Studies on the Reduction of 2,6-Dichlorophenolindophenol by 6-Substituted 3-Hydroxy-4-Pyrones,Using a Stopped-flow Method

The reduction of 2,6-dichlorophenolindophenol (DCIP) by 6-substituted 3-hydroxy-4-pyrones was studied in the pH range of 3.0 to 7.8, using a stopped-flow apparatus.

1) Kinetic characteristics of 6-substituted 3-hydroxy-4-pyrones are shown as the apparent first-order rate constant, K_app, or the second-order rate constant, k. The plot of log k against pH was different from those obtained for the reactions of L-ascorbic acid and triose reductone with DCIP.

2) The log (k/k_H) of 6-substituted 3-hydroxy-4-pyrone solutions at acidic pHs have been correlated with Δδ_C?3 values for 3-hydroxy-4-pyrone.     

Occurrence of 3-Hydroxy-3-methyl-glutaryl Coenzyme A Reductase in Sweet Potato

Illumination of the dark-grown Euglena gracilis, both the wild-green type and a permanently bleached mutant, for 4 hr at 2,000 lux caused about 6-fold increase of the cellular content of total l-ascorbic acid. The increase was mainly due to an increase of reduced-form l-ascorbic acid. From the action spectrum only blue light was found to be effective for the increase. Darkening stopped the increase and reillumination started a renewed increase. The activity of l-gulono-γ-lactone dehydrogenase, catalyzing the last step of l-ascorbic acid biosynthesis, was also increased two fold by illumination for 2 hr, and was changed in parallel to that of the cellular content of l-ascorbic acid depending on the presence or absence of illumination. The augmentation of l-ascorbic acid formation was markedly inhibited by various inhibitors and uncouplers, but not by dichlorophenyldimethylurea. The results in sum suggest that the light-dependent increase of l-ascorbic acid formation in E. gracilis is not primarily associated with photosynthesis, but is apparently related to the adaptation of the dark-grown cells to the illuminated state.     

Monodehydro-2,2′-iminodi-2(2′)-deoxy-l-ascorbic Acid,a Radical Product from the Reaction of Dehydro-l-ascorbic Acid with an α-Amino Acid

One of the radical species produced by the reaction of dehydro-l-ascorbic acid with an α-amino acid gave a very characteristic hyperfine structure in its electron spin resonance spectrum. The same spectrum was also obtained when l-scorbamic acid was oxidized with some oxidants, indicating the formation of the radical via the oxidation of l-scorbamic acid. From the results of deuterium exchange experiments, simulation spectra and the reduction of 2,2′-nitrilodi-2(2′)-deoxy-l-ascorbic acid monoammonium salt, the radical was concluded to be monodehydro-2,2′-iminodi-2(2′)-deoxy-l-ascorbic acid. Possible formation mechanism of the radical was also discussed.     

Crystallization and Characterization of Polyamine Oxidase from Aspergillus terreus

A thin-layer flow cell system for the determination of l-ascorbic acid by an ascorbate electrode was constructed and several components of this system were investigated. The most preferable conditions for optimum operation of the system were as follows: injection volume 150 μ1, delay coil length 60 cm, flow rate 1 ml/min, temperature 20°C, cell spacer thickness 0.2 mm. The linear response region was 0.2-3.0 mm and 0.02-0.5 mm (original l-ascorbic acid concentration) in the cases of pure oxygen and atmospheric oxygen bubbling, respectively. The relative standard variation at 1.5 mm l-ascorbic acid was 3.1 % for 20 successive assays. The measuring time was 2–3 min for each of these assays.     

Current studies on the enzymatic preparation 2-O-α-d-glucopyranosyl-l-ascorbic acid with cyclodextrin glycosyltransferase

2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) is one of the most important l-ascorbic acid derivatives because of its resistance to reduction and oxidation and its easy degradation by α-glucosidase to release l-ascorbic acid and glucose. Thus, AA-2G has commercial uses in food, medicines and cosmetics. This article presents a review of recent studies on the enzymatic production of AA-2G using cyclodextrin glycosyltransferase. Reaction mechanisms with different donor substrates are discussed. Protein engineering, physical and biological studies of cyclodextrin glycosyltransferase are introduced from the viewpoint of effective AA-2G production. Future prospects for the production of AA-2G using cyclodextrin glycosyltransferase are reviewed.     

Optimal Conditions for the Enzymatic Production of l-Aromatic Amino Acids from the Corresponding 5-Substituted Hydantoins

The reaction conditions for the production of l-tryptophan from dl-5-indolyl- methylhydantoin by Flavobacterium sp. AJ-3940, and the cultural conditions for the formation of the enzyme involved by this bacterium were investigated. The optimal pH of this reaction was around 8.5 and the optimal temperature was between 45 to 55°C. The amount of l-tryptophan produced was remarkably increased by the addition of inosine, which formed a water insoluble adduct with l-tryptophan, to the reaction mixture because of the release of end-product inhibition by l-tryptophan. This enzyme was inducibly and intracellularly produced by Flavobacterium sp. AJ-3940 in proportion to the increase in cell growth. Cells showing high activity were obtained using a medium containing 5 g glucose, 5 g (NH₄)₂SO₄, 1 g KH₂PO₄, 3 g K₂HPO₄, 0.1 g MgSO₄ · 7H₂O, 0.01 g CaCl₂ · 2H₂O, 50 ml corn steep liquor and 3.5 g dl-5-indolylmethylhydantoin in a total volume of 1 liter (pH 7.0). Under the best conditions, 43 mg/ml of l-tryptophan was produced from 50 mg/ml of dl-5-indolylmethylhydantoin with a molar yield of 97% in the presence of cells of Flavobacterium sp. AJ-3940. In addition, other l-aromatic amino acids such as l-phenylalanine, l-tyrosine, l-DOPA and related l-amino acids were also produced from the corresponding 5-substituted hydantoins by this bacterium containing the l-tryptophan-producing enzyme induced by dl-5-indolylmethylhydantoin.     

Micropolyamide Thin-layer Chromatography of Fluorescein-thiohydantoin-Amino Acids at Picomole Level

The formation of aromatic l-amino acid decarboxylase in bacteria was studied with intact cells in a reaction mixture containing the aromatic l-amino acids, 3,4-dihydroxy-l-phenyl-alanine, l-tyrosine, l-phenylalanine, l-tryptophan and 5-hydroxy-l-tryptophan. Activity was widely distributed in such genera as Achromobacter, Micrococcus, Staphylococcus and Sarcina. Bacterial strains belonging to the Micrococcaceae showed especially high decarboxylase activity toward l-tryptophan, 5-hydroxy-l-tryptophan and l-phenylalanine. M. percitreus AJ 1065 was selected as a promising source of aromatic l-amino acid decarboxylase. Results of experiments with this bacterium showed that the aromatic amine formed from l-tryptophan by the enzymatic method was identical with tryptamine. M. percitreus constitutively produced an enzyme which exhibited decarboxylase activity toward l-tryptophan. However, when large amounts of the aromatic l-amino acids listed above or the tryptamine formed from l-tryptophan were added, enzyme formation was repressed.

Cells with high enzyme activity were prepared by cultivating this bacterium at 30°C for 24 hr in a medium containing 0.5% glycerol, 0.5% yeast extract, 0.5% Polypepton, 3.0 vol % soybean protein hydrolyzate, 0.1% KH₂PO₄, 0.1% MgSO₄ · 7H₂O, 0.001% FeSO₄ · 7H₂O and 0.001% MnSO₄ · 5H₂O in tap water (pH 8.0).     

Resolution of the Chrysanthemic Acids with l-Lysine

A single-step synthesis of 3-O-ethyl-l-ascorbic acid was performed without the induction of protecting groups. Sodium l-ascorbate reacted with ethyl bromide in DMSO to give 3-O-ethylascorbic acid in a yield of 51.0%. 3-O-Ethylascorbic acid enhanced dibutyryl cyclic AMP-induced neurite outgrowth in PC12 cells.     

Inducibility of Rat Liver Drug-Metabolizing Enzymes as an Index of Food-Screeing for Safety Assessment

D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat?K _m) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s^?1, and 100 (mmol/L)^?1 s^?1 respectively.     

Degradation of Ovalbumin by L-Ascorbic Acid in the Presence of Cupric Ion

Ovalbumin, L-ascorbic acid and cupric sulfate were allowed to react at pH 3.0, 6.0, 6.8 and 7.5. Non-proteinous nitrogen compounds were formed from ovalbumin coupled with autoxidation of ascorbic acid, and a pronounced increase in their formation was observed in the reactions of neutral pH ranges. Non-proteinous nitrogen compounds contained peptides, free amino acids and ammonia. In the reactions of ovalbumin with triose reductone similar results to those with ascorbic acid were obtained. In the ovalbumin degraded with ascorbic acid at pH 6.8 was found an increase of N-terminal amino acid, which could react with carbonyl compounds resulting in browning.     

l-Glutamic Acid Fermentation with Molasses

When an l-Glutamic acid (l-GA)-forming bacterium, Microbacterium ammoniaphilum, was cultured in the molasses medium with the addition of penicillin to accumulate large quantity of l-GA extracellularly, no significant differences were observed in the phospholipid quantity and the fatty acid composition which were found between the l-GA-accumulating cells grown either in the molasses medium with addition of polyoxyethylene fatty acid ester (POEFE) or in the glucose medium with the addition of biotin.

Moreover, it was shown that, in the molasses-POEFE system, the amount of l-GA accumulated was nearly constant, independent of the extracellular osmotic pressure caused by the presence of NaNO₃ or β-alanine, while, in the molasses-penicillin system, the amount varied inversely to the osmotic pressure.

From these results, it is assumed that either chemical or mechanical process can eliminate the permeability barrier in the cell membrane, thus allowing the extracellular accumulation by l-GA-forming bacteria.     

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.     

Microbiological Studies of Coli-aerogenes Bacteria

α-Ketoglutarate was formed from the various carbohydrates including lactose, maltose, sucrose, d-glucose, d-fructose, d-galactose, d-mannose, d-mannitol, l-rhamnose, d-xylose, l-arabinose and glycerin. The influence of pH of the reaction mixture were tested, and inorganic phosphate was observed to be indispensable for α-ketoglutarate-fermentation. A cell of E. coli grown statically on glucose was found to reveal an ability of producing α-ketoglutarate under aerobic conditions. Optically dextro lactic acid was potent in the formation of a-ketoglutaric acid. The following reagents revealed the inhibiting effect on α-ketoglutarate-fermentation; CuSO₄, AgNO₃, iodoacetate, 2, 4-dinitrophenol, NaN₃, 3-sulfanilamido-6-methoxypyridazine and arsenite, while, kanamycin and 8-azaguanine has no inhibiting effect. When E. coli was grown in a glucose-medium, a small supply of air increased the yield of acetate against decreasing α-ketoglutarate.     

The Contribution of Arabidopsis Homologs of L-Gulono-1,4-lactone Oxidase to the Biosynthesis of Ascorbic Acid

To clarify the involvement of seven Arabidopsis homologs of rat L-gulono-1,4-lactone (L-GulL) oxidase, AtGulLOs, in the biosynthesis of L-ascorbic acid (AsA), transgenic tobacco cells overexpressing the various AtGulLOs were generated. Under treatment with L-GulL, the levels of total AsA in three transgenic tobacco cell lines, overexpressing AtGulLO2, 3, or 5, were significantly increased as compared with those in control cells.     

Substrate Specificity of Alkaline Proteinases from Aspergillus sydowi and Aspergillus sulphureus

l-Fucose (l-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1143 and purified about 380-fold with a yield of 23 %. The purification procedures were: treatment with polyethyleneimine, ammonium sulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of about 34,000. The optimum pH was at 9 — 10.5 and the isoelectric point was at pH 5.1. l-Fucose and l-galactose were effective substrates for the enzyme reaction, but d-arabinose was not so much. The anomeric requirement of the enzyme to l-fucose was the β-pyranose form, and the reaction product from l-fucose was l-fucono- lactone. The hydrogen acceptor for the enzyme reaction wasNADP⁺, and NAD ⁺ could be substituted for it to a very small degree. Km values were 1.9mm, 19mm, 0.016mm, and 5.6mm for l-fucose, l- galactose, NADP⁺, and NAD⁺, respectively. The enzyme activity was strongly inhibited by Hg^{2 +}, Cd^{2 +}, and PCMB, but metal-chelating reagents had almost no effect. In a preliminary experiment, it was indicated that the enzyme may be usable for the measurement of l-fucose.     

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp

Branched chain amino acid aminotransferase was partially purified from Pseudomonas sp. by ammonium sulfate fractionation, aminohexyl-agarose and Bio-Gel A-0.5 m column chromatography.

This enzyme showed different substrate specificity from those of other origins, namely lower reactivity for l-isoleucine and higher reactivity for l-methionine.

Km values at pH 8.0 were calculated to be 0.3 mm for l-leucine, 0.3 mm for α-ketoglutarate, 1.1 mm for α-ketoisocaproate and 3.2 mm for l-glutamate.

This enzyme was activated with β-mercaptoethanol, and this activated enzyme had different kinetic properties from unactivated enzyme, namely, Km values at pH 8.0 were calculated to be 1.2 mm for l-leucine, 0.3 mm for α-ketoglutarate.

Isocaproic acid which is the substrate analog of l-leucine was competitive inhibitor for pyridoxal form of unactivated and activated enzymes, and inhibitor constants were estimated to be 6 mm and 14 mm, respectively.     

Purification and Crystallization of d-Arabinose (l-Fucose) Isomerase from Aerobacter aerogenes by Polyethylene Glycol

d-Arabinose(l-fucose) isomerase (d-arabinose ketol-isomerase, EC 5.3.1.3) was purified from the extracts of d-arabinose-grown cells of Aerobacter aerogenes, strain M-7 by the procedure of repeated fractional precipitation with polyethylene glycol 6000 and isolating the crystalline state. The crystalline enzyme was homogeneous in ultracentrifugal analysis and polyacrylamide gel electrophoresis. Sedimentation constant obtained was 15.4s and the molecular weight was estimated as being approximately 2.5 × 10⁵ by gel filtration on Sephadex G-200.

Optimum pH for isomerization of d-arabinose and of l-fucose was identical at pH 9.3, and the Michaelis constants were 51 mm for l-fucose and 160 mm for d-arabinose. Both of these activities decreased at the same rate with thermal inactivation at 45 and 50°C. All four pentitols inhibited two pentose isomerase activities competitively with same Ki values: 1.3–1.5 mm for d-arabitol, 2.2–2.7 mm for ribitol, 2.9–3.2 mm for l-arabitol, and 10–10.5 mm for xylitol. It is confirmed that the single enzyme is responsible for the isomerization of d-arabinose and l-fucose.     

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.     

Production of l-Threonine by Analog-resistant Mutants

Mutants resistant to α-amino-β-hydroxyvaleri0c acid (AHV) were derived from various bacteria which belong to Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium, or Bacillus by mutational treatment with N-methyl-N′-nitro-N-nitrosoguanidine(NTG), and screened for their ability to produce l-threonine. A number of l-threonine producers were obtained from each group of bacteria. Among them, the mutants derived from C. glutamicum KY9159(Met^?) were further mutagenized with NTG to derive thialysine(S-Lys)-resistant mutants. An AHV-resistant mutant, KY10484 was proved to be much more sensitive to the growth inhibition by thialysine than the parent strain, KY9159. From KY10484, a number of AHV- and thialysine-resistant mutants were derived. Approximately a half of these mutants were found to produce more l-threonine than KY10484. Among these mutants, KY10440 (Met^?, AHV^R, s-Lys^R) was used to investigate the cultural conditions for l-threonine production. The growth of KY10440 decreased largely with addition of l-homoserine, a threonine precursor. l-Asparagine, l-cystine, l-glutamine or l-arginine partially reversed the inhibitory effect of l-homoserine. Addition of these amino acids at low level led to increase l-threonine production. The amount of l-threonine accumulation reached to a level of 14mg/ml with a medium containing 10% glucose and to a level of 10 mg/ml with a medium containing 5% molasses (as glucose).

Another AHV- and thialysine-resistant mutant, KY10251 which was also derived from KY9159 was found to produce both 9 mg/ml of l-threonine and 5.5 mg/ml of l-lysine in a culture broth.     

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.