(1975). Crystallization of 2-Ketogluconate Reductase from Gluconobacter liquefaciens. Agricultural and Biological Chemistry: Vol. 39, No. 11, pp. 2263-2264.
5-Ketogluconate reductase (5KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was partially purified about 120-fold by a procedure employing ammonium sulfate fractionation, and DEAE-cellulose-, hydroxylapatite- and DEAE-Sephadex A-50-column chromatographies. NADP was specifically required for the oxidative reaction of gluconic acid. The optimum pH for the oxidation of gluconic acid (GA) to 5-ketogluconic acid (5KGA) by the enzyme was 10.0 and for the reduction of 5KGA was 7.5. The optimum temperature of the enzyme was 50°C for both reactions of oxidation and reduction. The enzyme was considerably unstable and lost all of its activity within 3 days. The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ion, but remarkably stimulated by EDTA (1 × 10−3m). Apparent Km values were 1.8 × 10−2m for GA, 0.9 × 10−3m for 5KGA, 1.6 × 10−5 m for NADP, and 1.1 × 10−5 m for NADPH2.
The hydrogenase reaction in Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, aerobic hydrogen-oxidizing bacterium, was studied in the membrane fraction, methionaquinone-depleted membrane, and purified membrane-bound hydrogenase. Both b and c type cytochromes were involved in the hydrogen oxidation. Methionaquinone mediated an electron transport between membrane-bound hydrogenase and cytochrome b560. Methionaquinone was reduced directly by purified hydrogenase. From these results, we conclude that methionaquinone is a direct natural electron acceptor for the membrane-bound hydrogenase in the strain.
Journal Article Crystallization and Properties of Amine Dehydrogenase from Pseudomonas sp. Get access Emiko Shinagawa, Emiko Shinagawa Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan Search for other works by this author on: Oxford Academic Google Scholar Kazunobu Matsushita, Kazunobu Matsushita Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan Search for other works by this author on: Oxford Academic Google Scholar Koji Nakashima, Koji Nakashima Department of Laboratory Medicine, St. Luke’s College of Nursing, Akashi-cho, Chuo-ku, Tokyo 104, Japan Search for other works by this author on: Oxford Academic Google Scholar Osao Adachi, Osao Adachi Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan Search for other works by this author on: Oxford Academic Google Scholar Minoru Ameyama Minoru Ameyama Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan Search for other works by this author on: Oxford Academic Google Scholar Agricultural and Biological Chemistry, Volume 52, Issue 9, 1 September 1988, Pages 2255–2263, https://doi.org/10.1080/00021369.1988.10869019 Published: 01 September 1988 Article history Received: 22 March 1988 Published: 01 September 1988
Particulate alcohol dehydrogenase of acetic acid bacteria that is mainly participated in vinegar fermentation was purified to homogeneous state from Gluconobacter suboxydans IFO 12528. Solubilization of enzyme from the bacterial membrane fraction by Triton X-100 and subsequent fractionation on DEAE-Sephadex A-50 and hydroxylapatite was successful in enzyme purification. A cytochrome c-like component was tightly bound to the dehydro-genase protein and existed as an enzyme-cytochrome complex. It was also confirmed that the alcohol dehydrogenase is not a cytochrome component itself. The molecular weight of the enzyme was determined to be 150, 000, and gel electrophoresis showed the presence of three subunits having a molecular weight of 85, 000, 49, 000 and 14, 400. The smallest subunit was corresponded to the cytochrome c-like component. Ethanol was oxidized in the presence of dyes in vitro but NAD or NADP were not required as hydrogen acceptor. Unlike NAD-linked alcohol dehydrogenase in yeast or liver and other primary alcohol dehydrogenases in methanol utilizing bacteria, the enzyme from the acetic acid bacteria showed its optimum pH at fairly acidic pH.
Mammalian choline dehydrogenase (EC 1.1.99.1) has been proved to be a quinoprotein in which pyrroloquinoline quinone (PQQ) is involved as the prosthetic group. The enzyme was purified from dog liver mitochondria by solubilizing the enzyme with Brij 58 and chromatographically separating it almost to homogeneity. The absorption spectrum of mammalian choline dehydrogenase indicated the presence of PQQ with a typical shoulder at 320 nm. Since PQQ was attached to the enzyme by a covalent linkage, the chromophore was isolated with an acid hydrolysate and the isolated chromophore gave rise the identical spectroscopic characteristics to that obtained from the amine oxidase of Aspergillus niger in which PQQ is covalently linked. The isolated chromophore potently activated apo-D-glucose dehydrogenase (EC 1.1.99.17) supporting the presence of PQQ in mammalian choline dehydrogenase.
