Taurine Levels in Some Tissues of Yellowtail (Seriola quinqueradiata) and Mackerel (Scomber japonicus)

The mechanism of stereospecific production of l-amino acids from the corresponding 5-substituted hydantoins by Bacillus brevis AJ-12299 was studied. The enzymes involved in the reaction were partially purified by DEAE-Toyopearl 650M column chromatography and their properties were investigated. The conversion of dl-5-substituted hydantoins to the corresponding l-amino acids consisted of the following two successive reactions. The first step was the ring-opening hydrolysis to N-carbamoyl amino acids catalyzed by an ATP dependent l-5-substituted hydantoin hydrolase. This reaction was stereospecific and the N-carbamoyl amino acid produced was exclusively the l-form. N-Carbamoyl-l-amino acid was also produced from the d-form of 5-substituted hydantoin, which suggests that spontaneous racemization occurred in the reaction mixture. In the second step, N-carbamoyl-l-amino acid was hydrolyzed to l-amino acid by an N-carbamoyl-l-amino acid hydrolase, which was also an l-specific enzyme. The ATP dependency of the l-5-substituted hydantoin hydrolase was supposed to be the limiting factor in the production of l-amino acids from the corresponding 5-substituted hydantoins by this bacterium.     

Studies on Some New Thiazolidones as Potential Fungicides

The reaction conditions for the production of d-β-hydroxyphenylglycine (d-HPG) from dl-5-(β-hydroxyphenyl)hydantoin (dl-HPH) by cells of Pseudomonas sp. AJ-11220, and the cultural conditions for this bacterium for the formation of the d-HPG-producing enzyme involved by this bacterium were investigated. The optimal pH of this reaction was about 8.0 and the optimal temperature about 43°C. The d-HPG-producing enzyme was inducibly produced in Pseudomonas sp. AJ-11220 in proportion to the cell growth. Cells containing high activity were obtained when Pseudomonas sp. AJ-11220 was grown in a medium containing 20 g of glucose, 5g of (NH₄)₂SO₄,. 1 g of KH₂PO₄, 3g of K₂HPO₄, 0.5g of MgSO₄–7H₂O, 0.01 g of FeSO₄–7H₂O, 0.01 g of MnSO₄ -4H₂O, 10 g of yeast extract, 5g of dl-5-cyanoethylhydantoin and 20 g of CaCO₃ in a total volume of 1 liter (pH 7.0). Under the optimal conditions, 25 mg/ml of d-HPG was asymmetrically and directly produced from 30 mg/ml of dl-HPH with a molar yield of 92%. Various d-amino acids could also be effectively produced from the corresponding 5-substituted hydantoins.     

Occurrence of Plasmids in Zymomonas mobilis

A bacterium that stereospecifically produces l-valine from 5-isopropylhydantoin was isolated + from soil. It was identified as Bacillus brevis and given the number AJ-12299. l-Valine productivity from l-, d- or dl-5-isopropylhydantoin by B. brevis AJ-12299 was rather low because this bacterium had l-valine degrading-activity. In contrast, the productivity was improved by a mutant the l-valine degradation pathway of which was genetically blocked, and the 5-isopropylhydantoin consumed was stoichiometrically converted to l-valine. The optimal temperature and pH of the reaction were 30°C and 7.0~7.5. The enzyme involved in the reaction was inducible and was strongly induced by the addition of 5-isopropylhydantoin. In addition to l-valine production, this bacterium also produced various aliphatic and aromatic l-amino acids from the corresponding 5-substituted hydantoins.     

N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

Enzymatic Resolution of Racemic Amino Acids

The mold acylase of Aspergillus and Penicillium which hydrolyzes, asymmetrically, only the l-isomer of N-acylated dl-amino acids has been purified previously by the present authors. In this paper the application of asymmetric hydrolysis with the mold acylase to the resolution of N-acylated dl-amino acids, namely, acetylderivatives of dl-tryptophan, dl-leucine and dl-alanine is described. By this enzymatic procedure, the above amino acids were resolved in relatively good yields. It has been noted that the use of the mold acylase is suitable for general resolution of amino acid enantiomorphs of high optical purity.     

Biological Activities of Sex Hormone Produced by Tremella mesenteriea

d-Aminoacylase was found to be produced not only by S. olivaceus 62–3 isolated from soil but also by three strains of type culture of Streptomyces species. All four of these strains produced d-aminoacylase intracellularly only when an inducer was added to the culture medium. d-Amino acids or N-acetyl-d-amino acids were effective as inducers.

As S. tuirus showed the highest d-aminoacylase activity, the enzyme extract of this strain was subjected to further investigation to determine the optimal conditions for optical resolution of N-acetyl-dl-phenylglycine. Almost all contaminating l-aminoacylase in the enzyme extract could be eliminated by DEAE-Sephadex adsorption. d-Phenylglycine of 99.9% optical purity was obtained after complete hydrolysis of d-isomer with the use of d-aminoacylase solution.     

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.     

Protective Effect of dl-Threonine on Bacterial Cells during Freeeze-Drying and Ineffectiveness of its Optically Active Forms

dl-Threonine and dl-allothreonine showed a protective effect on various bacterial cells in the process of freeze-drying whereas l- and d-forms of them did not, probably owing to the difference in the physicochemical characteristics between l- (or d-) form and dl-form of the compounds in question. There was no difference in the protective activity between the optically active and inactive forms in the cases of serine, proline, tartaric acid and pyrrolidonecaboxylic acid.     

Effects of Glycine and d-Amino Acids on Growth of Various Microorganisms

Growth of various microorganisms in media containing high concentrations of glycine or d-amino acids was examined. Susceptibilities to glycine or d-amino acids differed among microorganisms, and the differences in susceptibility have no direct relation with Gram staining, morphological forms, and aerobic or anaerobic nature of the organisms. Certain glycine-resistant bacteria tested, which included Bacillus cereus, Staphylococcus aureus and Serratia marcescens, exhibited relatively high oxidative activities towards glycine. The inhibition of the growth of Escherichia coli by either glycine or d-amino acids, which included d-threonine, d-alanine and d-lysine, was reversed by l-alanine, partialy by l-serine, and not by l-lysine or l-threonine. These results suggest that the growth inhibition of microorganisms by d-amino acids was similar to that by glycine. The incorporation of l-alanine into E. coli cells which were preincubated with glycine was less than those of preincubated without glycine. Particularly, the incorporation into the cell wall fraction was most susceptible to glycine. An additive effect of penicillin and glycine was observed in the inhibition of cell wall biosynthesis as determined by the intracellular accumulation of N-acetylamino sugar compounds.     

Microbial Conversion of DL-5-Substituted Hydantoins to the Corresponding L-Amino Acids by Pseudomonas sp. Strain NS671

A bacterial strain, NS671, which converts DL-5-(2-methylthioethyl)hydantoin stereospecifically to L-methionine, was isolated from soil and was classified into the genus Pseudomonas. With growing cells of Pseudomonas sp. strain NS671, DL-5-(2-methylthioethyl)hydantoin was effectively converted to L-methionine. Under adequate conditions, 34g of L-methionine per liter was produced with a molar yield of 93% from DL-5-(2-methylthioethyl)hydantoin added successively. In addition to L-methionine, other amino acids such as L-valine, L-leucine, L-isoleucine, and L-phenylalanine were also produced from the corresponding 5- substituted hydantoins, but these L-amino acids produced were partially consumed by strain NS671. The hydantoinase, by which 5-substituted hydantoin rings are opened, was ATP-dependent. The N-carbamylamino acid amidohydrolase was found to be strictly L-specific, and its activity was inhibited by high concentration of ATP.     

Elimination,Replacement and Isomerization Reactions by Intact Cells Containing Tyrosine Phenol Lyase

Tyrosine phenol lyase catalyzes a series of α,β-elimination, β-replacement and racemization reactions. These reactions were studied with intact cells of Erwinia herbicola ATCC 21434 containing tyrosine phenol lyase.

Various aromatic amino acids were synthesized from l-serine and phenol, pyrocatechol, resorcinol or pyrogallol by the replacement reaction using the intact cells. l(d)-Tyrosine, 3,4-dihydroxyphenyl-l(d)-alanine (l(d)-dopa), l(d)-serine, l-cysteine, l-cystine and S-methyl-l-cysteine were degraded to pyruvate and ammonia by the elimination reaction. These amino acids could be used as substrate, together with phenol or pyrocatechol, to synthesize l-tyrosine or l-dopa via the replacement reaction by intact cells. l-Serine and d-serine were the best amino acid substrates for the synthesis of l-tyrosine or l-dopa. l-Tyrosine and l-dopa synthesized from d-serine and phenol or pyrocatechol were confirmed to be entirely l-form after isolation and identification of these products. The isomerization of d-tyrosine to l-tyrosine was also catalyzed by intact cells.

Thus, l-tyrosine or l-dopa could be synthesized from dl-serine and phenol or pyrocatechol by intact cells of Erwinia herbicola containing tyrosine phenol lyase.     

Metabolism of l-Theanine,d-Theanine and the Related Compounds in Bacteria

Some strains of Pseudomonas was found capable of utilizing l-theanine or d-theanine as a sole nitrogen and carbon source. The cell-free extract catalyzes the hydrolysis of the amide group of the compounds and the hydrolase activity was influenced remarkably by the nitrogen source in the medium. l-Theanine and d-theanine were hydrolyzed to yield stoichiometrically l-glutamic acid and d-glutamic acid, respectively, and ethylamine, which were isolated from the reaction mixture and identified.

The theanine hydrolase of Pseudomonas aeruginosa was purified approximately 200-fold. It was shown that the activities of l-theanine hydrolase, d-theanine hydrolase and the heat-stable l-glutamine hydrolase and d-glutamine hydrolase are ascribed to a single enzyme, which may be regarded as a γ-glutamyltransferase from the point of view of the substrate specificity and the properties. This theanine hydrolase catalyzed the transfer of γ-glutamyl moiety of the substrates and glutathione to hydroxylamine. l-Glutamine and d-glutamine were hydrolyzed by the theanine hydrolase and also by the heat-labile enzyme of the same strain of Pseudomonas aeruginosa, whose properties resembled the common glutaminase.     

Formation of 2-(5-Hyclroxymethyl-2-formylpyrrol-1-yl)alkyl Acid Lactones on Roasting Alkyl-α-amino Acid with d-Glucose

d-Glucose and several alkyl-α-amino acids (glycine, dl-α-alanine, dl-α-amino-n-butyric acid, l-valine, l-leucine and dl-α-amino-n-caproic acid) were roasted at 200°C or 250°C in a simple two components system. From the roasting products were newly isolated a series of 2-(5-hydroxymethyl-2-formylpyrrol-1-yl)alkyl acid lactones which were characterized by elementary analysis, UV, IR, MS (GC-MS) and NMR spectra.

These lactones have characteristic aroma which may contribute to the flavor produced by sugar-amino acid reaction. The subjective evaluation of aroma of the lactones obtained wrere as follows: 2-(5-hydroxymethyl-2-formyipyrrol-1-yl)propionic acid lactone, caramel and a little scorching; -n-butyric acid lactone, maple and strong sweet; isovaleric acid lactone and isocaproic acid lactone, miso, soy sauce and a little chocolate-like.     

Simultaneous determination of primary and secondary d- and l-amino acids by reversed-phase high-performance liquid chromatography using pre-column derivatization with two-step labelling method

This work describes a method for the simultaneous determination of primary d- and l-amino acids and secondary amino acids such as d- and l-proline. In order to remove interferences in the simultaneous determination of primary and secondary amines, the primary amines were derivatized with o-phthalaldehyde/N-acetyl-l-cysteine (OPA/NAC) and subsequently with 1-(9-fluorenyl)ethyl chloroformate (FLEC) for secondary amines, in a pre-column separation derivatization technique. These fluorescent diastereomers of the amino acids were obtained within 3 min at room temperature and determined simultaneously by changing wavelengths during analysis in a single eluting run in the high-performance liquid chromatography column. This method, referred to as the “two-step labelling method,” is effective for the simultaneous determination of d- and l-amino acids.     

Preparation of optically active 4-chlorophenylalanine from its racemate by deracemization technique using transformant Escherichia coli cells

We have developed a method for the preparation of l-4-chlorophenylalanine from its racemate with Escherichia coli cells expressing a single foreign gene. l-4-Chlorophenylalanine was obtained in a high optical yield by the inversion of configuration of its d-form via the tandem reactions catalyzed by d-amino acid dehydrogenase (DadA) and branched-chain amino acid aminotransferase (BCAAT). While we constructed a plasmid for BCAAT utilizing the gene from Sinorhizobium meliloti ATCC 51124, the first enzyme DadA was the dadA-gene product from E. coli host cell itself, which was activated by the addition of l-alanine in the growth medium.     

Enzymatic Production of l-Tryptophan from l- and dl-5-Indolyl-methylhydantoin by Newly Isolated Bacterium

Bacteria which can hydrolyze dl-5-indolylmethylhydantoin to l-tryptophan were isolated from various soils. The dl-5-indolylmethylhydantoin-hydrolyzing enzymes were found to be inducible and intracellular. With intact cells, 50 mg/ml as wet base, of newly isolated bacterial strain T-523, 10 mg/ml of dl-5-indolylmethylhydantoin dissapeared and 7.4 mg/ml of l-tryptophan in a molar yield of 82% was produced after 35 hr incubation. Tryptophan produced was confirmed to be l-form regardless of the stereoisomer of the substrates used. A mechanism of asymmetric hydrolysis of dl -5-indolyImethylhydantoin was discussed.     

A Growth Factor for Malo-lactic Fermentation Bacteria

A growth factor (TJF) for a malo-lactic fermentation bacterium has been isolated from tomato juice, and found to be a β-glucoside. The NMR spectra of TJF and its acetate revealed that the glucosyl residue linked to the hydroxyl group at C-2′ or C-4′ of d- or l-pantothenic acid moiety. Then, 2′-O-(β-d-glucopyranosyl)-dl-pantothenic acid (I), 4′-O-(β-d-glucopyranosyl)-dl-pantothenic acid (II) and 4′-O-(β-d-glucopyranosyl)-d(R)-pantothenic acid (II-a) were synthesized, and Il-a and 4′-O-(β-d-glucopyranosyl)-l-pantothenic acid (II-b) were obtained by the optical resolution of the acetate of II. Among the above compounds, II-a was identical with natural TJF regarding to the biological activity, NMR and ORD spectra, and thin-layer chromatography.     

Enzymatic Resolution of Racemic Amino Acids

The present paper is concerned with the availability of the acyl derivatives of lysine for the growth of young rats in the course of studying the enzymatic resolution of dl-lysine with mold acylase. The enzymatic resolution of dl-lysine to optically-active l and d-isomers was carried out in either of the following two ways, namely, the asymmetric hydrolysis of diacetyl-dl-lysine or that of ε-benzoyl-α-acetyl-dl-lysine.

The oral administration of ε-acetyl-l-lysine to rats fed on the lysine-deficient diet supported the growth of young rats at a rate approximately two-thirds of that observed when l-lysine was supplied. ε-Benzoyl-l-lysine proved to be quite ineffective while diacetyl lysine showed a slight but insignificant increase in body weight.     

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.     

Studies on the Metabolism of d-Amino Acid in Microorganisms

It is confirmed by a new method for the determination of d-glutamic acid, that Aerobacter strain A rapidly metabolizes d-glutamic acid, while it only shows feeble metabolic activity towards l-glutamic acid when it is grown on a dl-glutamate-K₂HPO₄ medium. A specific d-glutamic oxidase is demonstrated in the cell-free extracts of Aerobacter strain A. This enzyme seems to be different from d-glutamic-aspartic oxidase obtained from Aspergillus ustus by the authors, since the former has no activity towards d-aspartic acid.