Studies on Glucose Isomerase of Bacteria

A bacterial strain, HN-56, having an activity of d-glucose isomerization was isolated from soil, and was identified to be similar to Aerobacter aerogenes (Kruse) Beijerink. d-Glucose-isomerizing activity was induced when HN-56 was precultured in the media containing d-xylose, d-mannose, lactate, especially d-mannitol. Paper chromatography showed that the ketose formed in reaction system containing d-glucose was d-fructose alone. The optimum pH for the reaction was 6.5~7.0. Sulfhydryl reagents inhibit the reaction, but metal inhibitors affect little if any. With the washed living cells as enzyme source, only arsenate could accumulate d-fructose. In addition, the cells grown with d-mannitol and d-mannose showed no activity of d-xylose isomerase.     

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.

     

Polyol Dehydrogenases in the Soluble Fraction of Acetobacter melanogenum

Polyol dehydrogenases of Acetobacter melanogenum were investigated. Three polyol dehydrogenases, i. e. NAD⁺-linked d-mannitol dehydrogenase, NAD⁺-linked sorbitol dehydrogenase and NADP⁺-linked d-mannitol dehydrogenase, in the soluble fraction of the organism were purified 12-fold, 8-fold and 88-fold, respectively, by fractionation with ammonium sulfate and DEAE-cellulose column chromatography. NAD⁺-linked sorbitol dehydrogenase reduced 5-keto-d-fructose (5KF) to l-sorbose in the presence of NADH, whereas NADP⁺-linked d-mannitol dehydrogenase reduced the same substrate to d-fructose in the presence of NADPH. It was also shown that NAD⁺-linked d-mannitol dehydrogenase was specific for the interconversion between d-mannitol and d-fructose and that this enzyme was very unstable in alkaline conditions.     

Crystalline d-Mannitol:NAD Oxidoreductase from Leuconostoc mesenteroides

The crystalline d-mannitol dehyrogenase (d-mannitol:NAD oxidoreductase, EC 1.1.1.67) catalyzed the reversible reduction of d-fructose to d-mannitol. d-Sorbitol was oxidized only at the rate of 4% of the activity for d-mannitol. The enzyme was inactive for all of four pentitols and their corresponding 2-ketopentoses. The apparent optimal pH for the reduction of d-fructose or the oxidation of d-mannitol was 5.35 or 8.6, respectively. The Michaelis constants were 0.035 m for d-fructose and 0.020 m for d-mannitol. The enzyme was also found to be specific for NAD. The Michaelis constans were 1 × 10^?5 m for NADH₂ and 2.7 × 10^?4 m for NAD.     

d-Ribose Formation by Pseudomonas reptilivora

During the course of some works on sugar metabolism in bacteria, we could find out bacteria having producibility of free d-ribose. Among 1395 strains isolated from soil, only nine strains were found to be able to produce aldopentose which was identified chromato-graphically as ribose. From cultured broth of Pseudomonas reptilivora S-1104, a representative strain among these nine strains, d-ribose was isolated in crystalline form as aldopentose. It was also found that ribose was formed not only from glucose but also from d-fructose, d-arabitol, gluconic acid, etc., and that d-fructose and a glucoside (remained unknown) were also accumulated at the same time in the culture broth of Pseudomonas reptilivora S-1104.     

Studies on Sugar-isomerizing Enzyme

A glucose isomerase which reversibly catalyzes the reaction between d-glucose and d-fructose was demonstrated in the cell-free extracts of a strain of Streptomyces sp. isolated from soil. The enzyme was produced when the strain was grown in the medium containing xylan or xylan-containing material such as wheat bran. A medium which consists of 3% of wheat bran, 2% of corn steep liquor and 0.024% of CoCl₂·6H₂O is recommendabie for the production of the glucose isomerase enzyme with the strain. With the enzyme, some conditions for the conversion of d-glucose to d-fructose were also studied. The method is very useful for the production of invert sugar from d-glucose and is now on the way to be applied to the practical use.     

Kinetic and Equilibrium Studies on d-Mannose-d-Fructose Isomerization Catalyzed by Mannose Isomerase from Streptomyces aerocolorigenes

The equilibrium constant of the isomerization reaction between d-mannose and d-fructose which is catalyzed by a mannose isomerase from Streptomyces aerocolorigenes was obtained by using three methods over the temperature range from 1 to 40°C.

It was found that the equilibrium constant was scarcely dependent on temperature, ΔH, the heat of the formation of d-fructose from d-mannose, being approximately zero.

The standard free energy change, ΔG, and the standard entropy change, ΔS, of the reaction were calculated from the equilibrium constants at various temperatures and ΔH. The values of ΔG and ΔS at 25°C were ?650 cal/mole and + 2.2 cal/deg·mole, respectively.

By combining these thermodynamic data with those obtained for the isomerization reaction between d-glucose and d-fructose reported in the previous paper, ΔH, ΔG and ΔS for the isomerization between d-mannose and d-glucose were indirectly obtained to be +2220 cal/mole, +830 cal/mole and +4.6 cal/deg·mole at 25°C, respectively.     

Detection of Carboxymethyl Cellulase Activity in Acetobacter xylinum KU-1

We detected carboxymethyl cellulase activity in a crude extract of Acetobacter xylinum KU-1. The enzyme activity was detected when glycerol, d-fructose, d-mannitol, d-glucose, d-arabitol, d-sorbitol, or carboxymethyl cellulose was used as a carbon source. The optimum pH was found to be 4.0, while the optimum temperature was 50°C. The enzyme activity was inhibited characteristically by the addition of Hg²⁺.     

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.     

Studies on d-Glucose-isomerizing Activity of d-Xylose-grown Cells from Bacillus coagulans,Strain HN-68

d-Glucose-isomerizing enzyme has been extracted in high yield from d-xylose-grown cells of Bacillus coagulans, strain HN-68, by treating with lysozyme, and purified approximately 60-fold by manganese sulfate treatment, fractionation with ammonium sulfate and chromatography on DEAE-Sephadex column. The purified d-glucose-isomerizing enzyme was homogeneous in polyacrylamide gel electrophoresis and ultracentrifugation and was free from d-glucose-6-phosphate isomerase. Optimum pH and temperature for activity were found to be pH 7.0 and 75°C, respectively. The enzyme required specifically Co⁺⁺ with suitable concentration for maximal activity being 10^?3 m. In the presence of Co⁺⁺, enzyme activity was inhibited strongly by Cu⁺⁺, Zn⁺⁺, Ni⁺⁺, Mn⁺⁺ or Ca⁺⁺. At reaction equilibrium, the ratio of d-fructose to d-glucose was approximately 1.0. The enzyme catalyzed the isomerization of d-glucose, d-xylose and d-ribose. Apparent Michaelis constants for d-glucose and d-xylose were 9×10^?2 m and 7.7×10^?2 m, respectively.     

Mechanisms of Browning Degradation of d-Fructose in Special Comparison with d-Glucose-Glycine Reaction

Degradation mechanisms of d-fructose by the interaction with amino acids or organic acids in aqueous solution at initial pH 5.5 heated at 100°C were investigated and a substantial difference in mechanisms between fructose degradation and glucose-glycine reaction was presented. d-Fructose browned more intensely than did d-glucose in lower concentration of glycine and/or in earier stage of reaction period. By catalytic action of carboxylate anions without any condensation with amino groups, d-fructose was decomposed to 3-deoxy-d-erythrohexosulose, 5-(hydroxymelhyl)-2-furaldehyde, and a less amount of pyruval-dehyde through caramelization. It was considered that the main path of fructose degradation was 1,2-enolization but 2,3-enolization would also occur to a little extent.     

Inactivation of Bacteriophage ϕX174 by d-Fructose 6-Phosphate

The inactivation of bacteriophage ?X174 by d-fructose 6-phosphate was investigated. This inactivation was inhibited by EDTA or reducing agents, and stimulated by Cu²⁺ but other metal ions could not be substituted for Cu²⁺. The reaction was also inhibited by superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and various free radical scavengers.

No detectable changes were observed in adsorption capacity of phage and in the conformation of the virion. The viral DNA in the virion was, however, found to be cleaved. This strand scission was also enhanced by Cu²⁺ and protected by catalase. Similar results were obtained when ?X174 DNA was directly treated with d-fructose 6-phosphate.

It is concluded that the inactivation of ?X174 is due to DNA strand scission in the virion by the free radical of d-fructose 6-phosphate or oxygen radicals generated during autoxidation of d-fructose 6-phosphate.     

Prodigiosin-25 C

The equilibrium constant of the isomerization reaction between d-glucose and d-fructose which is catalyzed by a. glucose isomerase from Streptomyces sp. was obtained by both methods of chemical analysis and of kinetic study over the temperature range of 25° to 70°C.

It was found that the formation of d-fructose from d-glucose was an endothermic reaction with the heat of the reaction, ΔH, of +2220 cal/mole. The standard free energy change, ΔG, and the standard entropy change, ΔS, associated with the isomeric change were found to be +180 cal/mole and + 6.8 cal/deg. mole at 25°C, respectively. The values of these thermodynamic quantities at other temperature are also summarized.     

A Modified Procedure for the Synthesis of Sucrose

A modified procedure for synthesis of sucrose was carried out by using purified crystalline 1,3,4,6-tetra-O-benzoyl-d-fructofuranose in place of 1,3,4,6-tetra-O-acetyl-d-fructofuranose; namely, reaction of 1,2-anhydro-3,4,6-tri-O-acetyl-α-d-glucopyranose with tetrabenzoyl-d-fructose at 110~118°C for 3 hr followed by deacylation, and by chromatographic separation of the deacylated product afforded crude sucrose. Acetylation of the synthetic product gave octaacetate sucrose in the yield of 5.0%.     

Studies on Glucose-Isomerizing Enzyme of Bacteria

d-Glucose-isomerizing enzyme from Escherichia intermedia HN-500, which converts d-glucose to d-fructose in the presence of arsenate, was purified by treating with manganous sulfate, rivanol, and DEAE-Sephadex column chromatography. About 180-fold purified enzyme preparation was obtained by the above procedures. The purified preparation was free from the activities of d-glucose-, d-galactose-, glucose-6-phosphate-, mannitol-, and sorbitol-dehydrogenases and was homogeneous on polyacrylamide gel in zone electrophoresis. Optima of pH and temperature for the enzyme were found to be pH 7.0 and 50°C, respectively. The enzyme was completely inactivated by heating at 60°C for ten minutes and stable in the pH range of 7.0~9.0 at 30°C. Activation energy for the isomerizing enzyme was calculated to be 15,300 calories per mole degree from Arrhenius' equation. Either in the absence or presecne of arsenate, d-mannose, d-xylose, d-mannitol and d-sorbitol could not be isomerized by the purified enzyme at all, but the present enzyme isomerized exclusively glucose-6-phosphate and fructose-6-phosphate in the absence of arsenate.     

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.     

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.     

Studies on Isomerization of Sugars by Bacteria

An enzyme, which catalyzes the isomerization of d-glucose to d-fructose, has been found in a newly isolated bacterium which tentatively identified as Pacacolobacterum aerogenoides. The enzyme converts not only d-glucose but also d-mannose to d-fructose, and NAD and Mg⁺⁺ are required as cofactor for this isomerization. The properties of this enzyme were summarized as follows: (1) As a cofactor for the isomerization by this enzyme, NAD was absolutely necessary, whereas NADP, FMN and FAD were not. (2) The optimum pH was found to be at 7.5 and optinum temperature was at about 40°C. (3) The enzyme activity was markedly reduced by EDTA treatment and the reduced activity by EDTA was restored by the addition of Mg⁺⁺, Mn⁺⁺ or Co⁺⁺. (4) The enzyme activity was strongly inhibited by monoiodoacetate, p-chloromercuribenzoate, and Cu⁺⁺, however, the activity was recovered by adding cysteine or glutathione.     

Biological Activities of Sex Hormone Produced by Tremella mesenteriea

d-Aminoacylase was found to be produced not only by S. olivaceus 62–3 isolated from soil but also by three strains of type culture of Streptomyces species. All four of these strains produced d-aminoacylase intracellularly only when an inducer was added to the culture medium. d-Amino acids or N-acetyl-d-amino acids were effective as inducers.

As S. tuirus showed the highest d-aminoacylase activity, the enzyme extract of this strain was subjected to further investigation to determine the optimal conditions for optical resolution of N-acetyl-dl-phenylglycine. Almost all contaminating l-aminoacylase in the enzyme extract could be eliminated by DEAE-Sephadex adsorption. d-Phenylglycine of 99.9% optical purity was obtained after complete hydrolysis of d-isomer with the use of d-aminoacylase solution.     

A new pathway for the synthesis of oligosaccharides by the use of non-Leloir glycosyltransferases

The growing recognition of the roles of carbohydrates in fundamental biological processes and their potential application as functional foods and new therapeutics have generated a need for larger amounts of different carbohydrate structures. Leloir glycosyltransferases catalyze the synthesis of complex oligosaccharides. However they are difficult or expensive to obtain, and require expensive nucleotide activated sugars. In contrast non-Leloir pathway enzymes use sucrose, which is known to be a high energy donor of d-glucose for glucosyltransferases like dextransucrase, or a donor of d-fructose for fructosyltransferases like inulin- and levansucrases for the synthesis of polysaccharides. Here we present the synthesis and kinetic studies of oligosaccharides using non-Leloir glycosyltransferases and sucrose analogues as new substrates, like β-d-fructofuranosyl-α-d-galactopyranoside (Gal-Fru) by a fructosyltransferase (FTF) from B. subtilis NCIMB 11871. The sucrose analogues carry a high binding energy in the glycosidic bond similar to that of sucrose. Thus, β-d-Fructofuranosyl-α-d-galactopyranoside (Gal-Fru) and β-d-Fructofuranosyl-α-d-fucopyranoside (d-Fuc-Fru) have been shown to be substrates for fructosyltransferases, which produce oligo- or polysaccharides, also in the presence of acceptors.