Gluconate Dehydrogenase Research Articles

2-Ketogluconic acid production by acetobacter pasteurianus

Production of 2-ketogluconic acid in batch fermentation was investigated.Acetobacter pasteurianus ATCC 6438, which produces selectively 2-ketogluconic acid only, was used. The optimal pH for glucose dehydrogenation to gluconate by resting cells was 5.0 and for gluconate dehydrogenation to 2-ketogluconate 4.25. When glucose medium was used, the 89% yield was achieved after 90 h. For the optimal productivity, medium containing glucose and gluconate with the molar glucose:gluconate ratio 7.4 was proposed, and the yield of 92% after 56 h was achieved. This composition of medium led to the elevation of dissolved oxygen concentration during fermentation. It consequently resulted in elevated gluconate dehydrogenase activity being discussed as the rate-limiting activity of the batch production.

The physiological function of periplasmic glucose oxidation in phosphate-limited chemostat cultures of Klebsiella pneumoniae NCTC 418

Periplasmic oxidation of glucose into gluconate and 2-ketogluconate in Klebsiella pneumoniae occurs via glucose dehydrogenase (GDH) and gluconate dehydrogenase (GaDH), respectively. Since, as is shown here, in the presence of glucose, gluconate and 2-ketogluconate are not further metabolized intracellularly the physiological function of this periplasmic route was studied. It was found that periplasmic oxidation of glucose could function as an alternative production route of ATP equivalents. Instantaneous activation of either GDH or GaDH reduced the rate of degradation of glucose via glycolysis and the tricarboxylic acid (TCA) cycle in vivo. Furthermore, aerobic, magnesium- and phosphate-limited chemostat cultures with glucose as the carbon source showed high GDH plus GaDH activities in contrast to nitrogen- and sulphate-limited cultures. However, when fructose, which is not degraded by GDH, was the carbon source, specific oxygen consumption rates under these four conditions were essentially the same. The latter observation suggests that high transmembrane phosphate gradients which are supposedly present under phosphate-limited conditions do not cause high energetic demands due to futile cycling of phosphate ions. In addition, dissipation of the transmembrane phosphate gradient of phosphate-limited cells immediately increased the rate of intracellular glucose degradation. It is concluded that under phosphate-limited conditions (i) extensive futile cycling of phosphate ions is absent and (ii) low concentrations of phosphate ions limit intracellular degradation of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Gluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture.

The metabolism of gluconate by Klebsiella pneumoniae NCTC 418 was studied in continuous culture. Under all gluconate-excess conditions at low culture pH values (pH 4.5-5.5) the majority (70-90%) of the gluconate metabolized was converted to 2-oxogluconate via gluconate dehydrogenase (GADH), although specific 2-oxogluconate production rates under potassium-limited conditions were significantly lower than under other gluconate-excess conditions. At high culture pH values, metabolism shifted towards production of acetate. Levels of GADH were highest at low culture pH values and synthesis was stimulated by the presence of (high concentrations of) gluconate. An increase in activity of the tricarboxylic acid cycle was accompanied by a decrease in GADH activity in vivo and in vitro, suggesting that the GADH serves a role as an alternative energy-generating system. Anaerobic 2-oxogluconate production was found to be possible in the presence of nitrate as electron acceptor. Levels of gluconate kinase were highest when K. pneumoniae was grown under gluconate-limited conditions. Under carbon-excess conditions, levels of this enzyme correlated with the intracellular catabolic flux.

Determination of enzyme activities of energy metabolism in the maturing rat oocyte.

Enzyme activities were determined quantitatively in individual rat oocytes to study their energy metabolism during maturation. Low hexokinase activity and high activities of lactate dehydrogenase and enzymes in the phosphate pathway, i.e., glucose 6-P and 6-P gluconate dehydrogenases, were characteristic of immature oocytes. Hexokinase may be a rate-limiting enzyme that enables oocytes to use glucose as an energy source. During maturation, the activities of hexokinase, phosphofructokinase, and malate dehydrogenase increased significantly, suggesting that the glycolytic pathway, as well as the tricarboxylic acid cycle, developed as the first meiotic division proceeded. In contrast, the activities of glucose 6-P and 6-P gluconate dehydrogenases decreased in maturing oocytes. The observation that the enzyme pattern in mature oocytes resembles more closely that in somatic cells appears to be significant, especially in light of previous studies showing this developmental trend in preimplantation embryos.

Direct Bioelectrocatalysis at Metal and Carbon Electrodes Modified with Adsorbed d-Gluconate Dehydrogenase or Adsorbed Alcohol Dehydrogenase from Bacterial Membranes

Abstract d-Gluconate dehydrogenase, a flavohemoprotein, and alcohol dehydrogenase, a quinohemoprotein, are adsorbed strongly on carbon and metal electrodes. All the electrodes with the adsorbed enzymes produce anodic currents due to the electro-enzymatic oxidation of the substrates, where the adsorbed enzymes donate electrons directly to the electrodes; neither mediators nor promoters are necessary.

Changes in Metabolic Enzyme Activities during Thidiazuron-induced Lateral Budbreak of Apple

The activity of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphate gluconate dehydrogenase (6PGDH), isocitrate dehydrogenase (ICDH), pyruvate kinase (PK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were studied in apple (Malus domestics Borkh.) buds during dormancy and thidiazuron-induced budbreak. When buds were dormant, the activity of the glycolytic enzymes GAPDH and PK and the tricarboxylic acid (TCA) cycle enzyme ICDH was low compared to that in nondormant buds. The activity of these enzymes increased during budbreak, peaked when buds were in the green tip stage just before the start of rapid expansion (at 8 days after thidiazuron treatment), and declined thereafter. The activity of pentose-phosphate cycle enzymes G6PDH and 6PGDH was higher in dormant buds than in nondormant buds. 6PGDH was about twice as high as G6PDH. During budbreak and resumption of growth, G6PDH and 6PGDH activity decreased.

Proline metabolism in senescing leaves of horsegram ( Macrotyloma uniflorum Lam.)

Summary ABA, GA and KN treatments on proline metabolism during senescence were studied in leaf disks of Horsegram ( Macrotyloma uniflorum Lam.) incubated in the dark. Senescence, being accelerated by ABA treatments and retarded by GA and KN treatments, is evidenced from the relative depletion in chlorophyll and protein, and an increase in free amino acids and proline. The enzymes involved in oxidation of proline, proline dehydrogenase and proline oxidase showed no change; the enzymes involved in supplying NADPH for proline biosynthesis, glucose-6-phosphate dehydrogenase and 6-phospho gluconate dehydrogenase were inhibited, suggesting that the accumulated proline in senescing leaf disks of horsegram is due to loss of protein budget.

Influence of dissolved oxygen tension on the production rate of 2,5-diketogluconic acid byGluconobacter melanogenum

Increased constant dissolved oxygen tension (200% related to air saturation at 1 bar) has a positive influence on the direct oxidation of glucose byGluconobacter melanogenum. This effect is pronounced and apparent by the faster production of 2,5-diketogluconic acid, mediated through induction in activity of gluconic acid dehydrogenase.

The influence of the culture pH value on the direct glucose oxidative pathway in Klebsiella pneumoniae NCTC 418.

Klebsiella pneumoniae NCTC 418 was cultured aerobically in chemostat cultures (D = 0.3 h-1; 35 degrees C) under respectively carbon-, phosphate-, potassium-, sulphate-, and ammonia-limited conditions with glucose as the sole carbon and energy source. The effect of the external pH value on glucose metabolism and on the enzymes of the direct glucose oxidative pathway was examined. The pH value of the medium had a profound influence on both the activity and the synthesis of the glucose dehydrogenase and the gluconate dehydrogenase. At pH values ranging from pH 5.5 to pH 6.0 maximal activity and synthesis of these enzymes resulted in a more than 80% conversion of the glucose consumed into gluconate and 2-ketogluconate under potassium- or phosphate-limited conditions. On the other hand, no gluconate and/or 2-ketogluconate production could be detected when K. pneumoniae was cultured at pH 8.0. Whereas the synthesis of gluconate dehydrogenase seemingly was completely repressed, still some glucose dehydrogenase was present. The lack of glucose dehydrogenase activity at pH 8.0 was shown not to be due to the dissociation of the cofactor PQQ from the enzyme.

Taxonomic Diversity of the D-Glucose Oxidation Pathway in the Enterobacteriaceae

Tests allowing the screening of large numbers of enterobacterial strains for the presence of the glucose oxidation pathway (glucose, gluconate, and 2-ketogluconate dehydrogenases) were devised or adapted. A total of 506 strains representing 111 taxa (named species or subspecies and unnamed genomic groups) were studied. The members of the genera Budvicia, Edwardsiella, Leminorella, Providencia, and Xenorhabdus and the species Citrobacter freundii, Erwinia carnegeana, Erwinia carotovora, Erwinia chrysanthemi, Erwinia nigrifluens, Erwinia salicis, Moellerella wisconsensis, Proteus penneri, Proteus vulgaris, Yersinia intermedia, Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia ruckeri were negative in all tests. Five species, Erwinia cypripedii, Ewingella americana, Rahnella aquatilis, Serratia marcescens (at 20°), and Tatumella ptyseos produced 2,5-diketogluconate from glucose without a requirement for pyrroloquinoline quinone (PQQ). When PQQ was provided (required for glucose oxidation), Serratia grimesii and Serratia liquefaciens produced 2,5-diketogluconate from glucose at 20°. Escherichia blattae had gluconate- and 2-ketogluconate dehydrogenases without glucose dehydrogenase. The members of the genera Hafnia, Obesumbacterium, and Pragia had only gluconate dehydrogenase. Other species had glucose dehydrogenase (with or without a requirement for PQQ) with or without gluconate dehydrogenase. Classification and identification may take advantage of tests exploring the glucose oxidation pathway.

Direct Bioelectrocatalysis at Electrodes Modified with d--Gluconate Dehydrogenase

Direct Bioelectrocatalysis at Electrodes Modified with <scp>d-</scp>-Gluconate Dehydrogenase

The role of copper(I)-nicotinic acid complex on kojic acid biosynthesis by Aspergillus flavus.

Addition of cooper-monovalent-nicotinic acid complex to a synthetic medium specific for kojic acid production by Aspergillus flavus enhanced the production by about 47%. The substance is proposed to act via a biochemical utilization of the copper(I)-B3 complex in a manner similar to that of the naturally utilized nicotinic acid. NAD and NADP like carriers with higher reactivity have been predicted. According to this prediction the biosynthetic route of kojic acid has been interpreted on the basis of a model proposed by Bajpai et al. (1981). In this model the enzymes participating are dependent on NAD and NADP (glucose dehydrogenase and gluconate dehydrogenase) as well as on other reduction processes.

Enzymatic studies of bacteria used for distillery waste treatment

Six bacterial strains isolated from sewage waters, acclimatized on spentwash and purified were very effective in the treatment of distillery waste. Because of their apparently extensive metabolic capabilities, eight enzymes were assayed in cell free sonicates of these strains at 8, 20, 48 and 72 h during distillery waste treatment. Hexokinase (EC 2.7.1.1) was found to be a membrane bound enzyme in all the strains. Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), glucose dehydrogenase (EC 1.1.1.47) and gluconate dehydrogenase (EC 1.1.99.3) were found to be more active in the later phases of growth. Glutamate oxaloacetate aminotransferase ( l-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), glutamate pyruvate aminotransferase ( l-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and protease were active throughout the growth period.

Gluconate accumulation and enzyme activities with extremely nitrogen-limited surface cultures of Aspergillus niger.

Batch cultures of Aspergillus niger grown from conidia on a medium with high C/N ratio accumulated gluconate from glucose with a yield of 57%. During almost the whole time of accumulation there was no net synthesis of total protein in the mycelium but the activity per flask and the specific activity of glucose oxidase (EC 1.1.3.4) in mycelial extracts increased whereas both values decreased for glucose dehydrogenase (EC 1.1.99.10) 'gluconate 6-phosphatase' (cf. EC 3.1.3.1, 3.1.3.2), gluconokinase (EC 2.7.1.12), glucose 6-phosphate and phosphogluconate dehydrogenases (EC 1.1.1.49, EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), and most enzymes of the Embden-Meyerhof pathway and the tricarboxylic acid cycle. Gluconate dehydratase (EC 4.2.1.39), gluconate dehydrogenase (EC 1.1.99.3) and enzymes of the Entner-Doudoroff pathway could not be detected. By cycloheximide the increase of glucose oxidase activity was inhibited. It is concluded that the high yield of gluconate was due mainly to the net (de novo) synthesis of glucose oxidase which occurred during protein turnover after the exhaustion of the nitrogen source, and which was not accompanied by a net synthesis of the other enzymes investigated. Some gluconate may also have been formed by hydrolytic cleavage of gluconate 6-phosphate.

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.

Enzymes of gluconate metabolism and glycolysis in Penicillium notatum.

In addition to the ability of Penicillium notatum to grow on sucrose, glucose, fructose and gluconate, substantial growth occurred on 2-ketogluconate and 5-ketogluconate thereby indicating a diverse sugar metabolism. Cell-free extracts contained all the enzymes of the Embden-Meyerhof-Parnas pathway and for both oxidative and non-oxidative pentose phosphate metabolism. Despite inconsistencies in results between different assay methods for the conventional Entner-Doudoroff (ED) enzymes, the data indicated the route was enzymatically possible. Demonstrations of the activities of the enzymes of the non-phosphorylative equivalent of the ED pathway were achieved. No evidence was found of a phosphorylative linking enzyme between the two pathways. Both 2- and 5-ketogluconate reductases were detected along with gluconate dehydrogenase which suggested interconvertibility between the ketogluconates and gluconate. However, ketogluconokinase, responsible for the conversion of ketogluconate to 2-keto-6-phosphogluconate, was not detected. A scheme for the interrelationships of routes of gluconate metabolism is discussed.

Enzymes of glucose metabolism in Frankia sp.

Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures.

Utilization of Gluconate by Aspergillus niger. I. Enzymes of Phosphorylating and Nonphosphorylating Pathways

Gluconate which was produced in cultures of Aspergillus niger with glucose as the sole source of carbon, or which was added after exhaustion of glucose, was utilized by this mold. In cell-free extracts from gluconate degrading mycelia gluconokinase and five enzymes of the pentose phosphate pathway were identified. Enzymes of the Entner-Doudoroff pathway and of a modified (non-phosphorylating) Entner-Doudoroff system, as well as a gluconate dehydrogenase could not be detected. It is concluded that gluconate after its phosphorylation to 6-phosphogluconate is metabolized via the pentose phosphate pathway by the strain used.

Hyperinduction of enzymes of the phosphorylative pathway of glucose dissimilation inPseudomonas cepacia

Glucose-dehydrogenase-deficient (Gcd−) strains ofPseudomonas cepacia 249 compensated for loss of operation of the direct oxidative pathway by expanding the phosphorylative pathway. When grown on glucose, they had between two- and fourfold higher than normal levels of glucokinase and NAD-linked glucose-6-phosphate dehydrogenase activity and a comparable increase in capacity to transport glucose. Similar expansion of the phosphorylative pathway was noted when the wild type was grown on cellobiose or trehalose. Gcd− strains grew normally on cellobiose and trehalose, but not if also deficient in glucokinase; this indicates that the disaccharides were converted to glucose and metabolized via the phosphorylative pathway. The expansion of the phosphorylative pathway during growth of the wild type on disaccharides or of Gcd− mutants on glucose was a consequence of hyperinduction of pathway enzymes. Other compounds that promoted such hyperinduction included aromatic conjugates of glucose such as arbutin and salicin, and mannose. Under conditions leading to expansion of the phosphorylative pathway, enzymes related to the direct oxidative pathway, such as gluconate dehydrogenase and the 6-phosphogluconate dehydrogenase active with NAD, were not formed. The results indicate that intracellular glucose and extracellular glucose are metabolized to 6-phosphogluconate via different routes.

The Role of Oxygen in the Regulation of Glucose Metabolism, Transport and the Tricarboxylic Acid Cycle in Pseudomonas aeruginosa

The effect of dissolved oxygen concentration on the metabolism of glucose in Pseudomonas aeruginosa was studied with chemostat cultures using both single-step and gradual transitions from either ammonium or glucose limitation to oxygen limitation and studying transient and steady states. The pathway of glucose metabolism was regulated by the availability of oxygen. The organism responded to oxygen limitation by adjusting its metabolism of glucose from the extracellular direct oxidative pathway, which produces gluconate and 2-oxogluconate, to the intracellular phosphorylative route. This change was a consequence of decreased activities of glucose dehydrogenase and gluconate dehydrogenase and of the transport systems for gluconate and 2-oxogluconate, and an increased activity of glucose transport, while relatively high activities of hexokinase and glucose-6-phosphate dehydrogenase were maintained. Citrate synthase, isocitrate dehydrogenase and malate dehydrogenase activities responded to changes in dissolved oxygen concentration rather than to changes in the glucose or ammonium concentrations. The effect of oxygen limitation on the oxo-acid dehydrogenases and aconitase was probably due, wholly or in part, to repression by glucose consequent upon the increase in residual glucose concentration. Succinate dehydrogenase was repressed by an increase in ammonium concentration under an oxygen limitation.