Gluconobacter Melanogenus Research Articles

Purification and Characterization of Membrane-bound Quinoprotein Quinate Dehydrogenase

Several bacterial strains carrying quinoprotein quinate dehydrogenase (QDH) were screened through acetic acid bacteria and other bacteria. Strong enzyme activity was found in the membrane fraction of Gluconobacter melanogenus IFO 3294, G. oxydans IFO 3292, G. oxydans IFO 3244, and some strains of Acinetobacter calcoaceticus. Interestingly, in the membrane fraction of A. calcoaceticus AC3, which is unable to produce pyrroloquinoline quinone (PQQ), fairly large amounts of apo-QDH were formed, and were converted to holo-QDH only by the addition of PQQ. It was difficult to detach PQQ from the holo-QDH by EDTA treatment, and EDTA treatment with apo-QDH prior to PQQ addition gave no significant holo-QDH. For QDH purification, Gluconobacter strains were not suitable due to the presence of huge amounts of quinohemoprotein alcohol dehydrogenase (ADH) in the same membrane, which was co-solubilized with QDH and disturbed purification of QDH. Purification of holo-QDH was done with Acinetobacter sp. SA1 instead, which contained no ADH. Apo-QDH was purified from A. aclcoaceticus AC3. This is the first report dealing with QDH purification, and two different criteria of QDH purification were given. A combination of two steps using butyl-Toyopearl and hydroxyapatite columns gave a highly purified holo-QDH which was monodispersed and showed enough purity, though the specific activity did not increase as much as expected. When QDH purification was done with A. calcoaceticus AC3 in the absence of PQQ, purified apo-QDH appeared to be a dimer, which was converted to the monomer on addition of PQQ. Since QDH was highly hydrophobic, one-step chromatography on a DEAE-Sepharose column was tried. Purified holo-QDH of higher specific activity was obtained with a higher yield. The molecular mass of QDH was estimated to be 88 kDa. There was no characteristic absorption spectrum with the purified QDH except for a small bump around 420 nm. QDH oxidized only quinate and shikimate so far examined. The optimal QDH activity was found at pH 6-7 when assayed with artificial electron acceptors. QDH was formed in the presence or absence of quinate in the culture medium, although stronger induction was usually observed in the presence of quinate.

Crystallization and Properties of NADPH-Dependent L-Sorbose Reductase from Gluconobacter melanogenus IFO 3294.

NADPH-Dependent L-sorbose reductase (SORD, synonimously NADP-dependent D-srobitol dehydrogenase) was purified and crystallized for the first time from the cytosolic fraction of Gluconobacter melanogenus IFO 3294. The enzyme catalyzed oxidoreduction between D-sorbitol and L-sorbose in the presence of NADP or NADPH. Affinity chromatography by a Blue-dextran Sepharose 4B column was effective for purifying the enzyme giving about 770-fold purification with an overall yield of more than 50%. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 3.8 s. Gel filtration on a Sephadex G-75 column gave the molecular mass of 60 kDa to the enzyme, which dissociated into 30 kDa subunit on SDS-PAGE, indicating that the enzyme is composed of 2 identical subunits. Reduction of L-sorbose to D-sorbitol predominated in the presence of NADPH with the optimum pH of 5.0-7.0. Oxidation of D-sorbitol to L-sorbose was observed in the presence of NADP at the optimum pH of 7.0-9.0. The relative rate of L-sorbose reduction was more than seven times higher to that of D-sorbitol oxidation. NAD and NADH were inert for both reactions. D-Fructose reduction in the presence of NADPH did not occur with SORD. Since the reaction rate in L-sorbose reduction highly predominated over D-sorbitol oxidation over a wide pH range, the enzyme could be available for direct enzymatic measurement of L-sorbose. Even in the presence of a large excess of D-glucose and other substances, oxidation of NADPH to NADP was highly specific and stoichiometric to the L-sorbose reduced. Judging from the enzymatic properties, SORD would contribute to the intracellular assimilation of L-sorbose incorporated from outside the cells where L-sorbose is accumulated in huge amounts in the culture medium.

Isolation and Characterization of Membrane-boundl-Sorbose Dehydrogenase fromGluconobacter melanogenusUV10

Membrane-bound l-sorbose dehydrogenase was purified from the membrane fraction of Gluconobacter melanogenus UV10. The reaction with 2,6-dichlorophenolindophenol or phenazine methosulfate as acceptor was formulated as; l-sorbose + acceptor →l-sorbosone + reduced acceptor. The enzyme was purified by DEAE-Cellulose, DEAE-Sepharose, and Sephadex G-200 column chromatographies after differential solubilization with Triton X-100 and Tween 80, and appeared as a single band in a SDS-PAGE gel at the molecular weight of about 58,000. It was most active at pH 7.0 and 48°C, and rather stable at around 7.0 in a buffer containing 200 mm l-sorbose, below 30°C. It showed high substrate specificity for l-sorbose, and its activity was stimulated by Fe3 + and Co2 +, and inhibited by Cu2 +, quinine, mono-iodoacetate, and sodium fluoroacetate.

Isolation and characterization of NAD(P)-dependent L-sorbosone dehydrogenase from Gluconobacter melanogenus UV10.

NAD(P)-Dependent l-sorbosone dehydrogenase has been purified from the cytosol fraction of Gluconobacter melanogenus UV10. The enzyme was purified about 30-fold with an overall yield of 19% by column chromatographies on DEAE-Sepharose CL-6B, DEAE-Sephadex A-50, and Blue Sepharose CL-6B. The enzyme required NAD or NADP as a cofactor, and showed broad substrate specificity on various aldehyde compounds. Among them, l-sorbosone was the best substrate for the enzyme. In this respect, the enzyme is a kind of aldehyde dehydrogenase.

Purification and Properties of NADPH-Linked L-Sorbose Reductase from Gluconobacter melanogenus N44-1.

NADPH-Linked L-sorbose reductase from the cytosol fraction of Gluconobacter melanogenus N44-1, which is a 2-keto-L-gulonic acid producer from L-sorbose or D-sorbitol, was purified about 500-fold by DEAE-Cellulose, Blue Sepharose, and DEAE-Sepharose column chromatographies. The purified enzyme was entirely homogeneous on polyacrylamide gel and SDS gel electrophoresis. NADPH and NADP were specifically required for the reduction and oxidation of the substrate, respectively. The apparent molecular weight was 60, 000 by Sephadex G-200 column chromatography, and that of its subunit was 60, 000 by SDS-gel electrophoresis. The pH optimum for the reduction of L-sorbose and D-fructose was 7.0. The pH optimum for the oxidation of D-sorbitol to L-sorbose and D-mannitol to D-fructose was 10.0-10.5.

Isolation and characterization of membrane-bound L-sorbose dehydrogenase from Gluconobacter melanogenus UV10.

Membrane-bound L-sorbose dehydrogenase was purified from the membrane fraction of Gluconobacter melanogenus UV10. The reaction with 2, 6-dichlorophenolindophenol or phenazine methosulfate as acceptor was formulated as; L-sorbose + acceptor →-sorbosone + reduced acceptor. The enzyme was purified by DEAE-Cellulose, DEAE-Sepharose, and Sephadex G-200 column chromatographies after differential solubilization with Triton X-100 and Tween 80, and appeared as a single band in a SDS-PAGE gel at the molecular weight of about 58, 000. It was most active at pH 7.0 and 48°C, and rather stable at around 7.0 in a buffer containing 200 mM L-sorbose, below 30°C. It showed high substrate specificity for L-sorbose, and its activity was stimulated by Fe3+ and Co2+, and inhibited by Cu2+, quinine, mono-iodoacetate, and sodium fluoroacetate.

Microbial Production of 2-Keto-l-Gulonic Acid froml-Sorbose andd-Sorbitol byGluconobacter melanogenus

The strain SPO1 producing 13 g of 2-keto-l-gulonic acid (2KGA) per liter was isolated as a spontaneous mutant of Gluconobacter melanogenus IFO 3293. For the enhancement of 2KGA productivity, we did...

Microbial production of 2-keto-L-gulonic acid from L-sorbose and D-sorbitol by Gluconobacter melanogenus.

The strain SPO1 producing 13 g of 2-keto-l-gulonic acid (2KGA) per liter was isolated as a spontaneous mutant of Gluconobacter melanogenus IFO 3293. For the enhancement of 2KGA productivity, we did...

Metabolic pathway for 2-keto-L-gulonic acid formation in Gluconobacter melanogenus IFO 3293.

The l-sorbosone pathway for 2-keto-l-gulonic acid (2KGA) formation from l-sorbose in Gluconobacter melanogenus IFO 3293 was confirmed. l-Sorbose dehydrogenase, which catalyzes the conversion of l-sorbose to l-sorbosone, was found in the membrane fraction of the microorganism, and l-sorbosone dehydrogenase in the cytosol fraction. The latter enzyme requires NAD or NADP as a cofactor.2KGA was reduced to l-idonic acid by 2KGA reductase, but l -idonic acid was re-oxidized to 2KGA by membrane-bound l-idonate dehydrogenase.

L-sorbose dissimilation in 2-keto-L-gulonic acid-producing mutant UV10 derived from Gluconobacter melanogenus IFO 3293.

During the fermentation of 2-keto-l-gulonic acid (2KGA) from l-[U-14C]sorbose by the 2KGA-producing mutant UV10, derived from G. melanogenus IFO 3293, 40% of the metabolized substrate was converted to 14CO2. The CO2 evolved was mainly via the pentose phosphate pathway and partly via the intermediates such as l-sorbosone or the by-products of 2KGA formation. CO2 evolution from 2KGA was not observed.

Identification of the covalently bound flavins of d-gluconate dehydrogenases from Pseudomonas aeruginosa and Pseudomonas fluorescens and 2-keto-d-gluconate dehydrogenase from Gluconobacter melanogenus

Identification of the covalently bound flavins of <scp>d</scp>-gluconate dehydrogenases from <i>Pseudomonas aeruginosa</i> and <i>Pseudomonas fluorescens</i> and 2-keto-<scp>d</scp>-gluconate dehydrogenase from <i>Gluconobacter melanogenus</i>

Identification of the covalently bound flavins of D-gluconate dehydrogenases from Pseudomonas aeruginosa and Pseudomonas fluorescens and of 2-keto-D-gluconate dehydrogenase from Gluconobacter melanogenus.

An improved method is presented for the purification of 8 alpha-(N1-histidyl)riboflavin, 8 alpha-(N3-histidyl)riboflavin and their 2',5'-anhydro forms, which permits the isolation of sizeable quantities of each of these compounds from a synthetic mixture in pure form. Flavin peptides were isolated from the D-gluconate dehydrogenases of Pseudomonas aeruginosa and Pseudomonas fluorescens and from the 2-keto-D-gluconate dehydrogenase of Gluconobacter melanogenus. After conversion into the aminoacyl-riboflavin, the flavin in all three enzymes was identified as 8 alpha-(N3-histidyl)riboflavin. By sequential treatment with nucleotide pyrophosphatase and alkaline phosphatase, the flavin in each enzyme was shown to be in the dinucleotide form.

Purification and characterization of 2-keto-D-gluconate dehydrogenase from Gluconobacter melanogenus.

From membrane fraction of Gluconobacter melanogenus IFO 3293, 2-keto-D-gluconate dehydrogenase was solubilized and purified to an electrophoretically and ultracentrifugally homogeneous state of about 400-fold in high yields of 41%. Purification was achieved by solubilization with 2% cholate and 0.2 M KCl, subsequent precipitation with ammonium sulfate and polyethylene glycol 6000, and chromatographies on CM-cellulose and hydroxyapatite columns in the presence of Triton X-100. The purified enzyme was tightly bound to a c-type cytochrome existing as a dehydrogenase-cytochrome complex. The molecular weight of the enzyme was determined to be about 133,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of three components having molecular weights of 61,000 (flavoprotein), 47,000 (cytochrome c) and 25,000. The dehydrogenase was found to be a flavoprotein having a covalently bound flavin. Only 2-keto-d-gluconate was readily oxidized by the enzyme in the presence of dyes, such ...

Crystallization and properties of NADP-dependent aldehyde dehydrogenase from Gluconobacter melanogenus.

Crystallization and properties of NADP-dependent aldehyde dehydrogenase from Gluconobacter melanogenus.

Application of Oxygen-Enriched Aeration in the Conversion of Glycerol to Dihydroxyacetone by Gluconobacter melanogenus IFO 3293

Gluconobacter melanogenus 3293 converts glycerol to dihydroxyacetone(DHA) during exponential growth on a yeast extract-phosphate medium at pH 7. The efficiency of this conversion in 25-liter batch fermentations has been found to increase over threefold, when oxygen tension is controlled by increasing the partial pressure of oxygen in the aeration. Conversion of glycerol to DHA does not occur under oxygen-limited fermentation conditions. When the dissolved oxygen tension was maintained at 0.05 atmospheres (using oxygen-enriched air), quantitative conversion of up to 100 g of glycerol/liter to DHA was obtained in 33 h. The amount of glycerol converted can be increased without increasing impeller speed or aeration rate. This increase is not the result of increased production of cell mass. The specific conversion of glycerol to DHA increased from 12.2 g of DHA/g of cell mass at the point of maximum conversion to 35.8 with oxygen enrichment. This increased specific production occurred even though the specific growth rate during the period of oxygen enrichment decreased from 0.23 to 0.06/h.

Conversion of L‐sorbose to L‐sorbosone by immobilized cells of Gluconobacter melanogenus IFO 3293

Gluconobacter melanogenus IFO 3293 cells capable of converting L-sorbose to L-sorbosone were immobilized in polyacrylamide gel. The preferred polymer composition for high activity and stability was determined to contain a total monomer concentration of 7.2% and 16.6% crosslinking agent. No significant differences in optimal conditions for conversion, e.g., pH and temperature, were found in comparison with free cell suspensions. However, in the absence of L-sorbose, the thermal stability of immobilized cells was lower. After the initial loss, the conversion activity of immobilized cells increased, possibly due to lysis, and this increase was related to the polymerization conditions and the incubation temperature for the L-sorbose conversion. The enzymatic activity and stability of the immobilized cells also depended on the physical form of the gel and the aeration levels. Addition of electron acceptors or addition of L-sorbosone to the medium reduced, while addition of neomycin, ampicillin, chloramphenicol, and tetracycline increased the stability of the enzymatic activity.

Regulation of aspartokinase and homoserine dehydrogenase in acetic acid bacteria

The regulation of aspartokinase and homoserine dehydrogenase has been studied in three Acetobacter and two Gluconobacter species. Both enzymes were regulated by feedback inhibition. Aspartokinase was inhibited by L-threonine and concertedly inhibited by L-threonine plus L-lysine. The homoserine dehydrogenase was NADP-specific and was inhibited by L-threonine. Separation of the two enzymes by ammonium sulphate fractionation was possible in Acetobacter peroxydans, A. rancens and Gluconobacter melanogenus but not in A. liquefaciens or G. oxydans.

Stimulation by organic solvents and detergents of conversion of L‐sorbose to L‐sorbosone by Gluconobacter melanogenus IFO 3293

Treatment of Gluconobacter melanogenus IFO 3293 cells with benzene, carbon tetrachloride, cyclohexane, deoxycholate, toluene, or xylene stimulated their conversion of L-sorbose to L-sorbosone two- to threefold. The degree of stimulation depended upon the length of exposure time to the agent and the age of the G. melanogenus cells. A rapid decrease in viability of the cells and degradation of cell RNA was noted after treatment with the effective agents. The G. melanogenus cells were unable to absorb L-sorbose actively after toluene treatment.

New mechanisms for the biosynthesis and metabolism of 2-keto-L-gulonic acid in bacteria.

L-Sorbose is oxidized to 2-keto-L-gulonic acid (KGA) via the following sequence of reactions which we call the "sorbosone pathway": L-sorbose in equilibrium L-sorbosone leads to KGA. The first step is reversible and is mediated by enzymes found in a soluble fraction obtained from Pseudomonas putida ATCC 21812. Although no cofactor requirements were found for the forward reaction, the reverse reaction clearly required NADH. Enzymes for this NADH-dependent synthesis of L-sorbose could be differentiated on the basis of molecular weights. The second step in the sorbosone pathway is catalyzed by a particulate enzyme found in extracts from P. putida and Gluconobacter melanogenus IFO 3293. The rate limiting reaction in the sorbosone pathway is the synthesis of L-sorbosone. In addition to P. putida, Klebsiella pneumoniae (ATCC 27858) and Serratia marcescens (ATCC 27857) also contain the enzymes which catalyze the reactions of the sorbosone pathway. Two of the bacteria studied, P. putida and G. melanogenus, also contain an enzyme involved in the further metabolism of KGA to L-idonic acid. This enzyme, referred to as KGA-reductase, is found in the soluble fraction of cell-free extracts and is dependent on NADH or NADPH.

Conversion of L‐sorbose to L‐sorbosone by Gluconobacter melanogenus

AbstractGrowing cultures, washed cells, and cell‐free extracts of Gluconobacter melanogenus IFO 3293 were found to convert L‐sorbose to L‐sorbosone. The product was identified by thin layer chromatography of the 2, 4‐dinitrophenylhydrazone, and by paper partition chromatography using chemically prepared materials as standards. Factors influencing the conversion included incubation temperature and composition of the growth medium. Addition of betaine or choline to the growing cultures stimulated conversion of L‐sorbose to L‐sorbosone.