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.     

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.     

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.     

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.     

Acceptor Specificity of Cyclodextrin Glycosyltransferase from Bacillus stearothermophilus and Synthesis of α-D-Glucosyl O-β-D-galactosyl-(1→4)-β-D-glucoside

Bacillus stearothermophilus CGTase had a wider acceptor specificity than Bacillus macerans CGTase did and produced large amounts of transfer products of various acceptors such as D-galactose, D-mannose, D-fructose, D- and L-arabinose, d- and L-fucose, L-rhamnose, D-glucosamine, and lactose, which were inefficient acceptors for B. macerans CGTase. The main component of the smallest transfer products of lactose was assumed to be α-D-glucosyl O-β-D-galactosyl-(l→4)-β-D-glucoside.     

Transglucosylation Catalyzed by Sucrose Phosphorylase from Leuconostoc mesenteroides and Production of Glucosyl-xylitol

The synthesis of glucooligosaccharides from α-D-glucose-1-phosphate by transglucosylation with sucrose phosphorylase from Leuconostoc mesenteroides was studied using the purified enzyme and high performance liquid chromatography. The enzyme had a rather broad acceptor specificity and transferred glucosyl residues to various acceptors such as sugars and sugar alcohols. Especially, 5-carbon sugar alcohols (pentitols), D- and L-arabitol were acceptors equal to D-fructose, which was known as a good acceptor. The transfer product of xylitol formed by the enzyme was investigated. The structure of the product was found to be 4-O-α-D-glucopyranosyl-xylitol (G-X) by acid hydrolysis and ¹³C-nuclear magnetic resonance analysis. G-X is a probable candidate for a preventive for dental caries because it reduced the synthesis of water-insoluble glucan by Streptococcus mutans and kept a neutral pH in the cell suspension.     

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.     

Antitumor Activities and Immunochemical Properties of the Cell-wall Polysaccharides from Aureobasidium pullulans

Delipidated cell walls from Aureobasidium pullulans were fractionated systematically.

The cell surface heteropolysaccharide contains D-mannose, D-galactose, D-glucose, and D-glucuronic acid (ratio, 8.5:3.9:1.0:1.0). It consists of a backbone of (1→6)-α-linked D-mannose residues, some of which are substituted at O-3 with single or β-(1→6)-linked D-galactofuranosyl side chains, some terminated with a D-glucuronic acid residue, and also with single residues of D-glucopyranose, D-galactopyranose, and D-mannopyranose.

This glucurono-gluco-galactomannan interacted with antiserum against Elsinoe leucospila, which also reacted with its galactomannan, indicating that both polysaccharides contain a common epitope, i.e., at least terminal β-galactofuranosyl groups and also possibly internal β-(1→6)-linked galactofuranose residues.

It was further separated by DEAE-Sephacel column chromatography to gluco-galactomannan and glucurono-gluco-galactomannan.

The alkali-extracted β-D-glucan was purified by DEAE-cellulose chromatography to afford two antitumor-active (1→3)-β-D-glucans. One of the glucans (M_r, 1–2 × 10⁵) was a O-6-branched (1→3)-β-D-glucan with a single β-D-glucosyl residue, d.b., 1/7, and the other (M_r, 3.5–4.5 × 10⁵) had similar branched structure, but having d.b., 1/5. Side chains of both glucans contain small proportions of β-(1→6)-and β-(1→4)-D-glucosidic linkages.     

Desalting DNA by Drop Dialysis Increases Library Size upon Transformation

D-Mannitol dehydrogenase (EC 1.1.1.138) was purified and crystallized for the first time from the cell-free extract of Gluconobacter suboxydans IFO 12528. The enzyme was purified about 100-fold by a procedure involving ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, and gel filtration by a Sephadex G-75 column. The enzyme was completely separated from a similar enzyme, NAD-dependent D-mannitol dehydrogenase (EC 1.1.1.67), during enzyme purification. There being sufficient purity of the enzyme at this stage, the enzyme was crystallized, by the addition of ammonium sulfate, to fine needles. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 3.6 s. The molecular mass of the enzyme was estimated to be 50 kDa by SDS-PAGE and gel filtration chromatography. Oxidation of D-mannitol to D-fructose and reduction of D-fructose to D-mannitol were specifically catalyzed with NADP and NADPH, respectively. NAD and NADH were inert for the enzyme. Since the reaction equilibrium declined to D-fructose reduction over a wide pH range, the enzyme showed several advantages for direct enzymatic measurement of D-fructose. Even in the presence of a large excess of D-glucose and other substances, oxidation of NADPH to NADP was highly specific and stoichiometric to the D-fructose reduced.     

Fatty Acids as Inhibitors of Microbial Cell Wall Synthesis

The Maillard reaction of DNA with ketoses was investigated. Several days of incubation of d-fructose 6-phosphate with deoxyribonucleotides or with polymer DNA in an aqueous buffer resulted in the formation of chromophores and fluorophores. Aminoguanidine and sodium cvanoborohydride inhibited the formation of fluorophores. Transition metal ions such as Cu²⁺, Fe³⁺, Fe²⁺, or Mn^{2 +} promoted the formation of chromophores and fluorophores. Metal-chelating agents such as DETAPAC, citrate, and Desferal inhibited the formation of fluorophores. Superoxide dismutase and catalase also inhibited the formation of fluorophores. The transition metal ion-catalyzed autoxidation of d-fructose 6-phosphate or of the Heyns rearrangement products were to be partially involved in the glycation of DNA and subsequent formation of chromophores and of fluorophores.     

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.     

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.     

Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (K_m=12.5 mM), N-acetyl (K_m=2.52 mM), N-propionyl (K_m=0.194 mM), N-butyryl (K_m=0.033 mM), and N-glycyl (K_m =1.11 mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg²⁺, Ni²⁺, Cu²⁺) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.     

Properties of Tyrosinase from Pseudomonas melanogenum

The properties of the tyrosinase from Pseudomonas melanogenum was investigated with the crude enzyme preparation. Optimum temperature and pH of the enzyme were 23°C and 6.8, respectively. l-Tyrosine, d-tyrosine, m-tyrosine, N-acetyl-l-tyrosine and l-DOPA were utilized as a substrate by the enzyme. The value for Km obtained were as follows: l-tyrosine 6.90 × 10^?4 m, d-tyrosine 1.43 ×10^?3 m and l-DOPA 9.90 × 10^?4 m. The enzyme was inhibited by chelating agents of Cu²⁺ l-cysteine, l-homocysteine, thiourea and diethyl-dithiocarbamate and the inhibition was completely reversed by the addition of excess Cu²⁺ From these results it is concluded that the enzyme is a copper-containing oxidase.     

Further Characterization of Ureido Ring Synthetase from Pseudomonas graveolens

Detailed enzymatic properties of the ureido ring synthetase purified from Pseudomonas graveolens were investigated. Nucleotide specificity studies indicated that CTP, UTP, GTP, and ITP were each tenth to one-fifth as active as ATP. The effect of substrate concentration was examined. The Km values for 7,8-diaminopelargonic acid, biotin diaminocarboxylic acid, NaHCO₃, ATP, and MgCl₂ were 1 × 10^?4 M, 4 × 10^?5 M, 1 × 10^?2 m, 5 × 10^?5 M, and 3 × 10^?3 M, respectively. It was elucidated that only ADP was produced from ATP in both the reaction of desthiobiotin synthesis from 7,8-diaminopelargonic acid and biotin synthesis from biotin diaminocarboxylic acid. The reaction was remarkably inhibited by Ni²⁺, Cd²⁺, Cu²⁺, Ag⁺, and As³⁺, while Mn²⁺ remarkably enhanced the enzyme reaction. The reaction was remarkably inhibited by metal-chelating reagents. It was elucidated that ADP had a competitively inhibiting effect on this enzyme reaction. 7,8-DiaminopeIargonic acid, which is the substrate for the desthiobiotin synthesis, competitively inhibited the biotin synthesis from biotin diaminocarboxylic acid. The stoichiometry of the desthiobiotin synthesis indicated that the formation ratio of desthiobiotin to ADP was 1 to 1.     

Functional Characterization of D-Galacturonic Acid Reductase,a Key Enzyme of the Ascorbate Biosynthesis Pathway,from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38–39 kDa, as judged by SDS–PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5±4.5 μM and uronic acids, such as D-galacturonic acid (Km=3.79±0.5 mM) and D-glucuronic acid (Km=4.67±0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H₂O₂, suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Main Polyol Dehydrogenase of Gluconobacter suboxydans IFO 3255, Membrane-bound D-Sorbitol Dehydrogenase,That Needs Product of Upstream Gene,sldB, for Activity

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.     

Purification and Characterization of an Intracellular α-D-Xylosidase II from Aspergillus flavus MO-5

α-D-Xylosidase II activity from Aspergillus flavus MO-5 was increased roughly 5- to 10-fold by use of xylose instead of methyl α-D-xylopyranoside (α-MX) as a carbon source.

The enzyme was purified to an electrophoretically pure state by successive chromatography on Q-Sepharose, Phenyl Superose, PL-SAX, and TSK-gel G3000SWXL. The purified enzyme hydrolyzed isoprimeverose [α-D-xylopyranosyl-(1→6)-D-glucopyranose] and p-nitrophenyl α-D-xylopyranoside (α-p-NPX), but not α-MX or xyloglucan oligosaccharide. The apparent K_m and V_max of the enzyme for α-p-NPX and isoprimeverose were 0.97 mM and 28.0 µmol/min/mg protein, and 47.62 mM and 2.0 µmol/min/mg protein, respectively. This enzyme had an apparent molecular weight of 67,000 by SDS-polyacrylamide gel electrophoresis and 180,000 by gel filtration chromatography (TSK-gel G3000SWXL).

The enzyme showed the highest activity at pH 6.0 and 40°C, and was stable in the pH range from 6.0 to 7.0 and at the temperatures up to 40°C. The activity was inhibited by Cu²⁺, Zn²⁺, Hg²⁺, p-CMB, SDS, Fe³⁺, and N-ethylmaleimide.

This enzyme had nothing in common with α-D-xylosidase I and four α-D-xylosidases reported already.     

Isolation and Characterization of Pectin from Peel of Citrus tankan

A pectin was extracted from the peel of Citrus tankan with a yield of 2.75%. The uronic acid content was 80.0%, and the degree of methoxylation was 63.2%. The pectin was composed of D-GalA, D-Gal, L-Ara and L-Rha in the molar ratio of 100:11.3:3.6:2.6. The molecular weight was estimated to be approximately 9.2×10⁴. The pectin formed a gel by conventional procedures.