Gluconobacter Suboxydans Research Articles

Occurrence of old yellow enzyme in Gluconobacter suboxydans, and the cyclic regeneration of NADP.

Old yellow enzyme system has been found in the cytosol fraction of Gluconobacter suboxydans. This is the first time that the enzyme has been found in organisms other than yeast cells. Old yellow enzyme [EC 1.6.99.1], D-glucose-6-phosphate dehydrogenase [EC 1.1.1.49], and catalase were isolated and crystallized separately from the organism. The old yellow enzyme from G. suboxydans showed catalytic and physicochemical properties almost identical with those of the enzyme from yeast cells. NADPH was specifically oxidized by the old yellow enzyme and the reduced enzyme was spontaneously reoxidized by atmospheric oxygen. The old yellow enzyme from G. suboxydans also contained FMN as a prosthetic group, and two mol of FMN were found per mol of enzyme (molecular weight, 88,000 as determined by gel filtration). In the oxidation of D-glucose-6-phosphate to 6-phospho-D-gluconate, cyclic regeneration of NADP occurred smoothly in the presence of D-glucose-6-phosphate dehydrogenase and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.

Crystallization and properties of 5-keto-d-gluconate reductase from Gluconobacter suboxydans.

5-Keto-d-gluconate reductase (EC 1.1.1.69) was purified and crystallized for the first time from cell-free extract of Gluconobacter suboxydans IFO 12528. Purification of the enzyme was successfully performed by column chromatography on DEAE-Sephadex A–50, blue-dextran Sepharose 4B, followed by pH gradient chromatography on DEAE-Sephadex A–50. The enzyme was purified about 1200-fold with an overall yield of 40%. The enzyme was much stabilized against heating and storage by adding either d-gluconate or 5-keto-d-gluconate. 2-Keto-d-gluconate had no effect to stabilize the enzyme and the enzyme was confirmed to be a different entity from 2-keto-d-gluconate reductase stabilized by gluconate or 2-keto-d-gluconate but not 5-keto-d-gluconate. It was also confirmed with crystalline enzyme that 5-keto-d-gluconate reductase is considered to have a function to reduce intracellular 5-keto-d-gluconate to d-gluconate in combination with regeneration of NADP.

Crystallization and Properties of 5-Keto-d-gluconate Reductase fromGluconobacter suboxydans

5-Keto-d-gluconate reductase (EC 1.1.1.69) was purified and crystallized for the first time from cell-free extract of Gluconobacter suboxydans IFO 12528. Purification of the enzyme was successfully performed by column chromatography on DEAE-Sephadex A–50, blue-dextran Sepharose 4B, followed by pH gradient chromatography on DEAE-Sephadex A–50. The enzyme was purified about 1200-fold with an overall yield of 40%. The enzyme was much stabilized against heating and storage by adding either d-gluconate or 5-keto-d-gluconate. 2-Keto-d-gluconate had no effect to stabilize the enzyme and the enzyme was confirmed to be a different entity from 2-keto-d-gluconate reductase stabilized by gluconate or 2-keto-d-gluconate but not 5-keto-d-gluconate. It was also confirmed with crystalline enzyme that 5-keto-d-gluconate reductase is considered to have a function to reduce intracellular 5-keto-d-gluconate to d-gluconate in combination with regeneration of NADP.

Purification and Characterization of Particulate Alcohol Dehydrogenase fromGluconobacter suboxydans

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 dehydrogenase 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 othe...

Occurrence of Glutamine Synthetase/Glutamate Synthase Pathway in Gluconobacter suboxydans

Occurrence of Glutamine Synthetase/Glutamate Synthase Pathway in <i>Gluconobacter suboxydans</i>

Purification and characterization of particulate alcohol dehydrogenase from Gluconobacter suboxydans.

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.

Occurrence of glutamine synthetase/glutamate synthase pathway in Gluconobacter suboxydans.

The growth of Gluconobacter suboxydans was supported by addition of NH4C1 as sole nitrogen source. Glutamine synthetase and glutamate synthase activities were revealed in the cell-free extracts of NH4Cl-grown cells. The two enzymes were separated and partially purified about 120 and 200 fold, respectively.The optimum pH of glutamine synthetase was 8.0. The Km values for L-glutamine, ammonia and ATP were 6.7 mM, 3.3 mM and 0.56 mM, respectively. Mg2+ was indispensable for the activity. The enzyme activity was inhibited by glycine, L-alanine, L-hisdidine, ADP and AMP.Although glutamate synthase was strikingly unstable, it was reactivated 2~3 fold by addition of dithiothreitol. The optimum pH was 8.5. Only L-glutamine, α-ketoglutarate (α-KGA) and NADPH were the specific substrates and coenzyme. Their Km values were 0.71 mM, 2.2 µm and 20 µm, respectively. The enzyme activity was inhibited by various kinds of amino acids and α-keto acids or NADH.The effects of culture conditions on the enzyme activities were ...

Resolution and complementation of the labile l-leucinepyruvate transaminase. An intermediate during enzyme formation under nitrogen starvation in Gluconobacter suboxydans

l-Leucine-pyruvate transaminase (mol. wt. 70 000) in Gluconobacter suboxydans synthesized during nitrogen starvation contained a labile form which changed to the stable one later. The labile enzyme (mol. wt. 70 000) dissociated to the two proteinaceous components: a cationic one (mol. wt. 10 000–20 000) and an anionic one (mol. wt. 50 000–60 000), during column chromatography on DEAE-cellulose. The enzyme activity was reconstructed when they were mixed. The reconstructed enzyme had almost the same molecular size and enzymatic properties as the labile and the native stable enzymes.

Amino acid metabolism in acetic acid bacteria. III. Purification and some properties of L-leucine-pyruvate transaminase from Acetobacter suboxydans.

An enzyme, tentatively named l-leucine-pyruvate transaminase, was purified homogeneously by the criteria of ultracentrifugation and electrophoresis on a cellulose acetate membrane from Acetobacter suboxydans (Gluconobacter suboxydans IFO 3172). The molecular weight was about 70,000 and one mole of pyridoxal 5′-phosphate was bound per mole of the enzyme as coenzyme. The amino donor specificity was extremely broad and the optimum pH was 5.0. The enzyme exhibit absorption maxima at 280 nm and 332 nm.

Die biogenese von tartrat in der weinrebe

Summary Excised leaves and berries of the grapevine were fed 14 C-glucose and 14 C-glyoxylate and the radioactivity as well as the labelling pattern of the isolated tartrate determined. In both organs these substances proved to be about equally good tartaric acid precursors. When using glyoxylate-2- 14 C as a traces, the label is localized exclusively in the inner C-atoms of tartrate. Application of glucose-l- 14 C and glucose-6-14C to leaves yields tartrate labelled predominantly in the C1/C4 and the C2/C3 positions, respectively, whereas the inverse situation occurs in the fruits. Furthermore, the question of ascorbic acid being an extremely effective tartaric acid precursor in grapevine — as reported in the literature — was investigated. The differing distribution of radioactivity within the tartrate molecule after application of the above tracers reveals the existence of two, at least partly distinct, pathways for tartrate genesis in grapevine. The respective labelling patterns suggest that, in the leaf, glucose is transformed via gluconate to pretaric acid (1,2-dihydroxyethyl hydrogen L (+) tartrate), which undergoes cleavage between carbons 4 and 5, whereby tartaric acid and glycolalde-hyde are formed — an overall reaction sequence recently described to account for tartrate synthesis in Gluconobacter suboxydans . In the grape plant, two molecules of the glycoalde-hyde thus formed are condensed, yielding another tartaric acid molecule (cf. scheme). In the berry, on the other hand, the glucose is oxidized to glucuronic acid, and this compound transformed to pretaric acid with gulonic-, 5-keto-, enol- and 4-keto-gulonic acids as intermediates, in a way analogous to that in the leaf. Saito and Kasai (1969) reported that more than 70 % of the radioactivity of the aqueous HCl extract of young grape berries was localized in tartaric acid after administration of ascorbic acid-l- 14 C. A critical study of their working-up procedure shows, however, that the use of solutions permitting complete extraction of tartrate results in a rapid non-enzymatic conversion of ascorbate to tartrate. Therefore, at least the high incorporation rate of this compound — undoubtedly extreme for higher plants — must be considered an artefact.

Separation ofl-Leucine-pyruvate andl-Leucine-α-ketoglutarate Transaminases inAcetobacter suboxydansand Identification of Their Reaction Products

l-Leucine-pyruvate and l-leucine-α-ketoglutarate(α-KGA) transaminases were separated by DEAE-cellulose column chromatography and partially purified to 200- and 50-fold, respectively, from the cell-free extract of Acetobacter suboxydans (Gluconobacter suboxydans IFO 3172). The optimum pH range of the former was 5.0~5.5 and that of the latter was 8.5~9.0. l-Leucine, l-citrulline, and l-methionine were the most effective amino donors for the l-leucine-pyruvate transaminase. Basic amino acids as well as aromatic amino acids were able to be amino donors for the transamination with pyruvate. α-KGA was effective as an amino acceptor for this enzyme. The l-leucine-α-KGA transaminase had the typical properties of the branched-chain amino acid transaminase in its substrate specificity.The reaction products of the transaminations were identified. l-Alanine was formed from pyruvate and l-glutamate from α-KGA. α-Keto acids formed from various amino acids by the l-leucine-pyruvate transaminase were also identified.

Occurrence of Transaminations with Pyruvate and α-Ketoglutarate in Acetobacter suboxydans

Transaminations betweenL-amino acids and pyruvate orα-ketoglutarate(α-KGA) were observed in a cell-free extract ofAcetobacter suboxydans(Gluconobacter suboxydans IFO 3172). The level of the activities of transaminations with pyruvate was greatly influenced by the kinds and amounts of nitrogen sources for growth media. The enzymic activities of transaminations with pyruvate in glutamate-grown cells were extremely higher than in yeast ex- tract-grown cells. Nutritional components decreasing the activities were presumed to be some of amino acids and ammonium ion present in yeast extract. The activities of transaminations withα-KGA were not so variable in the presence or absence of the components.The optimum pH of transaminations with pyruvate was in the range of 5.0 ~ 5.5 and that of the reaction withα-KGA in 8.0 ~ 8.5. The optimum temperature of the former was 65°C and that of the latter about 45°C. Some other different properties were also recognized between the two kinds of reactions.

Separation of L-Leucine-pyruvate and L-Leucine-α-ketoglutarate Transaminases in Acetobacter suboxydans and Identification of Their Reaction Products

l-Leucine-pyruvate and l-leucine-α-ketoglutarate(α-KGA) transaminases were separated by DEAE-cellulose column chromatography and partially purified to 200- and 50-fold, respectively, from the cell-free extract of Acetobacter suboxydans (Gluconobacter suboxydans IFO 3172). The optimum pH range of the former was 5.0~5.5 and that of the latter was 8.5~9.0. l-Leucine, l-citrulline, and l-methionine were the most effective amino donors for the l-leucine-pyruvate transaminase. Basic amino acids as well as aromatic amino acids were able to be amino donors for the transamination with pyruvate. α-KGA was effective as an amino acceptor for this enzyme. The l-leucine-α-KGA transaminase had the typical properties of the branched-chain amino acid transaminase in its substrate specificity. The reaction products of the transaminations were identified. l-Alanine was formed from pyruvate and l-glutamate from α-KGA. α-Keto acids formed from various amino acids by the l-leucine-pyruvate transaminase were also identified.

Induction of Mutants from the Tartrate Producing Bacterium, Gluconobacter suboxydans and their Properties

Induction of Mutants from the Tartrate Producing Bacterium, Gluconobacter suboxydans and their Properties

Isolation Method of Highly Tartaric Acid Producing Mutants of Gluconobacter suboxydans

The present purpose is to improve tartaric acid productivity of Gluconobacter suboxydans IAM 1829, which is well known as a 5-ketogluconic acid producer, by mutation involving the use of newly developed isolation method. In the course of studies for recognizing the causes suppressing the yield of tartaric acid, it was revealed that hydrogen-ion concentration and glycolic acid accumulated during fermentation limited the tartaric acid formation by inhibiting the growth of the bacteria. From these point of view, isolation of acid tolerant mutants and glycolate tolerant mutants was carried out. The significant correlation was found between the tartaric acid productivity of these mutants and their tolerance to those inhivitory agents, and some desireable mutants were obtained.

Enzymatic studies on the oxidation of sugar and sugar alcohol. 8. Particle-bound L-sorbose dehydrogenase from Gluconobacter suboxydans.

Enzymatic studies on the oxidation of sugar and sugar alcohol. 8. Particle-bound L-sorbose dehydrogenase from Gluconobacter suboxydans.

Enzymatic Studies on the Oxidation of Sugar and Sugar Alcohol

During the course of studies on the oxidative metabolism of d-sorbitol by acetic acid bacteria, it was found that d-sorbitol was almost quantitatively converted to 5-keto-d-fructose via l-sorbose by a certain strain of Gluconobacter suboxydans. In addition to 5-keto-d-fructose, three γ-pyrone compounds, kojic acid, 5-oxymaltol, and 3-oxykojic acid, 2-keto-l-gulonate, and several organic acids such as succinic, glycolic, and glyceric acids were confirmed in the culture filtrate of this bacterium. The most suitable carbon source for 5-ketofructose fermentation by Gluconobacter suboxydans Strain 1 was confirmed to be d-sorbitol or l-sorbose using growing and resting cells. d-Fructose had little effect on the formation of this dicarbonylhexose.The optimal pH for the formation from l-sorbose by intact cells was found to be at 4.2.The activity of the pentose phosphate cycle in the resting cells was calculated as 13~17 μatoms/hr/mg of dry cells by the use of the manometric techniques.There was no strain tested s...

On the localisation of oxidase systems in Acetobacter cells

1. 1. The oxidation of several carbohydrates and related compounds by intact cells of Gluconobacter liquefaciens has been studied. 2. 2. Intact cells were disrupted by ultrasonic treatment and separated into several fractions by centrifugation. “Cell débris” and small particles oxidized glucose, gluconate and 2-ketogluconate to 2,5-diketogluconate and beyond; D-lactate and ethanol to the acetate stage; galactose, mannose, xylose and L-arabinose to the corresponding acids; sorbitol to sorbose; glycerol and meso-erythritol to the corresponding ketoderivatives. Acetate, 5-ketogluconate, fructose and mannitol were not attacked. 3. 3. Intact cells could be converted into “protoplasts” with an efficiency of about 95% by means of human serum in an appropriate medium. Pig serum affected 60–70% conversion. 4. 4. The “protoplasts” oxidized most of the substrates at about 60–80% of the rate of the intact cells. The oxidation rate of substrates, metabolized after an induction period by intact cells (fructose, mannitol, 5-ketogluconate and glycerol) was considerably slower by “protoplasts”. 5. 5. Only about 50% of the “protoplasts” lyzed in water or dilute buffer after several h at 30°. No lysis was observed after pig serum treatment. 6. 6. Several fractions were obtained from “protoplasts” after either lysis or short ultrasonic treatment. All the substrates which were oxidized by the small particles and “cell débris”, were likewise oxidized to the same extent by “ghosts” and “protoplasts débris”, with the exception of glycerol and meso-erythritol. The enzymes for these latter two substances were apparently inactivated at 37°. 7. 7. Several particle-linked oxidase from Gluconobacter (Acetobacter) suboxydans were constitutive. 8. 8. The oxidase-bearing particles from acetic acid bacteria apparently do not exist as such in the cytoplasm of the bacteria, but probably originate from “ghosts” through mechanical or ultrasonic disruption. 9. 9. The oxidative characteristics, typical for the acetic acid bacteria, are mainly determined by the enzymic activity of an outer cell envelope (probably the cytoplasmic membrane).