D-fructose dehydrogenase of Gluconobacter industrius: purification, characterization, and application to enzymatic microdetermination of D-fructose

Abstract

D-Fructose dehydrogenase was solubilized and purified from the membrane fraction of glycerol-grown Gluconobacter industrius IFO 3260 by a procedure involving solubilization of the enzyme with Triton X-100 and subsequent fractionation on diethylaminoethyl-cellulose and hydroxylapatite columns. The purified enzyme was tightly bound to a c-type cytochrome and another peptide existing as a dehydrogenase-cytochrome complex. The purified enzyme was deemed pure by analytical ultracentrifugation as well as by gel filtration on a Sephadex G-200 column. The molecular weight of the enzyme complex was determined to be about 140,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of three components having molecular weights of 67,000 (dehydrogenase), 50,800 (cytochrome c), and 19,700 (unknown function). Only D-fructose was readily oxidized by the enzyme in the presence of dyes such as ferricyanide, 2,6-dichlorophenolindophenol, or phenazine methosulfate. Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and oxygen did not function as electron acceptors. The optimum pH of D-fructose oxidation was 4.0. The enzyme was stable at pH 4.5 to 6.0 Stability of the purified enzyme was much enhanced by the presence of detergent in the enzyme solution. Removal of detergent from the enzyme solution facilitated the aggregation of the enzyme and caused its inactivation. An apparent Michaelis constant for D-fructose was observed to be 10(-2) M with the purified enzyme. D-Fructose dehydrogenase was shown to be a satisfactory reagent for microdetermination of D-fructose.

The Immobilization of Fructose Dehydrogenase on Nanoporous Gold Electrodes for the Detection of Fructose

“…The optimum pH of the free enzyme reported in the literature for the oxidation of d-fructose is 4.0 and FDH was reported to be stable between pH 4.5 and 6.0. [18] The observed shift in optimum pH may arise from the negatively charged carboxylic acid groups on the electrode surface as previously described for negatively charged acetate membranes [25] and carbon nanoparticles. [24a] At a fixed pH of 5.5 the current density increased from 20 to 35 8C, in good agreement with the reported optimum temperature of 37 8C [42] (Sorachim SA).…”

“…[17] FDH was first described by Adachi et al. [18] The enzyme (EC Nr. : 1.1.99.11) has an approximate molecular weight of 140 kDa FDH and is comprised of 3 subunits; a flavin adenine dinucleotide (FAD) subunit (MW: 67,000 Da), a heme C (cytochrome c) subunit (MW: 50,800 Da) and a peptide domain of unknown function (MW: 19,700 Da).…”

“…: 1.1.99.11) has an approximate molecular weight of 140 kDa FDH and is comprised of 3 subunits; a flavin adenine dinucleotide (FAD) subunit (MW: 67,000 Da), a heme C (cytochrome c) subunit (MW: 50,800 Da) and a peptide domain of unknown function (MW: 19,700 Da). [18][19] The FAD subunit of the enzyme is linked to the catalytic conversion of d-fructose to 5-dehydrod-fructose. The reduced form of FAD is then oxidized in an intramolecular electron transfer step to the heme subunit [19][20] Direct electron transfer (DET) of the heme subunit can then occur at an electrode.…”

Role of botrytized grape micro-organisms in SO2binding phenomena

“…According to Schrimsher et al (1988), this enzyme is linked to the membrane of Gluconobacter. The apoenzyme is an enzymatic complex, described by Ameyama et al (1981), composed of a dehydrogenase, a cytochrome c playing the role of electron conveyor and a third peptide of unknown function. The coenzyme allowing the oxidation of fructose, identi®ed by Yamada et al (1968), is the principal liposoluble quinone of Gluconobacter, the ubiquinone 10 or coenzyme Q10.…”

Heterologous Overexpression and Characterization of a Flavoprotein-Cytochrome c Complex Fructose Dehydrogenase of Gluconobacter japonicus NBRC3260

“…140 kDa, consisting of subunits I (67 kDa), II (51 kDa), and III (20 kDa). The enzyme, purified for the first time in 1981, is a flavoprotein-cytochrome c complex, since subunits I and II have covalently bound flavin adenine dinucleotide (FAD) and heme C as prosthetic groups, respectively (1).…”