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.     

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.     

Synthesis of l-Tryptophan from Pyruvate,Ammonia and Indole

The tryptophanase activity which synthesizes l-tryptophan from pyruvate, ammonia and indole, was found to be widely distributed in cells of bacteria belonging to Enterobacteriaceae, such genera as Escherichia, Kluyvera, Enterobacter, Erwinia and Proteus. With the cells of Proteus rettgeri, equilibrium of the elimination reaction of l-tryptophan in the presence of high concentration of ammonia was studied. It was found that the equilibrium inclines toward the synthetic state.

When 5-hydroxy- and 5-methyl-indole were substituted for indole, 5-hydroxy- and 5-methyl-l-tryptophan, respectively, were synthesized. The synthesis of l-tryptophan was also observed with indole and various amino acids, S-methyl-l-cysteine, S-ethyl-l-cysteine, l-cysteine, 5-fluoro-dl-tryptophan, or oxalacetic acid.     

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.     

Mechanism of Asymmetric Production of d-Amino Acids from the Corresponding Hydantoins by Pseudomonas sp.

The mechanism of asymmetric production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. AJ-11220 was examined by investigating the properties of the enzymes involved in the hydrolysis of dl-5-substituted hydantoins. The enzymatic production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. AJ-11220 involved the following two successive reactions; the d-isomer specific hydrolysis, i.e., the ring opening of d-5-substituted hydantoins to d-form N-carbamyl amino acids by an enzyme, d-hydantoin hydrolase (d-HYD hydrolase), followed by the d-isomer specific hydrolysis, i.e., the cleavage of N-carbamyl-d-amino acids to d-amino acids by an enzyme, N-carbamyl-d-amino acid hydrolase (d-NCA hydrolase).

l-5-Substituted hydantoins not hydrolyzed by d-HYD hydrolase were converted to d-form 5- substituted hydantoins through spontaneous racemization under the enzymatic reaction conditions.

It was proposed that almost all of the dl-5-substituted hydantoins were stoichiometrically and directly converted to the corresponding d-amino acids through the successive reactions of d-HYD hydrolase and d-NCA hydrolase in parrallel with the spontaneous racemization of l-5-substituted hydantoins to those of dl-form.     

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).     

l-Tryptophan Production by 5-Methyltryptophan-resistant Mutants of Glutamate-producing Bacteria

1. Some of 5-methyltrypotophan (5MT)-resistant mutants derived from glutamate-producing bacteria such as Brevibacterium flavum, Corynebacterium acetoglutamicum and Micrococcus glutamicus produced a small amount of l-tryptophan, while tyrosine and phenylalanine auxotrophs of B. flavum did not.

2. 5-MT-resistant mutant derived from the auxotroph for tyrosine and phenylalanine produced 390 mg/liter of l-tryptophan at most. A mutant resistant to a higher concentration of 5MT, which was derived from a tyrosine and phenylalanine auxotrophic mutant which was resistant to a low concentration of 5MT, produced 660 mg/liter of l-tryptophan. Using this mutant, the effects of the concentrations of components of the culture medium on the l-tryptophan production were examined. The high concentration of l-tyrosine, but not l-phenylalanine, inhibited the l-tryptophan production. Using the improved culture medium, this strain produced 1.9 g/liter of l-tryptophan.     

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.     

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.     

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.     

Purification,Crystallization and Properties of Tryptophanase from Proteus rettgeri

An inducible tryptophanase was crystallized from the cell extract of Proteus rettgeri grown in a medium containing l-tryptophan. The purification procedure included ammonium sulfate fractionation, heat treatment, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystals were obtained from solutions of the purified enzyme by the addition of ammonium sulfate.

The crystalline enzyme preparation was homogeneous by the criteria of ultracentrifugation and zone electrophoresis. The molecular weight was determined to be approximately 210,000.

The crystalline enzyme catalyzed the degradation of l-tryptophan into indole, pyruvate and ammonia in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from 5-hydroxy-l-tryptophan, 5-methyl-l-tryptophan, S-methyl-l-cysteine and l- cysteine. l-, d-Alanine, l-phenylalanine and indole inhibited pyruvate formation from these substrates.     

The Biosynthetic Control in Aromatic Amino Acid Producing Mutants of Corynebacterium glutamicum

Regulatory properties of the enzymes involved in aromatic amino acid biosynthesis in the mutant of Corynebacterium glutamicum which produces a large amount of aromatic amino acids were examined. A phenylalanine auxotrophic l-tyrosine producer, pr-20, had a 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthetase released from the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a two-fold derepressed chorismate mutase. A pair of l-phenylalanine and l-tyrosine still strongly inhibited the chorismate mutase activity, though the enzyme was partially released from the inhibition by l-phenylalanine alone. A tyrosine auxotrophic l-phenylalanine producer, PFP-19-31, had a DAHP synthetase sensitive to the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a prephenate dehydratase and a chorismate mutase both partially released from the feedback inhibition by l-phenylalanine. The mutant produced a large amount of prephenate as well as l-phenylalanine. A phenylalanine and tyrosine double auxotrophic l-tryptophan producer, Px-115-97, had an anthranilate synthetase partially released from the feedback inhibition by l-tryptophan and had a DAHP synthetase sensitive to the feedback inhibition. These data explained the mechanism of the production of aromatic amino acids by these mutants and supported the in vivo functioning of the control mechanisms of aromatic amino acid biosynthesis in C. glutamicum previously elucidated in vitro experiments.     

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.     

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.     

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.     

Isolation and Identification of a Sexual Hormone in Yeast

5-Fluorotryptophan (5FT), indolmycin (IM), 4-fluorotryptophan and 7-azatryptophan were found on screening to be tryptophan antagonists among various chemically synthesized and naturally occurring tryptophan analogues for the isolation of l-tryptophan (l-Trp) producing mutants of Bacillus subtilis K.

From among 5FT resistant mutants, potent l-Trp producers were obtained using an improved isolation medium. Growth of the isolated 5FT-resistant l-Trp producer, AJ 11709, was inhibited by IM. From among 5FT and IM resistant mutants, the best strain, AJ 11979, which produced 9.0 g/liter of l-Trp from 13% glucose on 120hr cultivation, was selected.     

Isolation of 6-hydroxy-L-tryptophan from the fruiting body of Lyophyllum decastes for use as a tyrosinase inhibitor

ABSTRACT

Tyrosinase is the key enzyme that controls melanin formation. We found that a hot water extract of the lyophilized fruiting body of the fungus Lyophyllum decastes inhibited tyrosinase from Agaricus bisporus. The extract was fractionated by ODS column chromatography, and an active compound was obtained by purification through successive preparative HPLC using an ODS and a HILIC column. Using spectroscopic data, the compound was identified to be an uncommon amino acid, 6-hydroxytryptophan. 6-Hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan were prepared through a Fenton reaction from L-tryptophan and D-tryptophan, respectively. The active compound was determined to be 6-hydroxy-L-tryptophan by comparison of their circular dichroism spectra and retention time on HPLC analysis of the N^α-(5-fluoro-2,4-dinitrophenyl)-L-leuciamide derivative with those of 6-hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan. A Lineweaver–Burk plot of the enzyme reaction in the presence of 6-hydroxy-L-tryptophan indicated that this compound was a competitive inhibitor. The IC₅₀ values of 6-hydroxy-L-tryptophan was 0.23 mM.     

Studies on Amino Acids. II

The enzymatic procedures for the resolution of dl-lysine such as asymmetric synthesis of acyl l-lysinc anilide and acyl dl-lysines have been studied. As a result, the procedure consisting in the enzymatic asymmetric hydrolysis of ε-benzoyl-α-acctyl-dl-lysine was found to be the most advantageous for the resolution of dl-lysine.     

Direction of Asymmetric Induction in the Reaction of Pseudooxazolone-(5) with l-Proline Ester

It was observed that the dl-dipeptide derivative was formed predominantly over the ll-compound, only when l-Pro-OR was allowed to react as amino-component to the pseudooxazolone-(5), in contrast to the other l-amino acid esters. From the observation of the influence of the solvent, the added base and H-Gly-OMe, some of the mechanism in this reaction was discussed. The preparative isolation of the dl-compound from the reaction product, the synthesis of Tfp-l-Ileu-OH and the corresponding pseudooxazolone-(5) compound were also described.     

Production of L-Tryptophan by 5-Fluorotryptophan Resistant Mutants of Bacillus subtilis

The growth of Bacillus subtilis TR–44, a prototrophic transductant from one of inosine producers, was completely inhibited by 200 µg/ml of 5-fiuorotryptophan, a tryptophan analogue, and the inhibition was reversed by the addition of L-tryptophan.

Several mutants resistant to 5FT* produced L-tryptophan in the growing cultures. The best producer, strain FT–39, which was selected on a medium containing 1500 µg/ml of 5FT, produced 2 g/liter of L-tryptophan, when cultured in a medium containing 8% of glucose but without any tryptophan precursors. In this mutant, anthranilate synthetase, a key enzyme of the tryptophan biosynthesis, had increased over 280-fold, presumably owing to a genetic derepression. From FT–39, mutants resistant to 7000 µg/ml of 5FT were derived. Among them, strain FF–25 produced 4 g/liter of L-tryptophan, twice as much as did the parental strain. Since this strain produced large amount of L-phenylalanine as well as L-tryptophan, the genetic alteration seemed to be involved in some metabolic regulation of common part of the aromatic amino acid biosynthetic pathway.

Further, some auxotrophs derived from these 5FT resistant mutants produced more L-tryptophan than did the parental strains.

Relationships between the accumulation of L-tryptophan and the regulation mechanisms of the L-tryptophan biosynthesis were discussed.