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.     

Properties of Revertants Appearing in l-Leucine Fermentation Culture Broth

The l-leucine productivity of an l-leucine producing strain, H-1204, of Corynebacterium glutamicum substantially decreased during a large-scale culture or repetitive subculturing. This instability was found to be due to the appearance of revertants with lower or no l-leucine productivity. Strains in the culture broth could be roughly classified into three types on the basis of their phenotypes: l-type, original l-leucine producing strain, Val^L Leu⁺ (valine leaky); M-type, Val⁺ Leu⁺ (prototroph); V-type, Val⁺ Leu^- (leucine auxotroph). The appearance of these revertants was determined to be caused by the distribution imbalance of α-ketoisovaleric acid, the common precursor for l-leucine and l-valine biosynthesis.     

Genetic Changes of Regulatory Mechanisms Occurred in Leucine and Valine Producing Mutants Derived from Brevibacterium lactofermentum 2256

The regulatory mechanisms in branched-chain amino acid synthesis were compared between 2-thiazolealanine (2-TA) resistant l-leucine and l-valine producing mutants and the 2-TA sensitive original strains of Brevibacterium lactofermentum 2256.

In the original strains, sensitive to 2-TA, α-isopropylmalate (IPM) synthetase, the initial enzyme specific for l-leucine synthesis, is sensitive to feedback inhibition and to repression by l-leucine, and α-acetohydroxy acid (AHA) synthetase, the common initial enzyme for synthesis of l-isoleucine, l-valine as well as l-leucine, is sensitive to feedback inhibition by each one of these amino acids, and to repression by them all. In strain No. 218, a typical l-leucine producer resistant to 2-TA, IPM synthetase was found to be markedly desensitized and derepressed, and AHA synthetase remained unaltered. On the contrary, in strain No. 333, l-valine producer resistant to 2-TA, AHA synthetase was found to be desensitized and partially derepressed, and IPM synthetase remained unaltered.

The genetic alteration of these regulatory mechanisms was discussed in connection with the accumulation pattern of amino acids.     

Studies on the the Isoleucine Fermentation

Accumulation of L-isoleucine and L-valine was studied on 14 genera, 47 species and 110 strains of aerobic bacteria using bacterial type cultures. A large amount of L-isoleucine and a small amount of L-valine accumulated when 1% of DL-α-aminobutyric acid was added to the culture medium. As a rule, facultative aerobes such as Aerobacter, Erwinia, Serratia and Bacillus showed good accumulation. In the absence of α-aminobutyric acid, powerful L-isoleucine accumulators produced a large amount of L-valine, although the accumulation of L- isoleucine was scarcely observed under that condition. In the presence of α-aminobutyric acid, the accumulation of L-valine was generally suppressed, but in several strains, on the contrary, the accumulation increased as well as that of L-isoleucine. When DL-threonine was used instead of α-aminobutyric acid, the amount of L-isoleucine accumulated was not as high as that with α-aminobutyric acid in almost all strains except Serratia marcescens. It was concluded that a distinct relationship between bacterial genera or species and accumulation of L-isoleucine did not exist, that is, powerful accumulators were limited to special strains, and that the addition of α-aminobutyric acid was necessary for the accumulation of a large amount of L-isoleucine.     

Production of l-Valine by 2-Thiazolealanine Resistant Mutants Derived from Glutamic Acid Producing Bacteria

Potent l-valine producers were screened among 2-thiazolealanine resistant mutants derived from three typical l-glutamic acid producing bacteria: Brevibacterium lactofermentum, Corynebacterium acetoacidophilum, Arthrobacter citreus. By strain No. 487, the best producer derived from Brevibacterium, 31 mg/ml of l-valine was produced after 72 hr when 10% glucose was supplied as a carbon source, thus giving the yield of 31% from glucose. Accumulation of the other amino acids was negligible. The addition of l-isoleucine and l-leucine in the culture medium did not reduce the l-valine production, indicating that the l-valine biosynthesis is insensitive to these end products in the l-valine producer.     

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.     

Production,Purification, and Characterization of D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

The best inducers for D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) were a poor substrate, N-acetyl-;-methyl-D-leucine, and an inhibitor, N-acetyl-D-alloisoleucine. The enzyme has been homogeneously purified. The molecular weight of the native enzyme was estimated to be 58,000 by gel filtration. A subunit molecular weight of 52,000 was measured by SD8–PAGE, indicating that the native protein is a monomer. The isoelectric point was 5.2. The enzyme was specific to the D-isomer and hydrolyzed N-acetyl derivatives of D-leucine, D-phenylalanine, D-norleucine, D-methionine, and D-valine, and also N-formyl, N-butyryl, and N-propionyl derivatives of D-leucine. The K_m for N-acetyl-D-leucine was 9.8mM. The optimum pH and temperature were 7.0 and 50°C, respectively. The stabilities of pH and temperature were 8.1 and 40°C. D-Aminoacylases from three species of the genus Alcaligenes differ in inducer and substrate specificities, but are similar with respect to molecular weight and N-terminal amino acid sequence.     

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.     

Bacterial Degradation of N-Lauroyl-L-valine

Studies were conducted on the degradation of N-lauroyl-L-valine by type cultured bacteria. Many strains could utilize sodium N-lauroyl-L-valinate as carbon and nitrogen sources for their growth. Metabolism of N-lauroyl-L-valine was investigated in detail using Ps. aeruginosa AJ2116. Laurie acid was identified by gas chromatography suggesting cleavage of N-acyl linkage in N-lauroyl-L-valine.

Laurie acid might be metabolized to capric acid (C₁₀) and caprylic acid (C₈) becuase the accumulated substances gave nearly identical peaks with those of authentic fatty acids on gas chromatograms. The experiment using N-lauroyl-L-valine (¹⁴C) indicated that ¹⁴CO₂ was produced as a final product. Valine was not detected because it might be metabolized very rapidly immediately after its release.

It was supposed that the enzymes or enzyme systems degrading N-lauroyl-L-valine might be constitutive from the experiment using two kinds of cells grown in the medium containing N-lauroyl-L-valine or nutrient broth.     

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.     

Occurrence of l-(−)-Pipecolic Acid in the Culture Medium of Rumen Ciliate Protozoa

Effect of phenoxazine derivatives (actinomycin D, l,8-dimethyl-3-aminophenoxazone-2, 3-aminophenoxazone-2, sodium resazurate, gallocyanine, fluorescent blue and capri blue) on amino acid transport in rat small intestine was investigated using tissue accumulation method. Capri blue (CI–51015) inhibited the accumulation of l-leucine, l-alanine and l-valine, and scarcely inhibited that of D-glucose. Kinetic analysis showed that the inhibition of amino acid accumulation by this pigment was non-competitive. Actinomycin D had no effect on amino acids and d-glucose accumulation in vitro at the high and low concentrations of these substances. High concentration of actinomycin D and long period of incubation had no effect, too. Accumulation of amino acids into the intestinal tissue was not significantly decreased or increased by the presence of other phenoxazine derivatives.     

Effects of a Feedback-Resistant Aspartokinase III Gene on L-Isoleucine Production in Escherichia coli K-12

An L-isoleucine-overproducing recombinant strain of E. coli, TVD5, was also found to overproduce L-valine. The L-isoleucine productivity of TVD5 was markedly decreased by addition of L-lysine to the medium. Introduction of a gene encoding feedback-resistant aspartokinase III increased L-isoleucine productivity and decreased L-valine by-production. The resulting strain accumulated 12 g/l L-isoleucine from 40 g/l glucose, and suppression of L-isoleucine productivity by L-lysine was relieved.     

Structure of Rugulovasine A,B and their Derivatives

Culture conditions for the preparation of cells containing high tyrosine phenol lyase activity were studied with Erwinia herbicola ATCC 21434. Adding pyridoxine to the medium enhanced enzyme formation, suggesting that it was utilized as a precursor of the coenzyme, pyridoxal phosphate. Glycerol plus succinic acid; amino acids, such as, DL-methionine, DL-alanine and glycine; and metallic ion, ferrous ion promoted enzyme formation as well as cell growth. Adding L-tyrosine, as inducer, to the culture medium was essential for enzyme formation. However, when large amounts of L-tyrosine were added, the enzyme formation was repressed by the phenol liberated from L-tyrosine. In fact, formation of the enzyme was enhanced by removing phenol during cultivation. L(D)-Phenylalanine or phenylpyruvic acid had a synergistic effect on the induction of enzyme by L-tyrosine.

Cells with high enzyme activity were prepared by growing cells at 28°C for 28 hr in a medium containing 0.2% L-tyrosine, 0.2% KH₂PO₄, 0.1% MgSO₄7H₂O, 0.001% FeSO_4·7H₂O, 0.01% pyridoxine-HC1, 0.6% glycerol, 0.5% succinic acid, 0.1% DL-methionine, 0.2% DL-alanine, 0.05% glycine, 0.1% L-phenylalanine and 120 ml/liter hydrolyzed soybean protein in tap water with the pH controlled at 7.5 throughout cultivation.     

Amino Acid Composition of Green Gram (Phaseolus radiatus L. var. typicus Prain)

N-Carboxymethyl-β-alanine and four γ-glutamyl peptides—γ-l-glutamyl-l-leucine, γ-l-glutamyl-l-methionine, γ-glutamylphenylalanine and γ-glutamyltyrosine—were isolated from green gram seeds. N-Carboxymethyl-β-alanine is a compound which is isolated from natural products for the first time. An amount of γ-glutamylmethionine was far more abundance than all others.     

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.     

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.     

Chemical Studies on the Antibiotic Esperin

Esperin is an acidic antibiotic with a molecular formula of C₃₉H₆₇N₅O₁₁ and, on hydrolysis with acid, it afforded l-aspartic acid, l-glutamic acid, l-valine, l-leucine, d-leucine and 2-tridecenoic acid. By treatment with alkali, esperin was transformed to esperinic acid, C₃₉H₆₉N₅O₁₂, which was shown to be β-hydroxytridecanoyl-glutamyl-aspartyl-valyl-leucyl-leucine. From chemical and physical studies, esperin was proved to be the lactone of esperinic acid, represented by the formula III.     

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.     

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.     

Syntheses of 14C-Labeled Pyrethrin I,Allethrin, Phthalthrin,and Dimethrin on a Submillimole Scale

Studies on the metabolic fate and degradation chemistry of pyrethroid insecticide chemicals are greatly facilitated by the use of compounds radiolabeled, in separate preparations, in the acid and alcohol moieties. Acid-labeled preparations were made by converting d-trans-chrysanthemic acid-1-¹⁴C (88 mg, 1.3 mCi/mm) into d-trans-d-pyrethrin-1-¹⁴C (68 mg, 1.3 mCi/mm), d-trans-d-allethrin-¹⁴C (43 mg, 1.3 mCi/mm), d-trans-dimethrin-¹⁴C (54 mg, 0.294 mCi/mm), and d-trans-phthalthrin-¹⁴C (47 mg, 0.294 mCi/mm), incorporating approximately 81% of the starting radiocarbon into the four pyrethroid preparations. Alcohol-labeled preparations were made by converting acetone-1,3-¹⁴C into d-trans-dl-ailethrin-¹⁴C (146 mg, 0.162 mCi/mm) and formaldehyde-¹⁴C into d-trans-phthalthrin-¹⁴C (299 mg, 0.276 mCi/mm). Each labeled compound had a high stereochemical purity and a radiochemical purity of greater than 99%. Detailed procedures were worked out for all conversions which took place in high yields except in one case: the synthesis of allethrin labeled in the alcohol moiety.