Abstract
NADP-Dependent D-glucose dehydrogenase (EC 1.1.1.47) was crystallized for the first time from cytosol fraction of Gluconobacter suboxydans IFO 12528. Purification of the enzyme was successfully performed by column chromatography on DEAE-Sephadex A-50 and affinity chromatography by blue-dextran Sepharose 4B. The enzyme was purified about 1, 800-fold with an overall yield of 30%. Crystalline enzyme preparation was homogeneous in disc gel electrophoresis and analytical ultracentrifugation. The enzyme was highly specific for NADP and completely inactive with NAD. NADPH yielded in D-glucose oxidation to D-glucono-δ-lactone was reoxidized to NADP by the old yellow enzyme which existed in the same cytosol fraction of the organism. Cyclic regeneration of NADP occurred smoothly in the presence of D-glucose dehydrogenase, old yellow enzyme and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.
