Enzymatic regeneration of NAD in enantioselective oxidation of secondary alcohols: Glutamate dehydrogenase versus NADH dehydrogenase

To improve yield and productivity of ketose in NAD-dependent polyol oxidati ons, two enzymatic methods for regeneration of the oxidized coenzyme form h ave been compared and partly optimized for the batch conversion of xylitol into D-xylulose and D-sorbitol into D-fructose. Polyol oxidation was cataly zed by xylitol dehydrogenase from the least Galactocandida mastotermitis. R eduction of O-2 (apparently to H2O) by partially purified NADH dehydrogenas e complex from Corynebacterium callunae could drive alcohol oxidations bett er than reductive amination of alpha-ketoglutarate by glutamate dehydrogena se. A fed-batch procedure was developed that overcame inhibition of glutama te dehydrogenase by a-ketoglutarate (K-is 25 mM). thus increasing the produ ctivity of ketose almost 2-fold, For D-fructuse production from osorbitol ( 0.1-0.3 M) yields of > 90% and productivities up to 1.30 g/(L.h) have been obtained. High conversion of up to 50 g/L xylitol into D-xylulose for which xylitol dehydrogenase exhibits an about 60-fold higher specificity constan t than for D-fructose required complexation of the ketose product with bora te. In comparison with reductive amination by glutamate dehydrogenase, adva ntages of using NADH-dehydrogenase catalyzed regeneration of NAD for ketose production are (i) avoidance of byproduct formation, (ii) cheaper substrat e (O-2 versus alpha-ketoglutarate), and (iii) easier process control (batch versus fed-batch).