The Determination of d-Glutamic Acid and d-Aspartic Acid by Means of a New Oxidase

The fractional determination of d-glutamic and d-aspartic acids using the enzyme preparation of Aspergillus ustus strain f. was studied. In the first part of this paper, the procedure of enzyme preparation, the effect of sodium chloride on enzyme activity, and a new device for the fractional determination of d-glutamic and d-aspartic acids are described. In the latter part, the contents of d-glutamic and d-aspartic acids of cancer and normal tissues are estimated. However, it was found that the cancer tissues are not characterized by the presence of d-glutamic acid in opposition to Kögl’s claim.     

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.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10^?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10^?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-¹⁴C revealed that l-glutamic acid-1-¹⁴C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme.     

Studies on the Synthesis of l-Amino Acids

l-Homoserine was prepared by the reduction of l-aspartic acid β-methyl ester with sodium borohydride in water solution without any racemization. The yield of l-homoserine was about 25% of the theoretical amount, and no product other than l-homoserine, l-aspartic acid and l-aspartic acid β-methyl ester was present in the reaction mixture. The low yield of l-homoserine was ascribed to the hydrolysis of the ester.

l-Azetidine-2-carboxylic acid could not be detected in the reaction mixture. In contrast with the reduction of l-glutamic acid γ-esters, the reduction of l-aspartic acid β-ester was not accompanied by the cyclization.     

Polyethylene Glycol–Modified Papain Catalyzed Oligopeptide Synthesis from the Esters of l-Aspartic and l-Glutamic Acids in Benzene

Comparative studies were made of the polymerization of l-aspartic and l-glutamic acid dialkyl esters using polyethylene glycol–modified papain as a catalyst in phosphate buffer (pH 7.5) and in benzene. Changes in the substrate specificity of papain and in the composition of oligomerized products were observed. In the buffer, the diethyl and di-n-propyl esters of l-glutamic acid were sufficiently converted to high molecular weight oligomers with the accumulation of dimer and trimer, but l-aspartic acid esters were very poor substrates. In benzene, l-aspartic acid esters became more reactive than L-glutamic acid esters. In particular, from l-aspartic acid dimethyl ester the product, which was mainly composed of heptamer to decamer, was obtained in a 90% yield. The reaction in benzene required desalted substrates and a small amount of water to proceed extensively.     

Synthesis of γ-Glutamyl Peptides Catalyzed by Transamidase from Bacillus natto

Crude ammonium sulfate fraction of a cell free extract from Bacillus natto contained an enzyme (or enzymes) which catalyzed the transamidation reaction specific for glutamine. Both l- and d-isomers of glutamine were active as substrate. On incubation of l- or d-glutamine with the enzyme preparation, two peptides consisting of glutamic acid and glutamine were formed. The main component of the peptides was readily isolated by ion-exchange chromatography and identified as γ-glutamylglutamine by paper chromatography and by paper electrophoresis using authentic peptides. The optical configuration of the amino acid residues in the dipeptide was determined by digestion of the acid hydrolyzate with l-glutamic acid decarboxylase, and the result showed that the dipeptide obtained from l-glutamine was a l-l isomer, while the dipeptide from d-glutamine was a d-d isomer.     

New Resolving Agents

Seven optical active 2-benzylamino alcohols were synthesized by reduction of N-benzoyl derivatives of L-alanine, L-valine, L-leucine, L-phenylalanine, L-aspartic acid, L-glutamic acid and L-lysine and applied for the resolution of (±)-trans-chrysanthemic acid. d-trans-Chrys-anthemic acid was obtained by resolution via the salts of 2-benzylamino alcohols derived from L-valine and L-leucine, while (?)-trans-chrysanthemic acid was prepared through the salts of the amino alcohols derived from L-alanine and L-phenylalanine.     

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.     

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 l-Glutamic Acid Fermentation

Micrococcus glutamicus, a glutamate-produeing bacterium, is known to have strong activity of l-glutamic acid dehydrogenase which requires NADP as co-enzyme. In this paper, the NADP-speeifie l-glutamic acid dehydrogenase was purified from M. glutamicus by means of heat treatment with sodium sulfate, precipitation with acetic acid and diethyl-amino-ethyl (DEAE) cellulose column chromatography. The activity of the purified enzyme preparation reached 200-fold as high as that of the crude extract. Some properties of the purified enzyme were investigated. As a result, it was found that the highly purified enzyme preparation acted not only on l-glutamic acid (l-GA) but also on α, ε-diaminopimelic acid (α, ε-DAP) in the presence of NADP. Some of the probable consideration for the dehydrogenation of l-GA and α, ε-DAP are noted.     

Studies on the Polymorphism of l-Glutamic Acid

The effects on the polymorphic crystallization of l-glutamic acid were examined of many substances including amino acids, inorganic salts, surface active agents, and sodium salt or hydrochloride of l-glutamic acid, when contained in the mother liquor.

The co-existence of amino acids, especially of l-aspartic acid, l-phenylalanine, l-tyrosine, l-lcucine and l-cystine contributed to the crystallization of l-glutamic acid in α-form, and these amino acid showed an inhibitory action on the transition of α-crystals as the solid phase in the aqueous solution, to β-crystals.

In the presence of a large amount of l-glutamate or the hydrochloride at the time of nucleation of l-glutamic acid, mostly β-crystals appeared even in the presence of the amino acids named above.     

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.     

Structure of the Hydrolyzed Product (F-2) Released from γ-Polyglutamic Acid by γ-Glutamyl Hydrolase YwtD of Bacillus subtilis

The structure of the hydrolyzed product (F-2) with a molecular mass of about 2 kDa released from γ-polyglutamic acid by the γ-glutamyl hydrolase YwtD of Bacillus subtilis was analyzed. The results showed that F-2 is an optically heterogeneous polymer consisting of D- and L-glutamic acid in an 80:20 ratio with D-glutamic acid on both the N- and C-terminal sides, suggesting that YwtD is an enzyme that cleaves the γ-glutamyl bond between D- and D-glutamic acid recognizing adjacent L-glutamic acid toward the N-terminal region.     

A New Antibiotic K-52B

A new antibiotic K-52B, different from K-52A, was isolated from the culture broth of Streptoverticillium roseoverticillatum subsp. albosporum, strain No. K-52. The antibiotic K-52B was thought to be a similar saccharide to K-52A from its physicochemical properties but differed from K-52A in the presence of nitrogen content. Antibiotic K-52B inhibited the growth of Gram-positive and Gram-negative bacteria, including Pseudomonas aeruginosa on a chemically defined medium. The growth inhibition was, however, reversed by l-glutamine, l-glutamic acid, l-asparagine and l-aspartic acid.     

Structure of Oligosaccharides Obtained by Controlled Degradation of Mung Bean Xyloglucan with Acid and Aspergillus oryzae Enzyme Preparation

A xyloglucan (MBXG) from the cell walls of etiolated mung bean hypocotyls was characterized by analyzing the fragment oligosaccharides from controlled degradation products of the polymer with acid and enzyme.

Cellobiose, cellotriose and cellotetraose were isolated from the partial acid hydrolyzate of MBXG. Isoprimeverose (6-O-α-d-xylopyranosyl-d-glucopyranose) and a pentasaccharide, α-l-fucosyl-(1 → 2)-β-d-galactosyl-(1 → 2)-α-d-xylosyl-(1 → 6)-β-d-glucosyl-(1 → 4)-d-glucose, were isolated from the hydrolyzate of MBXG with an Asp. oryzae enzyme preparation.     

Studies on Glucose-Isomerizing Enzyme of Bacteria

d-Glucose-isomerizing enzyme from Escherichia intermedia HN-500, which converts d-glucose to d-fructose in the presence of arsenate, was purified by treating with manganous sulfate, rivanol, and DEAE-Sephadex column chromatography. About 180-fold purified enzyme preparation was obtained by the above procedures. The purified preparation was free from the activities of d-glucose-, d-galactose-, glucose-6-phosphate-, mannitol-, and sorbitol-dehydrogenases and was homogeneous on polyacrylamide gel in zone electrophoresis. Optima of pH and temperature for the enzyme were found to be pH 7.0 and 50°C, respectively. The enzyme was completely inactivated by heating at 60°C for ten minutes and stable in the pH range of 7.0~9.0 at 30°C. Activation energy for the isomerizing enzyme was calculated to be 15,300 calories per mole degree from Arrhenius' equation. Either in the absence or presecne of arsenate, d-mannose, d-xylose, d-mannitol and d-sorbitol could not be isomerized by the purified enzyme at all, but the present enzyme isomerized exclusively glucose-6-phosphate and fructose-6-phosphate in the absence of arsenate.     

Some Properties of Three Proteolytic Enzymes Excreted by Bacillus cereus Kp 931

The crude enzyme preparation obtained from culture media of Bacillus cereus Kp 931 was fractionated into three active fractions by Sephadex G-100 gel filtration. These three enzymes had pH optima at between 10.5 and 11.0. One of them, the largest molecular weight species, the enzyme I, was purified extensively. The enzyme catalyzes the release of a number of free amino acids from casein. Large amounts of l-alanine and l-glutamic acid and small amounts of l-leucine, l-serine, glycine, l-cysteic acid and l-arginine were released from oxidized insulin B-chain by the action of the purified enzyme I. It is also suggested that the other two enzymes, II and III, belong to so-called bacterial proteninases.     

Relation between Fatty Acid Composition of Cellular Phospholipids and the Excretion of L-Glutamic Acid by a Glycerol Auxotroph of Corynebacterium alkanolyticum

Relation between fatty acid composition of cellular phospholipids and the excretion of L-glutamic acid was investigated using Corynebacterium alkanolyticum GL–21 (a glycerol auxotroph).

When grown on n-hexadecane, the proportion of unsaturated fatty acids was higher in L-glutamic acid-accumulating cells than in L-glutamic acid-nonaccumulating cells. When grown on fructose or acetic acid, the reverse relation was observed. Moreover, cells containing no oleic acid produced L-glutamic acid from n-pentadecane.

These results suggest that the membrane permeability to L-glutamic acid is not always controlled by the cellular content of unsaturated fatty acids.     

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.     

Change in Permeability to l-Glutamic Acid in the Glycerol Auxotroph GL-21

In this study, the mechanism of the extracellular accumulation of l-glutamic acid by the glycerol auxotroph was partially clarified. Whenever Corynebacterium alkanolyticum GL–21 (glycerol auxotroph) accumulated a large amount of l-glutamic acid in the fermentation broth, the content of its cellular phospholipids was not more than 50% of that of C. alkanolyticum No. 314 (prototroph).

Moreover, biotin, oleic acid or thiamine had no influence on the cellular phospholipid content of the auxotroph.

Under limited supply of glycerol, the efflux of l-glutamic acid in the auxotroph was extremely enhanced, but its enzyme activities participating in l-glutamic acid biosynthesis remained at the same level as those of the prototroph.

From the results, it is considered that the regulation of phospholipid content gave rise to the destruction of the permeability barrier to l-glutamic acid in the cell membrane.