5-Keto-D-fructose Reductase of Gluconobacter industrius: Purification, Crystallization and Properties

Abstract

5-Keto-D-fructose reductase was purified and crystallized from a cell-free extract of Gluconobacter industrius IFO 3260, grown on glycerol-yeast extract medium. After repeated chromatographies on DEAE-Sephadex A-50 and hydroxyapatite, the enzyme was purified about 440-fold. The enzyme was then separated from major impurities by ammonium sulfate fractionation, and crystallized. The yield of the crystallized enzyme was 25%, with a 750-fold purification. The crystallized enzyme was homogeneous in analytical ultracentrifugation, as well as in polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined to be 100, 000, and the enzyme was composed of four identical subunits, each having a molecular weight of 25, 000. The isoelectric point of the enzyme was found at 5.0. The enzyme was predominant in the reduction of 5-keto-D-fructose to D-fructose and strictly specific to NADPH.