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.     

Enzymatic Synthesis of α-d-Glucopyranosyl-2-deoxy-d-glucoses by Transglucosylation Reaction of Buckwheat α-Glucosidase

The transglucosylation reaction of buckwheat α-glucosidase was examined under the coexistence of 2-deoxy-d-glucose and maltose. As the transglucosylation products, two kinds of new disaccharide were chromatographically isolated in a crystalline form (hemihydrate). It was confirmed that these disaccharides were 3-O-α-d-glucopyranosyl-2-deoxy-d-glucose ([α]_d + 132°, mp 130 ~ 132°C, mp of ±-heptaacetate 151 ~ 152°C) and 4-O-±-d-glucopyranosyl-2-deoxy-d-glucose ([±]_d + 136°, mp 168 ~ 170°C), respectively. The principal product formed in the enzyme reaction was 3-O-±-d-glucopyranosyl-2-deoxy-d-glucose.     

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⁺.     

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.     

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 Conversion of d-Glucose to d-Fructose

Glucose isomerizing enzyme was partially purified after investigation on the properties of crude enzyme extract. The crude extract was partly inactivated by the contact with air. The addition of manganese was effective to improve the stability. Magnesium was essential to the enzyme action and cobalt accelerated the reaction.

The maximal activity was observed at pH about 7.6 and 50°C was optimal for the incubation time of 30 minutes. The enzyme solution reacted with d-xylose as well as d-glucose. The activity of the enzyme was inhibited at high glucose concentrations.

An enzyme which catalyzes the conversion of d-glucose to d-fructose has been demonstrated in cell-free extracts of Streptomyces phaeochromo genus grown in the presence of D-xylose. The enzyme preparation reacts with d-glucose and d-xylose, but not with other sugars tested. It appears to require magnesium for the maximal activity and the addition of cobaltous ion remarkably intensifies the heat tolerance of the enzyme. The maximal activity occurs at about pH 9.3~9.5. Equilibrium is reached when about 52% fructose is present in the reaction mixture. The enzyme has half-maximal activity when the concentration of d-glucose is about 0.3 M at pH 9 and 60°C.     

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.     

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.     

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.     

Characterization of α-Glucosyltransferase from Pseudomonas mesoacidophila MX-45

α-Glucosyltransferase was purified from Pseudomonas mesoacidophila MX-45. The molecular weight was estimated to be 63,000 by SDS–PAGE, and the isoelectric point was pi 5.4. For enzyme activity based on sucrose decomposition, the optimum pH and the optimum temperature were pH 5.8 and 40°C, respectively. The ranges of stable pH and temperature were pH 5.1–6.7 and below 40°C, respectively. The purified enzyme of MX-45 converted sucrose into trehalulose (1-O-α-d-glucopyranosyl-d-fructose) and isomaltulose (palatinose, 6–O-α-d-glucopyranosyl-d-fructose) simultaneously, and the ratio of trehalulose to isomaltulose increased at lower reaction temperatures. Therefore, optimum conditions for trehalulose production were pH 5.5–6.5 at 20°C. The yield of trehalulose from sucrose (20–40% solution) was 91%. The K_m for sucrose was 19.2 ± 3.3 mm estimated by the Hanes–Woolf plot. Product inhibition was observed, and the product inhibition constant was 0.17 m. Hg²⁺, Fe³⁺, Cu²⁺, Mg²⁺, Ag⁺, Pb²⁺, glucono-1,5-lactone, and Tris(hydroxymethyl)aminomethane inhibited the reaction.     

A New Trisaccharide, 2-α-Maltosyl-glucose,Synthesized by the Transglycosylation Reaction of Cyclodextrin Glycosyltransferase

The transglycosylation reaction of the cyclodextrin glycosyltransferase from Bacillus megaterium strain No. 5 was examined in the reaction system containing kojibiose and soluble starch. As the transglycosylation product, a new trisaccharide was chromatographically isolated. It was confirmed that the trisaccharide was 2-α-maltosyl-glucose ([α]d + 162.0°, α-undecaacetate: mp 105~106°C, [α]d + 163.0°), α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→2)-α-d-glucose (4²-α-glucosyl-kojibiose).

The transfer action to kojibiose occurred only to the C₄-hydroxyl group of the non-reducing end glucose unit of kojibiose, leading to the formation of 2-α-maltosyl-glucose.     

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.     

Enzymatic Conversion of d-Glucose to d-Fructose

It was found that a bacterial strain, KN-69, which was isolated from soil, converted d-glucose to d-fructose. From the results of investigations of characters, it seems reasonable to conclude that the strain is related to Aerobacter cloacae Bergey et al. The formation of d-fructose in the glucose containing reaction system was confirmed by isolation and characterization of the product.     

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.     

Two novel pyrrolooxazole pigments formed by the Maillard reaction between glucose and threonine or serine

Pyrrolothiazolate formed by the Maillard reaction between l-cysteine and d-glucose has a pyrrolothiazole skeleton as a chromophore. We searched for a Maillard pigment having a pyrrolooxazole skeleton formed from l-threonine or l-serine instead of l-cysteine in the presence of d-glucose. As a result, two novel yellow pigments, named pyrrolooxazolates A and B, were isolated from model solutions of the Maillard reaction containing l-threonine and d-glucose, and l-serine and d-glucose, respectively, and identified as (2R,3S,7aS)-2,3,7,7a-tetrahydro-6-hydroxy-2,5,7a-trimethyl-7-oxo-pyrrolo[2,1-b]oxazole-3-calboxylic acid and (3S,7aS)-2,3,7,7a-tetrahydro-6-hydroxy-5,7a-dimethyl-7-oxo-pyrrolo[2,1-b]oxazole-3-calboxylic acid by instrumental analyses. These compounds were pyrrolooxazole derivatives carrying a carboxy group, and showed the absorption maxima at 300–360 nm under acidic and neutral conditions and at 320–390 nm under alkaline conditions.     

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

A thermophilic spore-forming strain HN-68, only d-xylose grown cells of which have an activity of d-glucose isomerization, was isolated from soil, and identified to be similar to Bacillus coagulans Hammer. The conditions necessary for maximal production of the glucose isomerizing activity by the cells from shaken cultures in d-xylose media were studied. Much higher activities were observed with the cells grown from 14 ~ 16 hours at 40°C on d-xylose medium containing yeast extract, ammonium chloride, manganese sulfate and calcium carbonate. d-Glucose isomerizing activity was also developed inductively by exposing the washed cells grown on d-glucose to d-xylose within one hour. With the use of living cells as an enzyme source, the addition of both cobaltous ion and toluene in reaction system remarkably enhanced the reaction rate of d-glucose isomerization.     

Studies on Mannan in Wood Pulp

The glucomannan isolated from holocellulose pulp of Akamatsu (Pinus densiflora Sieb. et Zucc.) as its triacetate was methylated and the following methylated sugars were obtained by hydrolysis: the 2,3,4,6-tetra-O-methyl ethers of d-glucose and d-mannose (I part) and the 2,3,6-tri-O-methyl ethers of d-mannose and d-glucose (34–37 parts). Periodate oxidation of the glueomannan showed that 1.00 mole of periodate was consumed per mole of hexose unit and 3 moles of formic acid liberated for every 33 hexose units.     

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.     

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%.     

Cellotriose Synthesis Using d-Glucose as a Starting Material by Two Step Reactions of Immobilized β-Glucosidases

We tried to polymerize d-glucose to cellotriose, the smallest substrate for β-1,4-glucan synthesis by the β-transglycosylase of Trichoderma longibrachiatum, without participation of high energy compounds such as nucleotide sugars. A commercial β-glucosidase (sweet almond) showed a typical condensation reaction of d-glucose, producing cellobiose when it was entrapped in a visking tube and incubated in 30% d-glucose solution. The reaction was done with immobilized enzyme covalently bound to Polyacrylamide beads, and entrapped enzyme. Cellobiose (21.0 mg) was obtained from 30 g of d-glucose in a 3-day reaction, where 0.29 unit of the entrapped enzyme preparation was incubated with 100 ml of 30% d-glucose at pH 6.0 and 41°C. Gentiobiose was also produced in the mixture as a minor product. The immobilized β-glucosidase (Sumizyme C) preparation covalently bound to Polyacrylamide beads could catalyze a transglucosylation reaction to produce cellotriose from cellobiose in a good yield without production of gentiobiose. The transfer reaction was optimal at pH 4.8 and 30°C. Cellotriose (11.2 mg) was produced from the reaction mixture containing 68 mg of cellobiose and the enzyme preparation (0.1 unit) after 24-hr of incubation at the optimal conditions. Both immobilized β-glucosidases, sweet almond and Sumizyme C, may be used repeatedly without any loss of the initial activity.