L-Threonine Production by a Mutant of Arthrobactev paraffineus

An isoleucine leaky auxotroph of Arthrobacter paraffineus, which was isolated by Takayama et al.³⁾ as a mutant producing L-threonine and L-valine from n-paraffin, was subjected to further mutagenesis in an attempt to obtain better L-threonine producers. Some of the double auxotrophs derived from the isoleucine auxotroph and some of their revertants with respect to isoleucine requirement produced more L-threonine than the original isoleucine auxotroph. In contrast to the original isoleucine auxotroph, a revertant derived from a methionine plus isoleucine double auxotroph, KY7135, produced an increased amount of L-threonine and a decreased amount of L-valine. The optimum level of L-methionine for L-threonine production in KY7135 was much higher (1000 ~ 2000 μg/ml) with n-paraffin medium than with sorbitol or mannitol medium (10 ~ 50 μg/ml). L-Threonine production reached a maximum level (11.5 mg/ml) in 7 days incubation with the medium containing 10% n-paraffin (C₁₂ ~ C₁₄ rich). Several mutants which produce L-threonine more than 12 mg/ml were obtained from KY 7135 by monocolony isolation procedure.     

Preparation of Optically Active Allothreonine by Separating from a Diastereoisomeric Mixture with Threonine

A simple procedure is described to obtain D- and L-allothreonine (D- and L-aThr). A mixture of N-acetyl-D-allothreonine (Ac-D-aThr) and N-acetyl-L-threonine (Ac-L-Thr) was converted to a mixture of their ammonium salts and then treated with ethanol to precipitate ammonium N-acetyl-L-threoninate (Ac-L-Thr·NH₃) as the less-soluble diastereoisomeric salt. After separating Ac-L-Thr·NH₃ by filtration, Ac-D-aThr obtained from the filtrate was hydrolyzed in hydrochloric acid to give D-aThr of 80% de, recrystallized from water to give D-aThr of >99% de. L-aThr was obtained from a mixture of the ammonium salts of Ac-L-aThr and Ac-D-Thr in a similar manner.     

Formations of Oligopeptides from Dipeptides by the Protease from Streptomyces cellulosae

The protease from Streptomyces cellulosae formed more turbidity in a 16% soybean protein hydrolysate in the initial stage of the reaction than α-chymotrypsin did, when the proteolytic activity of the protease was same as that of α-chymotrypsin. In highly concentrated solutions (2.5%) of various dipeptides, oligopeptides were produced by condensation by the protease. The oligopeptides formed were (l-Leu-Gly)₂ and (l-Leu-Gly)₃ from l-Leu-Gly, (l-Phe-l-Val)₂ from l-Phe-l-Val, (l-Val-l-Phe)₂ and (l-Val-l-Phe)₃ from l-Val-l-Phe, and (l-Leu-l-Met)₂ and (l-Leu-l-Met)₃ from l-Leu-l-Met.     

Starch-hydrolyzing Enzymes in Germinating Kidney Bean

Enzymatic production of D-Glu was investigated by the succesive reactions of a glutamate racemase (EC 5.1.1.3) and a glutamate decarboxylase (EC 4.1.1.15) on L-Glu.Lactobacillus brevis ATCC8287 was chosen as a source of glutamate racemase. This strain produced a glutamate decarboxylase simultaneously. The glutamate racemase activity in the cell free extracts was 0.035 units/mg protein. The enzyme kept its activity even at 500 Mm of L-Glu (74g/liter). The optimum pHs of the racemase and the decarboxylase were at around 8.5 and below 4.0, respectively. Both enzymes had no activity at the optimum pH for the other enzyme. L-Glu was racemized first by the glutamate racemase at pH 8.5, then the pH was shifted to 4.0 at which L-Glu was decarboxylated by the glutamate decarboxylase. Starting from 100 g/liter of L-Glu, 50 g/liter of D-Glu was produced and no L-Glu remained in the reaction mixture.     

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.     

Enzyme-Substrate Complex of p-Hydroxybenzoate Hydroxylase; One of the Activated Intermediates of the Overall Reaction

The transglucosidation reaction of brewer’s yeast α-glucosidase was examined under the co-existence of l-sorbose and phenyl-α-glucoside. As the transglucosidation products, three kinds of new disaccharide were chromatographically isolated. It was presumed that these disaccharides consisting of d-glucose and l-sorbose were 1-O-α-d-glucopyranosyl-l-sorbose ([α]_D+89.0), 3-O-α-d-glucopyranosyl-l-sorbose ([α]_D+69.1) and 4-O-α-d-glucopyranosyl-l-sorbose ([α]_D+81.0). The principal product formed in the enzyme reaction was 1-O-α-d-glucopyranosyl-l-sorbose.     

5′-Nucleotidase from Yeast,Having Special Affinity for 5′-AMP

Three kinds of diketopiperazines which have retarditive activity for the growth of plant seedlings and plant roots at concentrations ranging from 1 : 2,500 to 1 : 100,000, were isolated from the neutral fraction by extracting the cultured broth of Rosellinia necatrix. These three diketopiperazines have been proved to be l-prolyl-l-leucine anhydride, l-prolyl-l-valine anhydride and l-prolyl-l-phenylalanine anhydride respectively, and the last one seems to be a new diketo-piperazine.

Furthermore, a crystalline wax having m.p. 52°C, a physiologically inactive substance, was also isolated from the same neutral fraction and presumed to be the saturated hydrocarbon of n-pentacosane C₂₅H₅₂.     

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.     

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.     

Regulatory Properties of Chorismate Mutase from Corynebacterium glutamicum

Regulatory properties of chorismate mutase from Corynebacterium glutamicum were studied using the dialyzed cell-free extract. The enzyme activity was strongly feedback inhibited by l-phenylalanine (90% inhibition at 0.1~1 mm) and almost completely by a pair of l-tyrosine and l-phenylalanine (each at 0.1~1 mm). The enzyme from phenylalanine auxotrophs was scarcely inhibited by l-tyrosine alone but the enzyme from a wild-type strain or a tyrosine auxotroph was weakly inhibited by l-tyrosine alone (40~50% inhibition, l-tyrosine at 1 mm). The enzyme activity was stimulated by l-tryptophan and the inhibition by l-phenylalanine alone or in the simultaneous presence of l-tyrosine was reversed by l-tryptophan. The Km value of the reaction for chorismate was 2.9 } 10^?3 m. Formation of chorismate mutase was repressed by l-phenylalanine. A phenylalanine auxotrophic l-tyrosine producer, C. glutamicum 98–Tx–71, which is resistant to 3-amino-tyrosine, p-aminophenylanaine, p-fluorophenylalanine and tyrosine hydroxamate had chorismate mutase derepressed to two-fold level of the parent KY 10233. The enzyme in C. glutamicum seems to have two physiological roles; one is the control of the metabolic flow to l-phenylalanine and l-tyrosine biosynthesis and the other is the balanced partition of chorismate between l-phenylalanine-l-tyrosine biosynthesis and l-tryptophan biosynthesis.     

Enzymatic activity of L-amino acid oxidase from snake venom Crotalus adamanteus in supercritical CO2

L-amino acid oxidase (L-AAO) from snake venom Crotalus adamanteus was successfully tested as a catalyst in supercritical CO₂ (SC-CO₂). The enzyme activity was measured before and after exposure to supercritical conditions (40°C, 110 bar). It was found that L-AAO activity slightly increased after SC-CO₂ exposure by up to 15%. L-AAO was more stable in supercritical CO₂ than in phosphate buffer under atmospheric pressure, as well as in the enzyme membrane reactor (EMR) experiment. 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) oxidation was performed in a batch reactor made of stainless steel that could withstand the pressures of SC-CO₂, in which L-amino acid oxidase from C. adamanteus was able to catalyze the reaction of oxidative deamination of L-DOPA in SC-CO₂. For the comparison L-DOPA oxidation was performed in the EMR at 40°C and pressure of 2.5 bar. Productivity expressed as mmol-s of converted L-DOPA after 3?h per change of enzyme activity after 3?h was the highest in SC-CO₂ (1.474?mmol?U^?1), where catalase was present, and the lowest in the EMR (0.457?mmol?U^?1).     

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.     

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).     

Dihydrosterigmatocystin and Dihydrodemethylsterigmatocystin,New Metabolites from Aspergillus versicolor

L-Arabinose isomerase (L-arabinose ketol-isomerase, EC 5.3.1.4) was demonstrated from the L-arabinose-grown cells of Streptomyces sp. which was isolated from sea water. The enzyme was purified by MnCl₂ treatment, fractionation by polyethylene glycol and by column chromatographies on Sephadex G-150 and DEAE-cellulose. The purified enzyme was specific only for L-arabinose and the Michaelis constant for L-arabinose was 40 mM at pH 7.5. Manganese or cobalt ions were effective for the enzyme activity after dialysis against EDTA. The enzyme activity was inhibited competitively by L-arabitoI, ribitol and xylitol, of which inhibition constants were 1.1, 1.0, and 15 mM, respectively.     

Regulation of l-Glutamate Biosynthesis by Copper Ions

A specific regulatory effect of copper ions on the microbiological synthesis of l-glutamate from acetate was found. The minimal concentration of copper ions necessary for the maximal production of l-glutamate was about 0.025 µg/ml at which the yield of l-glutamate was four times greater than that in the absence of copper ions. This effect of copper was demonstrated only when acetate was the substrate; it was not observed when the substrate was glucose ethanol, lactate or n-paraffin.

The physiological features of the l-glutamate production from acetate were examined in the presence or absence of copper ions. The most striking features of the culture without added copper ions were the increase in Q_O2 and NADH oxidase and the marked reduction of succinate oxidase accompanied with the reduction of l-glutamate formation. In addition, the regulation of l-glutamate synthesis by copper ions proved to have no relation to the wellknown regulatory factor, cell permeability. These facts suggest that the l-glutamate biosynthesis from acetate is regulated through unknown factors related to the respiratory activities.     

Structures of the Oligosaccharides from the Enzymic Hydrolyzate of Rice-straw Arabinoxylan by a Xylanase of Aspergillus niger

A trisaccharide consisting of two d-xylose units and one l-arabinose unit, and a tetrasaccharide consisting of three d-xylose units and one l-arabinose unit were isolated from the hydrolyzate of rice-straw arabinoxylan by the xylanase I produced by Asp. niger.

The structures of the trisaccharide and the tetrasaccharide were determined to be 3¹-α-l-arabinofuranosylxylobiose ([α]_d^? 80°) and 3¹-α-l-arabinofuranosylxylotriose ([α]_d^? 84°), respectively, by chemical and enzymic methods.

According to the structures of two arabinose-xylose mixed oligosaccharides, it was shown that the rice-straw arabinoxylan is composed of chain of 1,4-linked βd-xylopyranose residues and some of xylose residues have side-chain of 1,3-linked α-l-arabinofuranose.     

Radiation Chemistry of Foods

An intermediate radical, ?H₂OH, was produced in aqueous methanol solution containing nitrous oxide by γ-irradiation. Yields of ethylene glycol and formaldehyde, the major and the minor product from ?H₂OH, respectively, changed on the addition of some solutes. Cysteine lowered the both product yields to zero even at a low concentration of 5 × 10^?5m. Oxygen of low concentrations (2.5~7.5 × 10^?5 m) changed effectively the major product from ethylene glycol to formaldehyde. k _(CySH+?H2OH)/k_(O2+?H2OH) was calculated as 0.5.

Ascorbic acid (5 × 10^?5 m) lowered ethylene glycol yield to 48%, cystine (10^?3m) to 15%, methionine (10^?3m) to 31%, histidine (10^?3m) to 42%, tryptophan (10^?3m) 46%, tyrosine (10^?3m) to 77%, phenylalanine (10^?3m) to 73%, hypoxanthine (10^?3m) to 37%, adenine (10^?3m) to 52%, uracil (10^?3m) to 20%, thymine (10^?3m) to 10%, cytosine (10^?3 m) to 49%, rutin (10^?3m) to 23%, pyrogallol (10^?3m) to 41%, and gallic acid (10^?3m) to 78% of the control. These results suggest that the reactions of the secondary radicals such as ?H₂OH perform an important role in material change of foods irradiated with γ rays.     

Amino Acid Metabolism in Microorganisms

Certain Streptomyces strains were found to accumulate an unknown substance in culture broth when the microorganisms were grown in the medium containing dl-methionine. The substance was isolated from the culture broth as hydrochloride and was identified as 3-methylthiopropylamine (MTPA), decarboxylated product of methionine, from its melting point, chemical composition, infrared spectrum, and other properties. Cultural conditions for MTPA formation in Streptomyces sp. K 37 were investigated. The yield of MTPA from l-methionine reached about 90% with a culture medium containing corn steep liquor. Namely, 6.47 mg of MTPA per millilitre of culture broth was produced from 10 mg of l-methionine per millilitre of the growth medium. The transforming activity was found in the cells of the early culture period. MTPA-producing activity was induced by l- methionine in the medium. d-Methionine was not utilized as a substrate of the reaction with intact cells. Optimum pH for the reaction appeared to be 6.0~8.0.     

Amino Acid Metabolism in Microorganisms

Certain strains of Streptomyces were found to convert l-methionine into 3-methylthio-propylamine (MTPA), but not d-methionine. Now, optical resolution of DL-methionine was attempted using this phenomenon. Streptomyces sp. K37 was cultured in a medium containing DL-methionine (10 mg/ml). The culture filtrate was applied to a column of Diaion SA-21A (OH form). MTPA was recovered from the effluent by ether exraction. The Diaion SA-21A was eluted with 1N HCl and the eluate was applied to a column of Diaion SK-1 (H form). d-Methionine was eluted from the column with 1N NH₄OH and recovered after concentration, decolorization with active carbon, and precipitation with ethanol. The yields of MTPA and d-methionine from the broth were 69.5% and 89.5%, respectively.     

Studies on the Chemical Structure of Konjac Mannan

The electrophoretically homogeneous glucomannan isolated from konjac flour was composed of d-glucose and d-mannose residues in the approximate ratio of 1: 1.6. Controlled acid hydrolysis gave 4-O-β-d-mannopyranosyl-d-mannose, 4-O-β-d-mannopyranosyl-d-glucose_T 4-O-β-d-glucopyranosyl-d-glucose(cellobiose), 4-O-β-d-glucopyranosyl-d-mannose(epicellobiose), O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-glucopyranosyl- (1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-mannopyranosyl-(1→4)-O-β-d-glucopy- ranosyl-(1→4)-d-mannose and O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-d-mannose.