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 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.     

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 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.     

Change in the Regulation of Enzyme Synthesis under Catabolite Repression in Bacillus subtilis Pleiotropic Mutant Lacking Transketolase

The regulation of enzyme synthesis has changed in Bacillus subtilis pleiotropic mutant lacking transketolase (tkt). The tkt mutant is hypersensitive to d-glucose repression of the synthesis of d-mannitol catabolic enzymes, such as d-mannitol-1-phosphate dehydrogenase and d-mannitol transport system. d-Gluconate, d-xylose and l-arabinose are also effectors for repression in the tkt mutant. In contrast, the synthesis of sorbitol catabolic enzymes, such as sorbitol permease and sorbitol dehydrogenase, are almost insensitive to d-glucose repression. These changes in the regulation of enzyme synthesis seem to be related to some defect in the cell surface structure of the tkt mutant by which other pleiotropic properties are also generated.     

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.     

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.     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

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-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.     

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 an Enzyme Capable of Splitting β-d-1,6-Glucosidic Linkage

In the previous paper, it was reported that the higher molecular weight luteose which consisted of β-1,6-linked d-glucose units was made by Pen. aculeatutn var. apiculatum. The isolation of the microorganism which is able to produce β-d-1,6-glucanase and some properties of this glucanase were described in this paper. The optimum reaction temperature for this enzyme was at about 40°C, and the optimum pH was at about 6.7. At the maximum degree of the hydrolysis, only d-glucose and gentiobiose were detected, and at this point, the hydrolysis degree of higher molecular weight luteose was about 55% as d-glucose. The formation of β-d-1,6-glucanase was induced by the addition of higher molecular weight luteose. From the property of this enzyme, the authors proposed the name “lutease” for this enzyme.     

Production of l-rhamnulose,a rare sugar,from l-rhamnose using commercial immobilized glucose isomerase

Abstract

A commercial immobilized d-glucose isomerase from Streptomyces murines (Sweetzyme) was used to produce l-rhamnulose from l-rhamnose in a packed-bed reactor. The optimal conditions for l-rhamnulose production from l-rhamnose were determined as pH 8.0, 60?°C, 300?g L^?1 l-rhamnose as a substrate, and 0.6?h^?1 dilution rate. The half-life of the immobilized enzyme at 60?°C was 809?h. Under the optimal conditions, the immobilized enzyme produced an average of 135?g L^?1 l-rhamnulose from 300?g L^?1 l-rhamnose after 16 days at pH 8.0, 60?°C, and 0.6?h^?1 dilution rate, with a productivity of 81?g/L/h and a conversion yield of 45% in a packed-bed reactor.     

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.     

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.     

Isopullulanase from Arthrobacter globiformis T6

The physico-chemical properties of the purified glucose isomerases [d-xylose ketol isomerase, EC 5.3.1.5] of Streptomyces olivochromogenes and Bacillus stearothennophilus were examined. The molecular size and shape of both enzymes were similar. The molecular weights, sedimentation coefficients, partial specific volumes, diffusion constants and Stokes’ radii of the Streptomyces and Bacillus enzymes were determined to be 120,000 and 130,000, 7.55 S and 9.35 S, 0.725 and 0.736 ml/g, 5.87 × 10^-7 and 6.82 × 10^-7 cm²/sec, and 51 and 53 Å, respectively. The Streptomyces glucose isomerase was found to consist of two subunits, each having a molecular weight of 56,000. Large differences were found in the amino acid compositions of these two enzymes, especially in their serine, proline, tyrosine, lysine and arginine contents. The enzymatic properties of both these purified glucose isomerases were also examined, and it was seen that they both displayed activity on d-xylose, d-xylulose, d-glucose, d-fructose, d-arabinose and d-ribose. The smaller Km values and the larger molecular activities for d-xylose and d-xyluIose indicated that both enzymes are essentially d-xylose isomerases. The optimum temperature was 80°C for both enzymes. The optimum pH was 8 to 10 for the Streptomyces enzymes and 7.5 to 8.0 for the Bacillus enzyme. The Bacillus enzyme was more thermostable than the Streptomyces enzyme, but required cobalt ions in addition to magnesium ions for the full expression of its activity.     

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.     

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.     

Production of l-allose and d-talose from l-psicose and d-tagatose by l-ribose isomerase

l-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert l-psicose and d-tagatose to l-allose and d-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce l-allose and d-talose. Conversion reaction was performed with the reaction mixture containing 10% l-psicose or d-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of l-allose and d-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert l-psicose to l-allose without remarkable decrease in the enzyme activity over 7 times use and d-tagatose to d-talose over 37 times use. After separation and concentration, the mixture solution of l-allose and d-talose was concentrated up to 70% and crystallized by keeping at 4 °C. l-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% l-allose and 7.30% d-talose that were obtained from l-psicose and d-tagatose, respectively.     

The Studies on the Action of Yeast Hexokinase upon the α- and β-Diasteromers of d-Glucose

The yeast hexokinase is highly specific for α-isomer of d-glucose. The relative rate of phosphorylation of β-d-glucose, catalyzed by the purified yeast hexokinase, is observed to be 60~70 (α-d-glucose=100). The average Michaelis constants of yeast hexokinase are found to be 1.8 × 10^?4 and 2.4 × 10^?4 for α-d-glucose and (β-d-glucose respectively, therefore the difference between the two constants is considered to be negligible.