis

Article abstract of DOI:10.1021/ja00299a044

Procedures are described for regeneration of reduced nicotinamide cofactors NADH and NADPH from NAD(P) based on glucose and a thermostable glucose dehydrogenase from Bacillus cereus immobilized in polyacrylamide gels.The turnover number for NAD in a synthesis of 200 mmol of D-lactate is 40000.Application of this system to other syntheses is demonstrated with preparations of ethyl (R)-4-chloro-3-hydroxybutanoate, (R)-2,2,2-trifluoro-1-phenylethanol, ethyl (S)-3-hydroxyvalerate, (S)-lactaldehyde dimethyl acetal, and (S)-3-hydroxybutanal dimethyl acetal.Further investigation of the kinetics regarding the thermoresistance of glucose dehydrogenase in the presence of NaCl has been carried out, and it appears that the enhancement by NaCl of the thermal stability of the enzyme is approximately third order.The immobilized glucose dehydrogenase incubated at 55 deg C, pH 7.5, for 7 days is still fully active while many other enzymes are completely inactivated in 1-2 days.Addition of NaCl enhances the thermal stability more significantly than the immobilization does, and a remarkable increase in thermal stability was observed with these two combined factors.The half-life of the immobilized glucose dehydrogenase at 55 deg C in a buffer (pH 7.0-7.5) containing 1 M NaCl is more than 30 days compared to 3 min for the free enzyme, corresponding to an overall ca. 50000-fold increase in thermal stability.