Synthesis of L-Tyrosine or 3,4-Dihydroxyphenyl-L-alanine from DL-Serine and Phenol or Pyrocathechol

Reaction conditions for the synthesis of L-tyrosine or L-dopa from DL-serine and phenol or pyrocatechol were studied with intact cells of Erwinia herbicola (ATCC 21434) containing high tyrosine phenol lyase activity. The optimum pH for this reaction was around 8.0, and the optimum temperature range was between 37~40°C for the synthesis of L-tyrosine and between 15~25°C for that of L-dopa. Sodium sulfite and EDTA were added to protect the synthesized L-dopa from decomposition. As high concentrations of phenol or pyrocatechol denatured the enzyme, each substrate was fed to maintain the optimum concentration during incubation.

The reaction mixture (100 ml) containing 4.0 g of DL-serine, 1.0 g of phenol or 0.7 g of pyrocatechol, 0.5 g of ammonium acetate and the cells, was incubated. During incubation, phenol or pyrocatechol was fed at intervals to maintain the substrate at the initial concentration. 5.35 g of L-tyrosine or 5.10 g of L-dopa was synthesized in 100 ml of the reaction mixture.     

Purification,Crystallization and Properties of Tyrosine Phenol Lyase from Erwinia herbicola

Crystalline tyrosine phenol lyase was prepared from the cell extract of Erwinia herbicola grown in a medium supplemented with l-tyrosine. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be approximately 259,000. The crystalline enzyme catalyzed the conversion of l-tyrosine into phenol, pyruvate and ammonia, in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from d-tyrosine, S-methyl-l-cysteine, 3, 4-dihydroxyphenyl-l-alanine, l- and d-serine, and l- and d-cysteine, but at lower rates than from l-tyrosine. l-Phenyl-alanine, l-alanine, phenol and pyrocatechol inhibited pyruvate formation from l-tyrosine.

Crystalline tyrosine phenol lyase from Erwinia herbicola is inactive in the absence of added pyridoxal phosphate. Binding of pyridoxal phosphate to the apoenzyme is accompanied by pronounced increase in absorbance at 340 and 425 mμ. The amount of pyridoxal phosphate bound to the apoenzyme was determined by equilibrium dialysis to be 2 moles per mole of enzyme. Addition of the substrate, l-tyrosine, or the competitive inhibitors, l-alanine and l-phenyl-alanine, to the holoenzyme causes appearance of a new absorption peak near 500 mμ which disappears as the substrate is decomposed but remains unchanged in the presence of the inhibitor.     

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.     

New Polar Constituents in the Pupae of the Silkworm Bombyx mori L. (II): Developmental Changes of the Constituents

A correlation between the quantitative changes in L-methionine analogs, the ratio of D-serine/L-serine during the pupal stage, and metamorphosis was observed. The glycoside appearing at low blood sugar values during the pupal stage was isolated and characterized as D-glucosyl-L-tyrosine. ¹H-NMR indicated the appearance and increase of this glycoside, and Mirrorcle Ray CV4 equipment was used to take X-ray pictures of the pupal bodies. The results indicate that γ-cyclic di-L-glutamate and L-methionine sulfone might be concerned with ammonia assimilation in the pupae, and that D-glucosyl-L-tyrosine served as a switch for the fatty acid (pupal oil) dissimilation hybrid system.     

Production of 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) and Its Derivatives by Vibrio tyrosinaticus

Six strains of bacteria belonging to Vibrio and Pseudomonas were selected as good producers of L-DOPA from L-tyrosine out of various bacteria. The condition for the formation of L-DOPA by Vibrio tyrosinaticus ATCC 19378 was examined and the following results were obtained. (1) Intermittent addition of L-tyrosine in small portions gave higher titer of L-DOPA than single addition of L-tyrosine. (2) Higher amount of L-DOPA was produced in stationary phase of growth than in logarithmic phase. (3) Addition of antioxidant, chelating agent or reductant such as L-ascorbic acid, araboascorbic acid, hydrazine, citric acid and 5-ketofructose increased the amount of L-DOPA formed. (4) L-Tyrosine derivatives such as N-acetyl-L-tyrosine amide, N-acetyl-L-tyrosine, L-tyrosine amide, L-tyrosine methyl ester and L-tyrosine benzyl ester were converted to the corresponding L-DOPA derivatives.

In the selected condition about 4 mg/ml of L-DOPA was produced from 4.3 mg/ml of L-tyrosine.     

L-Tyrosine Production by Analog-resistant Mutants Derived from a Phenylalanine Auxotroph of Corynebacterium glutamicum

Mutants resistant to various phenylalanine- or tyrosine-analogs were isolated from a phenylalanine auxotroph of Corynebacterium glutamicum KY 10233 by treatment with N- methyl-N′-nitro-N-nitrose guanidine (NTG) and screened for L-tyrosine production. A mutant, 98–Tx–71, which is resistant to 3-aminotyrosine, p-aminophenylalanine, p-fluoro-phenylalanine, and tyrosine hydroxamate was found to produce L-tyrosine at a concentration of 13.5 mg/ml in the cane molasses medium containing 10% of sugar calculated as glucose. A tyrosine-sensitive mutant, pr–20 which was derived from 98–Tx–71 produced L-tyrosine at a concentration of 17.6 mg/ml. L-Tyrosine formation in the strain pr–20 was found to be still inhibited by L-phenylalanine though it was not inhibited by L-tyrosine. The L-tyrosine formation in the mutant was repressed neither by L-phenylalanine nor by L-tyrosine.     

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.     

Synthesis of l-Tyrosine or 3,4-Dihydroxyphenyl-l-alanine from Pyruvic Acid,Ammonia and Phenol or Pyrocatechol

The synthesis of l-tyrosine or 3,4-dihydroxyphenyl-l-alanine (l-dopa) from pyruvate, ammonia and phenol or pyrocatechol was studied with intact cells of Erwinia herbicola ATCC 21434 containing high tyrosine phenol lyase activity. By elemental analyses and determination of optical activity, the tyrosine or dopa synthesized was confirmed to be entirely of l-form. Maximum amount of l-tyrosine (60.5 g/liter) or l-dopa (58.5 g/liter) was formed using this enzymatic method by feeding sodium pyruvate and phenol or pyrocatechol. However, large amounts of by-products were formed in the l-dopa synthetic reaction mixture. By-products were proved to be formed from l-dopa and pyruvate by a nonenzymic reaction. pH and the temperature of reaction had intensive effects on the formation of by-products. A simple method using a boric acid-pyrocatechol complex was devised, as the feeding procedure of substrates was complicated.     

Distribution of Tyrosine Phenol Lyase in Microorganisms

The distribution of tyrosine phenol lyase activity in microorganisms was studied with intact cells in a synthetic reaction mixture containing l-serine and phenol or pyrocatechol. This activity was found in various bacteria, most of which belonged to the Enterobacteriaceae; especially to the genera Escherichia, Proteus and Erwinia. Cells of Erwinia herbicola ATCC 21434 were selected as a promising source of enzyme.

Intact cells of Erwinia herbicola ATCC 21434 prepared from a broth cultured for 24 hr contained markedly high enzymic activity and catalyzed the synthetic reaction of l-tyrosine or 3,4-dihydroxyphenyl-l-alanine (l-dopa) from l-serine and phenol or pyrocatechol in significantly high yields.

Results of the isolation and identification of the products showed that the amino acid synthesized by this enzymatic method was identical with l-tyrosine or l-dopa.     

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.     

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.     

Gene Cloning of α-Methylserine Aldolase from Variovorax paradoxus and Purification and Characterization of the Recombinant Enzyme

The α-methylserine aldolase gene from Variovorax paradoxus strains AJ110406, NBRC15149, and NBRC15150 was cloned and expressed in Escherichia coli. Formaldehyde release activity from α-methyl-L-serine was detected in the cell-free extract of E.coli expressing the gene from three strains. The recombinant enzyme from V. paradoxus NBRC15150 was purified. The V _max and K _m of the enzyme for the formaldehyde release reaction from α-methyl-L-serine were 1.89 μmol min^?1 mg^?1 and 1.2 mM respectively. The enzyme was also capable of catalyzing the synthesis of α-methyl-L-serine and α-ethyl-L-serine from L-alanine and L-2-aminobutyric acid respectively, accompanied by hydroxymethyl transfer from formaldehyde. The purified enzyme also catalyzed alanine racemization. It contained 1 mole of pyridoxal 5′-phosphate per mol of the enzyme subunit, and exhibited a specific spectral peak at 429 nm. With L-alanine and L-2-aminobutyric acid as substrates, the specific peak, assumed to be a result of the formation of a quinonoid intermediate, increased at 498 nm and 500 nm respectively.     

L-Tyrosine production by Polyauxotrophic Mutants of Corynebacterium glutamicum

Polyauxotrophic mutants of Corynebacterium glutamicum which have additional requirements to L-phenylalanine were derived from L-tyrosine producing strains of phenylalanine auxotrophs, C. glutamicum KY 9189 and C. glutamicum KY 10233, and screened for L-tyrosine production. The increase of L-tyrosine production was noted in many auxotrophic mutants derived from both strains. Especially some double auxotrophs which require phenylalanine and purine, phenylalanine and histidine, or phenylalanine and cysteine produced significantly higher amounts of L-tyrosine compared to the parents, A phenylalanine and purine double auxotrophic strain LM–96 produced L-tyrosine at a concentration of 15.1 mg per ml in the medium containing 20% sucrose. L-Tyrosine production by the strain decreased at high concentrations of L-phenylalanine.     

Kinetics of Thermostable Alanine Racemase of Bacillus stearothermophilus

Unlabeled D- and L-alanine were racemized in deuterium oxide with an alanine racemase of Bacillus stearothermophilus at saturated concentration of substrate, and various p²H and temperature. Samples of the solution were taken at intervals, and all alanine isomers in the samples were transformed into a mixture of diastereomeric derivatives of methyl N-(–)-camphanylalaninate. Their ratio was measured on a GC-Mass, and the relative rate was calculated at the initial stage of the reaction. There was little difference in the decrease rate of the optical rotation between the enantiomers. Internal proton-transfer to the antipode was almost zero for either substrate. The α-hydrogen was abstracted 1.2–2.3 times faster from D-alanine than from L-alanine. D-Alanine gave an almost even mixture of deuterium labeled D- and L-alanine, while L-alanine gave a mixture of labeled D- and L-alanine at a ratio of 3:1. These results suggest the racemase builds two different bases in the active site. The base for D-alanine may be closer to the enzyme surface, and that for L-alanine inside.     

Free Amino Acid Contents of Plasma and Urine,and Liver Enzyme Activities in Rats Fed High Glycine Diets

The contents of plasma free amino acids, the amounts of urinary excreted amino acids and urea, and the activities of liver serine dehydratase, glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase were determined in weanling rats fed ad libitum a 10% casein diet (control), a 10% casein diet containing 7% glycine and 10% casein diets containing 7% glycine supplemented with 1.4% L-arginine and/or 0.9% L-methionine for 14 days.

The remarkable increase of glycine and the moderate increase of serine in the plasma of animals fed excess glycine diets were observed. The amount of excreted glycine in the urine of animals fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than that of animals given the excess glycine diet. Urinary excreted urea of rats fed the excess glycine diet was a little greater and that of rats fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than the control. Liver serine dehydratase activity of animals given the excess glycine diets with or without L-arginine was higher than the control and the highest activity was observed in the liver of animals fed the excess glycine diet containing L-arginine and L-methionine. The activity of liver glutamic-oxalacetic transaminase of rats fed the excess glycine diet containing L-arginine and L-methionine was a little higher than that of rats given the other diets. Liver glutamic-pyruvic transaminase activity was a little higher in animals given the excess glycine diets with or without L-arginine and further higher in animals fed the excess glycine diet containing L-arginine and L-methionine than the control.     

Screening of 3,4-Disubstituted Phenyl-L-alanine-producible Microorganisms

Microbial transaminase which catalyzes the reaction between 3,4-disubstituted phenyl pyruvate and certain amino acids to produce 3,4-disubstituted phenyl-L-alanine was investigated. Wide distribution of this enzyme among the various kinds of microorganisms was confirmed.

3,4-Dihydroxyphenyl-L-alanine (L-Dopa) or 3,4-dimethoxyphenyl-L-alanine was isolated from corresponding keto acids using three strains of selected microorganisms.     

L-Tyrosine Production by Analog-resistant Prototrophic Mutants of Glutamic Acid Producing Bacteria

p-Fluorophenylalanine (PFP) and m-fluorophenylalanine were the most effective inhibitors on the growth of Corynebacterium glutamicum ATCC 13032 among the analogs of phenylalanine and tyrosine tested. Their inhibitory effects were released by L-phenylalanine, and slightly by L-tyrosine and L-tryptophan. 3-Aminotyrosine (3AT), p-aminophenylalanine, o-fluorophenylalanine, and β-2-thienylalanine were weak inhibitors.

Resistant mutants of C. glutamicum isolated on the medium containing both PFP and 3AT or PFP and L-tyrosine were found to accumulate both L-tyrosine and L-phenylalanine, while resistant mutants isolated on the medium containing only PFP were found to produce only L-phenylalanine. Resistant mutants from other glutamic acid producing bacteria isolated on the medium containing both PFP and 3AT or both PFP and L-tyrosine were found to accumulate L-tyrosine and L-phenylalanine.     

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.     

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).     

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).