Purification and characterization of D-sorbitol dehydrogenase from membrane of Gluconobacter suboxydans var. .ALPHA..

Abstract

D-Sorbitol dehydrogenase was solubilized from the membrane fraction of Gluconobacter suboxydans var. α IFO 3254 by a procedure involving solubilization of the enzyme with Triton X-100 in the presence of KCl and D-sorbitol. Purification of the enzyme was performed by fractionation with polyethylene glycol 6000, and column chromatographies on DEAE- and CM-cellulose in the presence of Triton X-100. The enzyme was purified about 240-fold from the membrane fraction of the organism. The purified enzyme was tightly bound to a c-type cytochrome existing as a dehydrogenase-cytochrome complex. The dehydrogenase was found to be a flavoprotein, and its flavin moiety was covalently bound to the dehydrogenase protein. The optimum pH of the enzyme activity was 4.5, and the optimum temperature was 25°C. D-Sorbitol was specifically oxidized by the purified enzyme, and D-mannitol was also oxidized to 5% of the reaction rate with D-sorbitol.