Biochemical Aspects of 2-Keto-l-gulonate Accumulation from 2,5-Diketo-d-gluconate by Corynebacterium sp. and Its Mutants

Corynebacterium sp. SHS 0007 accumulated 2-keto-l-gulonate and 2-keto-d-gluconate simultaneously with 2,5-diketo-d-gluconate utilization. This strain, however, possibly metabolized 2,5- diketo-d-gluconate through two pathways leading to d-gluconate as a common intermediate: via 2- keto-d-gluconate, and via 2-keto-l-gulonate, l-idonate and 5-keto-d-gluconate. A polysaccharide- negative, 2-keto-l-gulonate-negative and 5-keto-d-gluconate-negative mutant produced only calcium 2-keto-l-gulonate from calcium 2,5-diketo-d-gluconate, in a 90.5 mol% yield. The addition of a hydrogen donor such as d-glucose was essential for its production. This mutant possessed the direct oxidation route of d-glucose to d-gluconate, the pentose cycle pathway and a possible Embden-Meyerhof-Parnas pathway, indicating that d-glucose was metabolized through these three pathways and provided NADPH for the reduction of 2,5-diketo-d-gluconate.     

Production of 4-Keto-D-arabonate by Oxidative Fermentation with Newly Isolated Gluconacetobacter liquefaciens

Production of 4-keto-D-arabonate (4KAB) was confirmed in a culture medium of Gluconacetobacter liquefaciens strains, newly isolated from water kefir in Argentina. The strains rapidly oxidized D-glucose, D-gluconate (GA), and 2-keto-D-gluconate (2KGA), and accumulated 2,5-diketo-D-gluconate (25DKA) exclusively before reaching the stationary phase. 25DKA was in turn converted to 4KAB, and 4KAB remained stable in the culture medium. The occurrence of 4KAB was assumed by Ameyama and Kondo about 50 years ago in their study on the carbohydrate metabolism of acetic acid bacteria (Bull. Agr. Chem. Soc. Jpn., 22, 271–272, 380–386 (1958)). This is the first report confirming microbial production of 4KAB.     

d-Gluconate Dehydrogenase, 2-Keto-d-gluconate Yielding,from Gluconobacter dioxyacetonicus: Purification and Characterization

d-Gluconate dehydrogenase catalyzing the oxidation of d-gluconate to 2-keto-d-gluconate was solubilized with Triton X-100 from the membrane of Gluconobacter dioxyacetonicus IFO 3271 and purified to an almost homogeneous state by chromatographies on DEAE-cellulose and CM-Toyopearl in the presence of 0.1% Triton X-100. The enzyme had three subunits with molecular weights of 64,000, 45,000 and 21,000, and contained approximately 2 mol of heme per mol of the enzyme. The prosthetic group of the dehydrogenase was found to be a flavin covalently bound to the enzyme protein. The substrate specificity of the purified enzyme was very strict for d-gluconate and the apparent Michaelis constant for d-gluconate was 2.2 mm. The optimum pH and temperature of the purified enzyme were 6.0 and 40°C, respectively.     

2-Keto-l-gulonic Acid Fermentation

A number of bacterial strains from type culture collections and natural sources were examined in their metabolic characteristics toward sorbitol and l-sorbose.

Paper chromatographic analyses of sorbitol and l-sorbose metabolites obtained from the cultures of various bacteria revealed that the organisms producing 2-keto-l-gulonic acid from sorbitol were merely found in the genera Acetobacter, Gluconobacter and Pseudomonas, whereas those producing the acid from l-sorbose were distributed in the twelve genera of bacteria: Acetobacter, Alcaligenes, Aerobacter, Azotobacter, Bacillus, Escherichia, Gluconobacter, Klebsiella, Micrococcus, Pseudomonas, Serratia and Xanthomonas.

G. melanogenus, which was characterized by excellent production of 2-keto-l-gulonic acid from sorbitol, also formed several other sugars and sugar acids as the sorbitol metabolites. These compounds were identified to be d-fructose, l-sorbose, d-mannonic acid, L-idonic acid, 2-keto-d-gluconic acid and 5-keto-d-mannonic acid, respectively, by means of two-dimensional paper chromatography.

Bacteria producing 2-keto-l-gulonic acid from sorbitol were usually isolated from fruits but not from soil.     

Preparation of Enzymes Required for Enzymatic Quantification of 5-Keto-D-gluconate and 2-Keto-D-gluconate

For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA.     

Screening of Thermotolerant Gluconobacter Strains for Production of 5-Keto-d-Gluconic Acid and Disruption of Flavin Adenine Dinucleotide-Containing d-Gluconate Dehydrogenase

We isolated thermotolerant Gluconobacter strains that are able to produce 5-keto-d-gluconic acid (5KGA) at 37°C, a temperature at which regular mesophilic 5KGA-producing strains showed much less growth and 5KGA production. The thermotolerant strains produced 2KGA as the major product at both 30 and 37°C. The amount of ketogluconates produced at 37°C was slightly less than the amount produced at 30°C. To improve the yield of 5KGA in these strains, we disrupted flavin adenine dinucleotide-gluconate dehydrogenase (FAD-GADH), which is responsible for 2KGA production. Genes for FAD-GADH were cloned by using inverse PCR and an in vitro cloning strategy. The sequences obtained for three thermotolerant strains were identical and showed high levels of identity to the FAD-GADH sequence reported for the genome of Gluconobacter oxydans 621 H. A kanamycin resistance gene cassette was used to disrupt the FAD-GADH genes in the thermotolerant strains. The mutant strains produced 5KGA exclusively, and the final yields were over 90% at 30°C and 50% at 37°C. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase, which is responsible for 5KGA production, increased in response to addition of PQQ and CaCl₂ in vitro when cells were grown at 37°C. Addition of 5 mM CaCl₂ to the culture medium of the mutant strains increased 5KGA production to the point where over 90% of the initial substrate was converted. The thermotolerant Gluconobacter strains that we isolated in this study provide a promising new option for industrial 5KGA production.Gluconobacter is a genus of acetic acid bacteria that are able to oxidize a broad range of sugars, sugar alcohols, and sugar acids, and large amounts of the corresponding oxidized products accumulate in the culture medium. Such “incomplete” oxidation is carried out by membrane-bound enzymes, whose catalytic sites face the periplasm. These enzymes catalyze the dehydrogenization of d-glucose, d-sorbitol, d-mannitol, glycerol, d-gluconate, and the keto-d-gluconates. All of these enzymes are firmly attached to the cytoplasmic membrane, and the electrons abstracted from the substrates are passed on to ubiquinone and then to terminal ubiquinol oxidases, forming simple respiratory chains which create the membrane potential necessary to produce biological energy for these microorganisms.The oxidation of d-glucose to ketogluconates is known to be catalyzed by a series of enzymes. Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase oxidizes d-glucose to glucono-δ-lactone, and then gluconolactonase converts the glucono-δ-lactone to d-gluconate. The formation of ketogluconates in Gluconobacter strains has been reported to be catalyzed by two types of membrane-bound gluconate dehydrogenases (GADH) (10). One type is flavin adenine dinucleotide (FAD)-GADH, an FAD-containing, 2-keto-d-gluconate (2KGA)-producing enzyme, and the other type is a PQQ-containing, 5-keto-d-gluconate (5KGA)-producing enzyme. The former enzyme has three subunits: an FAD-containing dehydrogenase, a c-type cytochrome subunit containing three hemes, and a small subunit of unknown function (17). The latter enzyme, which produces 5KGA, is identical to the PQQ-containing polyol dehydrogenase (9), which is known as d-arabitol dehydrogenase (1), d-sorbitol dehydrogenase (20), or PQQ-dependent glycerol dehydrogenase (PQQ-GLDH) (2). PQQ-GLDH has broad substrate specificity but high regio- and stereospecificity, and it catalyzes reactions as predicted by the Bertrand-Hudson rule. This enzyme can oxidize d-gluconate only at the C-5 position to produce 5KGA from d-gluconate; however, the affinity of the enzyme for d-gluconate is quite low. The gene encoding this enzyme was cloned from Gluconobacter suboxydans IFO 3255 (11), and two open reading frames (ORFs) were found. One of these ORFs is believed to encode a hydrophobic protein with five membrane-spanning regions, and the other encodes a dehydrogenase subunit similar to that found in several PQQ-dependent enzymes, particularly the PQQ domain of membrane-bound glucose dehydrogenase. In contrast, 2KGA reductase and 5KGA reductase, the NADPH-dependent enzymes located in the cytoplasm, are thought to be involved in gluconate metabolism in the assimilation of 2KGA and 5KGA.5KGA is a useful raw material for the production of tartaric acid and xylaric acid and is used as a precursor for the synthesis of a number of flavor compounds, including 4-hydroxy-5- methyl-2,3-dihydrofuranone-3 (15). Moreover, it has been reported that 5KGA can be used to produce vitamin C by Gray''s method (6, 7), which is different from Reichstein''s method, which is now commonly used in industry. Reichstein''s method requires the use of high temperatures and an organic solvent in processing; however, Gray''s method does not.Most Gluconobacter strains produce both 2KGA and 5KGA from d-gluconate. Thus, production of 5KGA by Gluconobacter species generates 2KGA as a major by-product, and production of the two ketogluconates is competitive in vivo. Recently, an FAD-GADH-defective mutant strain of Gluconobacter oxydans 621 H which produced almost exclusively 5KGA from d-glucose was discovered (5). However, the optimum temperature for production of 5KGA in this mesophilic strain was around 20°C (19). For cost-effective industrial synthesis of 5KGA, we sought to develop a Gluconobacter strain which is able to produce 5KGA at higher temperatures, such as 37°C, in order to reduce the cost of cooling during fermentation.We successfully isolated thermotolerant Gluconobacter strains that are able to produce 5KGA at 37°C. We cloned the FAD-GADH gene and constructed FAD-GADH-defective mutants that produced almost exclusively 5KGA from d-gluconate at both ambient temperatures and higher temperatures up to 37°C. We believe that the thermotolerant strains reported in this study should be useful for industrial 5KGA production.     

Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1-phosphate. The K _m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k _cat was 5.4 s^-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s^-1) k _cat followed by 6-deoxy-D-glucose (17 s^-1) and 2-deoxy-D-glucose (16 s^-1). The natural substrate, D-glucose with the k _cat of 8.0 s^-1 had the highest (1.1×10⁴ M^-1 s^-1) k _cat/K _m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.     

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 so far which could accumulate a large amount of 5- keto-d-fructose from d-sorbitol except this bacterium.

• The experimental results shown in this paper makes the prediction that a certain dehydrogenating system of l-sorbose is functional in the organism, and the metabolic pathways of d-sorbitol via l-sorbose and 5-keto-d-fructose is proposed.

     

Characterization of an Extracellular Polysaccharide Elaborated by TX-1, a New Strain of Beijerinckia indica

An extracellular polysaccharide elaborated by a new species of Beijerinckia indica, named TX-1, was composed of D-glucose, L-fucose, D-glycero-D-manno-heptose, and D-glucuronic acid in a molar ratio of 5.0:1.0:2.0:0.9, in addition to 16.2% of the acetyl group. Among the polysaccharides of the Beijerinckia species, the present polysaccharide might be the first acidic type having an L-fucose residue. A methylation analysis, Smith degradation study and fragmentation analysis show that this polysaccharide consisted of non-reducing terminal D-glucose, O-4 substituted D-glucose, O-2 substituted D-glycero-D-manno-heptose, O-4 substituted D-glucuronic acid, O-3 and O-4 substituted D-glucose, and O-3 substituted L-fucose residues. A D-glucuronic acid residue was linked to the O-3 position of the L-fucose residue by an α-glycosidic linkage. Most of the D-glucose residues in the backbone chain were substituted at the O-3 position, with the side chain having non-reducing terminal D-glucose residues. It is suggested by the reaction with Con A that the anomeric configuration of the terminal D-glucose residues was β.     

Isolation and Characterization of Thermotolerant Gluconobacter Strains Catalyzing Oxidative Fermentation at Higher Temperatures

Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1% D-sorbitol or 1% D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10°C to 37°C and had average optimum growth temperature between 30-33°C. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37°C. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36-99.79%. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37°C, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264^T was unable to do such fermentation at 37°C. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100% within 24 h at 30°C. Even oxidative fermentation of D-fructose done at 37°C, the strain CHM16 still accumulated D-fructose at 80% within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37°C was superior to that observed at 30°C. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.     

Preparation of [1-13C]D-Glucose 6-Phosphate from [13C]Methanol and D-Ribose 5-Phosphate with Methylotrophic Enzymes

[¹³C]Formaldehyde was selectively incorporated into the C-1 position of D-fructose 6-phosphate by condensation with D-ribulose 5-phosphate catalyzed by a partially purified enzyme system for formaldehyde fixation in Methylomonas aminofaciens 77a. Much of the [1-¹³C]D-fructose 6-phosphate produced in this reaction was converted to [1-¹³C]D-glucose 6-phosphate by the addition of glucose-6-phosphate isomerase. A fed-batch reaction with periodic additions of the substrates afforded 56.2 g/liter D-glucose 6-phosphate and 26.8g/liter D-fructose 6-phosphate. When [¹³C]methanol was used as the C₁-donor, the yield of [1-¹³C]D-glucose 6-phosphate was high when alcohol oxidase was added. The optimum conditions for sugar phosphate production in the fed-batch reaction gave 45.6g/liter [1-¹³C]D-glucose 6-phosphate and 16.4g/liter [1-¹³C]D-fructose 6-phosphate in 165min. The molar yield of the total sugar phosphates to methanol added was 95%. The addition of H₂O₂ and catalase to the reaction system supplied molecular oxygen for methanol oxidation to formaldehyde by alcohol oxidase.     

Purification,Characterization, and Molecular Cloning of a Pyranose Oxidase from the Fruit Body of the Basidiomycete,Tricholoma matsutake

A new H₂O₂-generating pyranose oxidase was purified as a strong antifungal protein from an arbuscular mycorrhizal fungus, Tricholoma matsutake. The protein showed a molecular mass of 250 kDa in gel filtration, and probably consisted of four identical 62 kDa subunits. The protein contained flavin moiety and it oxidized D-glucose at position C-2. H₂O₂ and D-glucosone produced by the pyranose oxidase reaction showed antifungal activity, suggesting these compounds were the molecular basis of the antifungal property. The V _max, K _m, and k _cat for D-glucose were calculated to be 26.6 U/mg protein, 1.28 mM, and 111/s, respectively. The enzyme was optimally active at pH 7.5 to 8.0 and at 50°C. The preferred substrate was D-glucose, but 1,5-anhydro-D-glucitol, L-sorbose, and D-xylose were also oxidized at a moderate level. The cDNA encodes a protein consisting of 564 amino acids, showing 35.1% identity to Coriolus versicolor pyranose oxidase. The recombinant protein was used for raising the antibody.     

Efficient Production of 2,5-Diketo-d-Gluconate via Heterologous Expression of 2-Ketogluconate Dehydrogenase in Gluconobacter japonicus

2,5-Diketo-d-gluconate (2,5DKG) is a compound that can be the intermediate for d-tartrate and also vitamin C production. Although Gluconobacter oxydans NBRC3293 produces 2,5DKG from d-glucose via d-gluconate and 2-keto-d-gluconate (2KG), with accumulation of the product in the culture medium, the efficiency of 2,5DKG production is unsatisfactory because there is a large amount of residual d-gluconate at the end of the biotransformation process. Oxidation of 2KG to 2,5DKG is catalyzed by a membrane-bound flavoprotein-cytochrome c complex: 2-keto-gluconate dehydrogenase (2KGDH). Here, we studied the kgdSLC genes encoding 2KGDH in G. oxydans NBRC3293 to improve 2,5DKG production by Gluconobacter spp. The kgdS, kgdL, and kgdC genes correspond to the small, large, and cytochrome subunits of 2KGDH, respectively. The kgdSLC genes were cloned into a broad-host-range vector carrying a DNA fragment of the putative promoter region of the membrane-bound alcohol dehydrogenase gene of G. oxydans for expression in Gluconobacter spp. According to our results, 2KGDH that was purified from the recombinant Gluconobacter cells showed characteristics nearly the same as those reported previously. We also expressed the kgdSLC genes in a mutant strain of Gluconobacter japonicus NBRC3271 (formerly Gluconobacter dioxyacetonicus IFO3271) engineered to produce 2KG efficiently from a mixture of d-glucose and d-gluconate. This mutant strain consumed almost all of the starting materials (d-glucose and d-gluconate) to produce 2,5DKG quantitatively as a seemingly unique metabolite. To our knowledge, this is the first report of a Gluconobacter strain that produces 2,5DKG efficiently and homogeneously.     

Biochemical characteristics of maltose phosphorylase MalE from Bacillus sp. AHU2001 and chemoenzymatic synthesis of oligosaccharides by the enzyme

ABSTRACT

Maltose phosphorylase (MP), a glycoside hydrolase family 65 enzyme, reversibly phosphorolyzes maltose. In this study, we characterized Bacillus sp. AHU2001 MP (MalE) that was produced in Escherichia coli. The enzyme exhibited phosphorolytic activity to maltose, but not to other α-linked glucobioses and maltotriose. The optimum pH and temperature of MalE for maltose-phosphorolysis were 8.1 and 45°C, respectively. MalE was stable at a pH range of 4.5–10.4 and at ≤40°C. The phosphorolysis of maltose by MalE obeyed the sequential Bi–Bi mechanism. In reverse phosphorolysis, MalE utilized d-glucose, 1,5-anhydro-d-glucitol, methyl α-d-glucoside, 2-deoxy-d-glucose, d-mannose, d-glucosamine, N-acetyl-d-glucosamine, kojibiose, 3-deoxy-d-glucose, d-allose, 6-deoxy-d-glucose, d-xylose, d-lyxose, l-fucose, and l-sorbose as acceptors. The k_cat(app)/K_m(app) value for d-glucosamine and 6-deoxy-d-glucose was comparable to that for d-glucose, and that for other acceptors was 0.23–12% of that for d-glucose. MalE synthesized α-(1→3)-glucosides through reverse phosphorolysis with 2-deoxy-d-glucose and l-sorbose, and synthesized α-(1→4)-glucosides in the reaction with other tested acceptors.     

Efficient Production and Purification of Extracellular 1,2-α-L-Fucosidase of Bacillus sp. K40T

When Bacillus sp. K40T was cultured in the presence of L-fucose, 1,2-α-L-fucosidase was found to be produced specifically in the culture fluid. The enzyme was purified to homogeneity from a culture containing only L-fucose by chromatography on hydroxylapatite and chromatofocusing. The molecular weight of the enzyme was estimated to be 200,000 by gel filtration on Sephadex G-200. The enzyme was optimal at pH 5.5–7.0 and was stable at pH 6.0–9.0. The enzyme hydrolyzed the α(1 → 2)-L-fucosidic linkages in various oligosaccharides and glycoproteins such as lacto-N-fucopentaose (LNF)-I 〈O-α-L-fucose-(1 → 2)-O-β-D-galactose-(1 → 3)-N-acetyl-O-β-D-glucosamine-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉, porcine gastric mucin, and porcine submaxillary mucin. The enzyme also acted on human erythrocytes, which was confirmed by the hemagglutination test using Ulex anti-H lectin. The enzyme did not hydrolyze α(1 → 3)-, α-(1 → 4)- and α-(1 → 6)-L-fucosidic linkages in LNF-III 〈O-β-D-galactose-(1 → 4)[O-α-L-fucose-(1 → 3)-]-N-acetyl-O-β-D-glucosamine-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉, LNF-II 〈O-β-D-galactose-(1 → 3)[O-α-L-fucose-(1 → 4)-]-N-acetyl-O-β-D-galactose-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉 or 6-O-α-L-fucopyranosyl-N-acetylglucosamine.     

Practical Production of 6-O-Octanoyl-D-allose and Its Biological Activity on Plant Growth

The transesterification of D-allose (the C-3 epimer of D-glucose) with vinyl octanoate using Candida antarctica lipase in tetrahydrofuran proceeded with high regioselectivity to produce 6-O-octanoyl-D-allose with nearly complete conversion. The growth-inhibiting activity of 6-O-octanoyl-D-allose on lettuce seedlings was about 6-fold greater than that of D-allose.     

2-Keto-l-gulonic Acid Fermentation

l-Sorbose metabolism in Pseudomonas aeruginosa IFO 3898 was studied. When the strain was cultivated in l-sorbose medium, l-idonic and 2-keto-l-gulonic acids were detected in the culture broth.

From the results on the metabolism of various sugars and sugar acids with the cell suspension and the metabolites accumulated, the following pathway was proposed for the l-sorbose metabolism in Ps. aeruginosa IFO 3898.

l-Sorbose → l-idose → l-idonic acid → 2-keto-l-gulonic acid.     

Studies on Glucose Isomerase of Bacteria

A bacterial strain, HN-500, having an activity of d-glucose isomerization was newly isolated from soil, and was identified to be similar to Escherichia intermedia (Werkman and Gillen) Vaughn and Levine. The strain, grown on wide varieties of carbon sources, shows definitely d-glucose isomerizing activity in the presence of arsenate. d-Fructose formed in reaction mixture was identified by paper chromatography and was isolated in crystalline form from calcium-fructose complex. In order to increase the production of d-glucose isomerase, d-glucose and ammonium nitrogen were effective carbon and nitrogen sources, respectively, but none of the metallic ions tested were effective, furthermore manganese, ferrous and ferric ions present mOre than 10^-5m in growth medium fully repressed the enzyme formation. The cells grown on carbon sources other than d-xylose showed no activity of d-xylose isomerase.     

A single membrane-bound enzyme catalyzes the conversion of 2,5-diketo-d-gluconate to 4-keto-d-arabonate in d-glucose oxidative fermentation by Gluconobacter oxydans NBRC 3292

4-Keto-d-arabonate synthase (4KAS), which converts 2,5-diketo-d-gluconate (DKGA) to 4-keto-d-arabonate (4KA) in d-glucose oxidative fermentation by some acetic acid bacteria, was solubilized from the Gluconobacter oxydans NBRC 3292 cytoplasmic membrane, and purified in an electrophoretically homogenous state. A single membrane-bound enzyme was found to catalyze the conversion from DKGA to 4KA. The 92-kDa 4KAS was a homodimeric protein not requiring O₂ or a cofactor for the conversion, but was stimulated by Mn²⁺. N-terminal amino acid sequencing of 4KAS, followed by gene homology search indicated a 1,197-bp open reading frame (ORF), corresponding to the GLS_c04240 locus, GenBank accession No. CP004373, encoding a 398-amino acid protein with a calculated molecular weight of 42,818 Da. An Escherichia coli transformant with the 4kas plasmid exhibited 4KAS activity. Furthermore, overexpressed recombinant 4KAS was purified in an electrophoretically homogenous state and had the same molecular size as the natural enzyme.     

Isolation of Oligosaccharides Corresponding to the Branching-point of Konjac Mannan

Ultracentrifugically homogeneous glucomannan acetate derived from konjac mannan was subjected to acetolysis. Besides β-1,4-linked oligosaccharides composed of D-mannose and/or D-glucose, three oligosaccharides corresponding to the branching point of the polysaccharide were isolated and identified as (1) 3-O-β-D-mannopyranosyl-D-mannose, (2) O-β-D-mannopyranosyl-(1→4)-O-β-D-mannopyranosyl-(1→3)-D-mannose, and (3) O-β-D- mannopyranosyl-(1→3)-O-β-D-mannopyranosyl-(1→4)-D-glucose. The average chain length (CL) was, moreover, determined to be about 46 by methylation analysis. The structural pattern of the glucomannan, including the branching point, is discussed.