Extracellular Accumulation of Phospholipids,UDP-N-Acetylhexosamine Derivatives and L-Glutamic Acid by Penicillin-treated Corynebacterium alkanolyticum

The addition of penicillin to cells of Corynebacterium alkanolyticum No. 314 growing on n-paraffins medium caused the simultaneous excretion of phospholipids, UDP-N-acetylhexosamine derivatives and L-glutamic acid.

Among many antibiotics which inhibit cell wall synthesis, only the inhibitors of peptideglycan transpeptidase such as penicillin G and cephaloridine were effective for inducing the excretion of phospholipids, UDP-N-acetylhexosamine derivatives and L-glutamic acid, while the others promoted only the excretion of UDP-N-acetylhexosamine derivatives.

From the close relationship between the excretion of L-glutamic acid and the excretion of phospholipids, it was suggested that the action of penicillins and cephalosporins on the cell membrane resulted in the excretion of L-glutamic acid.     

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.     

Culture Conditions for the Production of l-Glutamic Acid from n-Paraffins by Glycerol Auxotroph GL-21

A novel process for the microbial production of l-glutamic acid on an industrial scale was successfully established by using a glycerol auxotroph.

The most suitable carbon source for producing L-glutamic acid was n-paraffins (C₁₃–C₁₅). The production of L-glutamic acid was not affected by a large amount of biotin or oleic acid in the absence of penicillin, and occurred maximally at the glycerol concentration of 0.02% at pH 6.6. The most effective temperature was 28°C.

Under optimal conditions in a 200 liter fermentor, the mutant produced 72 g/liter of L-glutamic acid. On the other hand, the parent produced 53 g/liter of L-glutamic acid in the presence of penicillin.

It is believed that the low productivity of L-glutamic acid by the parent strain was mainly due to the occurrence of the marked decrease in the viable cell counts at the later phase of the fermentation caused by the action of penicillin added.     

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.     

Binding of Pepsin Inhibitor (S-PI) and Pepsin

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

When strain GL–21 was cultured in glycerol-limited medium which contained n-hexadecane, acetic acid or fructose as carbon source, there occurred the limitation of cellular phospholipid content and the over-accumulation of L-glutamic acid in the broth. Two-dimensional thin-layer chromatograms provided evidence that both the parent and the mutant strains contained the same phospholipids such as cardiolipin, phosphatidylethanolamine, phosphadityl-glycerol, phosphatidylinositol and phosphatidic acid. Limited supply of glycerol to the mutant did not greatly alter the proportions of the individual phospholipids.     

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.     

Effect of Penicillin on Amino Acid Fermentation

The effect of penicillin G(k) was first investigated on l-homoserine production by Micrococcus glutamicus 534-Co 147 (a threonine requiring mutant). The addition of 4 u/ml of penicillin, 7 to 9 hours after inoculation, brought about the conversion of l-homoserine to l-glutamic acid production. Similar phenomena were observed in l-lysine and l-valine fermentations. In these cases, a homoserine requiring and a leucine requiring mutant of M. glutamicus were used respectively. A marked conversion from lysine and valine to glutamate accumulation occured by penicillin addition. However, in l-isoleucine fermentation with Brevibacterium ammoniagenes ATCC 6871, no glutamate accumulation took place and isoleucine yields were remarkably decreased.     

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.     

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.     

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.     

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

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.     

l-Glutamate Formation from Hydrocarbons by Microorganisms

Large quantities of l-glutamic acid from liquid paraffins by microorganisms were produced with an addition of penicillin to the growing culture, and the action of penicillin to the glutamate production was studied. One of main effects of penicillin seems to exist in the cellular permeability of l-glutamic acid.     

l-Glutamic Acid Fermentation

Structure of a sugar lipid produced by an oleic acid-requiring mutant of Brevibacterium thiogenitalis was studied and established as (I).

Relation between biotin and oleic acid was studied using a biotin-requiring organism accumulating l-glutamic acid and its blocked mutants lacking the biosynthetic system of biotin or/and oleic acid. The results support the following considerations. Biotin is not formed from oleic acid and does not substantially affect the growth of l-glutamic acid-accumulating bacteria and their productivity of l-glutamic acid.

Consequently, biotin serves only for the synthesis of fatty acids in the present organisms. The essential factor for their growth and metabolism is an unsaturated fatty acid like oleic acid and not biotin. And also, saturated fatty acids have substantially no relation with their growth and metabolism like accumulation of l-glutamic acid.     

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.     

Induction of Antibiotic Formation in Streptomyces sp. No. 362 by the Change of Cellular Fatty Acid Spectrum

Microorganisms which require oleic acid for the formation of antibiotics were screened. Streptomyces sp. No. 362, one of the selected organisms, produced antimicrobial substances only when oleic acid, palmitic acid or the high concentration of l-glutamic acid (or l-glutamine) was supplemented to the medium. The cellular fatty acid composition was changed by the supplement of these fatty acids, but not by l-glutamic acid (or l-glutamine). Antibiotic-producing cells had about 4 to 10 times larger amino acid pools, especially l-glutamic acid pool, and hexosamine pools. The ability for l-glutamate uptake of cells grown in the oleic or palmitic acid supplemented medium was markedly enhanced and the efflux of the accumulated l-glutamate was reduced. The antibiotic produced by this strain was identified as one of the streptothricin-group antibiotics and the role of these additives in the antibiotic formation is discussed.     

Accumulation of l-Glutamic Acid from Dibasic Carboxylic Acids by a Hydrocarbon Utilizing Bacterium

In order to know the substrate specificity in a hydrocarbon utilizing bacterium, the following materials were examined: n-alkanes, n-alkenes, monohydric alcohols, aldehydes, monobasic carboxylic acids, dihydric alcohols and dibasic carboxylic acids.

It was found that dibasic carboxylic acids were well utilized, and a great deal of l-glutamic acid was accumulated from them. Then suberic acid, which is C₈ dibasic carboxylic acid, was compared with n-dodecane in the effects of thiamine, penicillin, C/N ratio and substrate concentration on l-glutamic acid accumulation and cell growth.     

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.     

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.     

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.