Antibacterial Activity of l-Lysine α-Oxidase against rec+ and rec− Strains of Bacillus subtilis

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker’s yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker’s yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

Same Advantages of Using Low Speed Centrifugation for the Purification of Rat Liver Mitochondria

Twelve strains of lactose-fermenting yeast isolated from raw milk were evaluated on β-galactosidase producing ability. The enzymes from the four strains (Tolulopsis versatilis M6, Tolulopsis sphaerica J28, Candida pseudotropicalis B57 and A60), selected by high productivity, showed very similar properties and were characterized by a pH optimum of 7.0 or 7.5 and a relatively low optimal temperature of 30°C. The molecular weights were estimated by gel filtration to be 200,000-233,000. The Km values for o-nitrophenyl-β-d-galactopyranoside were 3.45 mm, 2.09 mm, 3.45 mm and 2.82 mm for enzymes from M6, J28, B57 and A60, respectively. All enzymes were activated by Mn²⁺ and inhibited by Mg²⁺, Zn²⁺ and Ca²⁺. The enzymes are sulfhydryl dependent and were completely inhibited by Hg²⁺ and sulfhydryl reagents. The yeasts may be a potential source for the enzyme for industrial use.     

Reconstitution of Pronase-Susceptible Flocs of Flavobacterium sp.

A component responsible for the aggregation of cells was extracted from Flavobacterium strain B by treatment of cells with 5 m guanidine hydrochloride and partially purified by gel filtration. The guanidine hydrochloride-extracted cells were reaggregated with the component after dialysis against 0.3mm of CaCl₂. Various divalent cations were effective in place of Ca²⁺, but Ca²⁺ was most effective for reconstitution. The reconstituted flocs were deflocculated by the treatment of Pronase or ethylenediaminetetraacetic acid indicating that reconstituted flocs closely resemble natural flocs.     

Effect of Ca2+ Ions on the Viscometric Behavior of F-actin

Changes in the viscosity of the F-actin solutions which occur on addition of Ca²⁺ ions were investigated. The viscosity of F-actin decreased on addition of Ca²⁺ ions. The amount of Ca²⁺ ions needed to decrease the viscosity changed with pH of the solution, namely, 20~30 mm at pH 7, 15~20 mm at pH 6 and 5~10mm at pH 5.5. Other divalent cations had the same action on F-actin, but monovalent cations did not affect the F-actin viscosity even at the concentration as high as 1 m. Intrinsic viscosity of F-actin with and without Ca²⁺ions was 250 ±40 (ml/g) and 670 ±80 (ml/g), respectively. The cause of this viscosity change was discussed from the results of electron microscopic observation and light scattering measurements.     

Calcium-dependent Properties of Peptidylarginine Deiminase from Rabbit Skeletal Muscle

Peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, required Ca²⁺ as an essential cofactor and the half-maximal activity was attained at 40—60 μm Ca²⁺. Other divalent cations were practically inactive except for Sr²⁺, which was about 50% as active as Ca²⁺ when tested at 10 mm. However, Sr²⁺ at less than the concentration of 100 μm had little or no activity. The direct Ca²⁺-binding for the enzyme showed a sigmoidal curve with a transition midpoint of about 110 μm, indicating that the binding is cooperative. Analysis of Hill plots of the data revealed that the enzyme binds 3 mol of Ca²⁺/mol of protein with an apparent dissociation constant of llO μm. A conformational change upon Ca²⁺-binding was also described for the enzyme using UV-difference spectra. The alteration could be attributed to an increased exposure of the aromatic residues to a more aqueous environment, as has been described for Ca²⁺-binding proteins such as calmodulin. Phosphatidylserine enhanced the reaction velocity and concomitantly reduced the Ca²⁺-requirement for the enzyme. These effects were stimulated by the addition of diacylglycerol. Diacylglycerol alone had little or no effect. On the other hand, calmodulin had no effect on the enzymatic activity over a wide range of Ca²⁺ concentrations. These suggest that the activity and Ca²⁺-sensitivity of peptidylarginine deiminase is increased at the cell membrane.     

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.     

Kinetic Studies on Crystalline Yeast Phosphoglyceric Acid Mutase

Effects of the substrate and the coenzyme on the crystalline yeast phosphoglyceric acid mutase activity have been investigated. Lineweaver-Burk plots at different concentrations of the substrate (d-3-phosphoglyceric acid: 3×10^?7 to 8×10^?3m) and the coenzyme (d-2, 3-diphosphoglyceric acid: 8×10^?7 to 10^?5m) change in such a way to indicate the involvement of an enzyme-substrate-coenzyme ternary complex as an active intermediate in the enzymic reaction process. It is concluded that the reaction catalyzed by the yeast enzyme follows the sequential pathway and that a phosphorylated enzyme does not participate as an obligatory intermediate in the reaction mechanism, if it occurs. Kinetic studies indicate Km values of 6×10^?4m for d-3-phosphoglyceric acid and 8×10^?7m for d-2, 3-diphosphoglyceric acid. The substrate is a competitive inhibitor of the coenzyme with a Ksi (inhibition constant) of 3.2×10^?3m. The coenzyme inhibition is not observed at concentration tested. A kinetic treatment to determine the mechanism of the enzyme reaction from the experimental data which are obtaind in the range of inhibitory substrate concentrations is presented.     

Utilization of Amino Acids as a Sole Source of Nitrogen by Obligate Chemolithoautotroph Thiobacillus ferrooxidans

An obligate chemolithoautotroph, Thiobacillus ferrooxidans API 9–3, could utilize amino acids, other than glycine, methionine and phenylalanine, as a sole source of nitrogen. However, both the growth rate and growth yield were lower than those in Fe²⁺-NH4 -salts medium, suggesting that the ammonium ion was a superior nitrogen source for the strain compared to amino acids. Methionine and phenylalanine strongly inhibited the cell growth on Fe²⁺-NH4-salts medium at 10 mm. [¹⁴C]Glycine could not be taken up into the cells, and this meant the strain could not use glycine as a sole source of nitrogen. The uptake of [¹⁴C]leucine into the cells was dependent on the presence of Fe^{2 +}. When the strain was cultured on Fe^{2 +} - leucine (lOmm)-salts medium lacking an inorganic nitrogen source for 5 days at 30°C, 83.5% and 16.5% of the cellular carbon were derived from carbon dioxide and leucine, respectively, indicating that carbon dioxide was a superior carbon source for the bacterium compared to leucine. The ammonium ion did not inhibit the utilization of leucine for cellular carbon. Leucine uptake was markedly inhibited by inhibitors of protein synthesis, such as chloramphenicol (94.3% at 1 mm), streptomycin (57.2% at 5mm) and rifampin (77.2% at 0.1 mm), respectively. Carbon dioxide uptake was also completely inhibited by chloramphenicol at 4mm. These results suggest that the transport of both amino acids and carbon dioxide into the cells was dependent on protein synthesis.     

Studies on the Autolysis of Aspergillus oryzae

In was found that an intracellular ribonuclease was present as an inactive form in the fresh mycelium of Asp. oryzae. It was about 3 times activated either by 3 m urea or by the autolysis of mycelium at 30°C for 20 hr. The optimum pH of the ribonuclease activity was 8.3. It was heat sensitive (60°C, 10 min), and completely inhibited by 5 mm EDTA. It was activated by 1 mm Mg²⁺ and inhibited by Zn²⁺, Ca²⁺, Cd²⁺, Co²⁺ and Cu²⁺.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

The present investigation is concerned with l-glutamic acid production in the presence of pyrrolidone carboxylic acid and glucose in Bacillus megaterium st. 6126. This strain does not grow on dl-pyrrolidone carboxylic acid (dl-PCA)¹⁾ as the sole source of carbon and nitrogen. The optimal concentration of yeast extract required for the maximal production of l-glutamic acid was 0.005% under the conditions used. As the yeast extract concentration was increased, growth increased proportionally; but the l-glutamic acid production did not exceed the control’s to which glucose and ammonium chloride had been added. l-Glutamic acid produced by both growing cultures and resting cells was derived from glucose and ammonium salt of dl-PCA. Isotope experiments suggested that the l-glutamic acid produced was partially derived from ammonium salt of dl-PCA in the growing culture which had been supplemented with d-glucose-U-¹⁴C or dl-PCA-1-¹⁴C and that ammonium salt of dl-PCA was consumed as the source of nitrogen and carbon for l-glutamic acid.     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.     

Production of O-Acetyl-l-homoserme by Methionine Analogresistant Mutants and. Regulation of Homosenne-O-transacetylase in Gorynebacterium glutamicum

A large amount of O-acetyl-l-homoserine (OAH) was found to be produced by trifluo-romethionine-resistant mutants derived from Corynebacterium glutamicum ESLR–146 (Thr?,ethionine^R, selenomethionine^R) and ET_zR–606(Thr?,ethionine^R, 1,2,4-triazole^R) by mutational treatment with ethyl methanesulfonate. Some cultural conditions for OAH production were examined with one of the mutants, ESLFR-736, which was derived from ESLR–146. Addition of l-methionine or l-serine decreased OAH production. Optimal level of l-threo- nine, a growth factor in ESLFR–736, for OAH production was about 200 μg/ml, and further addition of excess l-threonine repressed OAH production. Corn steep liquor (CSL) and yeast extract added simultaneously enhanced OAH production to a great extent. Thus, the amount of OAH production reached to a level of 10.5 mg/ml with a medium containing 10% glucose and 0.01 % of both CSL and yeast extract after 2 days incubation.

Cell-free extract of C. glutamicum catalyzed the formation of OAH from acetyl CoA and l-homoserine, while a corresponding reaction with succinyl CoA was hardly detected. These observations indicate that OAH but not O-succinyl-l-homoserine is an intermediate of l-methionine biosynthesis in C. glutamicum.

The regulation of homoserine-O-transacetylase was examined in a methionine requiring mutant of C. glutamicum. The enzyme activity was not inhibited by l-methionine, S-adenosyl-methionine and S-adenosylhomocysteine, separately or in combination. The synthesis of homoserine-O-transacetylase was strongly repressed by l-methionine. The enzyme level in an OAH producer, ESLFR–736, increased to about 2-fold of that in ESLR–146, the parental strain.     

Studies on the Mechanism of Strengthening Fish Gel by Basic Amino Acids

An electro-energizing fermentation (E-E F) method has been developed. In this method, a direct electrical current is applied to a microbial culture to accelerate the reductive metabolism of microorganisms or to impart profitable effects to microbial cells. This E-E F method was applied to l-glutamic acid fermentation by Brevibacterium flavum No. 2247. When glucose was used as a substrate, the addition of 0.01 mm neutral red (NR), redox dye (electron carrier), to the fermentation broth at the beginning of cultivation was effective for l-glutamate (l-Glu) production. A direct current of 200~300 μA/cm² at 1.5 V was applied through out the cultivation of this bacterium. This resulted in about a 10% increase in yield of l-Glu.     

Synthesis and Biological Activities of (±)-Deoxy-abscisic Acid Isomers

Phosphodiesterase production with bis-p-nitrophenyl phosphate as a substrate by alkalophilic Bacillus No. A-40-2 increased with increasing Mn²⁺ concentration, showing maximum productivity at 10 mm. The enzyme production was negligible in the medium without Mn²⁺. The simultaneous addition of 10 mm Mn²⁺ and one of the several cations Mg²⁺, Co²⁺, Mo⁶⁺, and Pb²⁺ at suitable concentrations stimulated the enzyme production 1.8-fold at most over that with only 10 mm Mn²⁺. Inorganic phosphate hardly repressed the enzyme production. The enzyme was purified homogeneously. The purified enzyme had the optimum pH of 7.5 and was fairly stable from pH 7–11. The enzyme hydrolyzed 2′,3′-cyclic-nucleotides and 3′-nucleotides, but did not hydrolyze 3′,5′-cyclic-nucleotides or 5′-nucleotides, indicating it to be a 2′,3′-cyclic-nucleotide 2′-phosphodiesterase (EC 3.1.4.16). The enzyme had activity without metals, but Mg²⁺, Ca²⁺, Ba²⁺, and Mo⁶⁺ activated the enzyme reaction.     

Theanine Formation by Tea Suspension Cells

The theanine (THE: γ-glutamylethylamide) content and the growth rate of cultured cells of tea (Camellia sinensis L.) were increased greatly to 22.3%, in dry wt. with a medium containing 60 mM nitrate and 25 mM ethylamine as a nitrogen source. The optimum concentrations of nitrate, Mg²⁺, and K⁺ for the growth and formation of THE in suspension cells were 40mM, 3mM, and 104mM, respectively. The yield of THE accumulated in the cultured cells with the medium modified for THE formation was increased greatly due to a great increase of the growth rate.     

Stimulation of Nerve Growth Factor (NGF) Synthesis by Tryptophan and Its Metabolites in Cultured Mouse Astroglial Cells

Effects of l-tryptophan and its metabolites were evaluated on synthesis of nerve growth factor (NGF) in cultured mouse astroglial cells. l-Tryptophan stimulated NGF production in a dose-dependent fashion. Serotonin and quinolinic acid slightly increased NGF synthesis. l-Kynurenine had a marked stimulatory effect on NGF synthesis at a dose of 100 μm. In contrast, kynurenic acid had no effect.     

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.     

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.     

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.