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.     

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 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.     

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.     

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.     

Studies on Oxidation-Reduction Potentials (ORP) of Microbial Cultures

At maximum production of l-glutamic acid, the oxidation-reduction potential of the culture broth in l-glutamic acid fermentation showed a stable value of 9.0 to 9.6 as rH value. When biotin concentration in the medium was high (40γ/liter), the production of l-glutamic acid decreased, and the rH was 8.0 and it was out of accordance with that of the control (biotin-poor; 2γ/liter). Under “less-aerobic” conditions, its rH rose to 10.4.

From these results, it was concluded that the rH during maximum production of l-glutamic acid showed a stable value affected actively by the redox system, l-glutamic acid/α-ketoglutaric acid and      

Utilization of Hydrocarbons by Microorganisms

As already reported, strain S1OB1 was found to accumulate l-glutamic acid in a thiamine-deficient medium at the sole expense of hydrocarbon. In order to elucidate the biosynthetic pathway of l-glutamic acid, first of all, the incorporation of molecular oxygen into l-glutamic acid was examined. l-Glutamic acid accumulated under ¹⁸O-enriched atmosphere was separated, purified, identified and found to have been enriched with ¹⁸O. This results indicate the occurrence of oxygenase reaction involving addition of molecular oxygen. From a postulated biosynthetic pathway of l-glutamic acid, theoretical ¹⁸O content was calculated and compared with experimental one. ¹⁸O content of cells grown on n-alkane or glucose was also examined.     

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.     

Conversion of the Extracellular Dextransucrase Isoenzymes from Leuconostoc mesenteroides NRRL B-1299

Effect of oxygen tension on l-lysine, l-threonine and l-isoleucine accumulation was investigated. Sufficient supply of oxygen to satisfy the cell’s oxygen demand was essential for the maximum production in each fermentation. The dissolved oxygen level must be controlled at greater than 0.01 atm in every fermentation, and the optimum redox potentials of culture media were above ?170 mV in l-lysine and l-threonine and above ?180 mV in l-isoleucine fermentations. The maximum concentrations of the products were 45.5 mg/ml for l-lysine, 10.3 mg/ml for l-threonine and 15.1 mg/ml for l-isoleucine. The degree of the inhibition due to oxygen limitation was slight in the fermentative production of l-lysine, l-threonine and l-isoleucine, whose biosynthesis is initiated with l-aspartic acid, in contrast to the accumulation of l-proline, l-glutamine and l-arginine, which is biosynthesized by way of l-glutamic acid.     

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.     

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.     

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 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.     

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.     

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.     

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.     

Microbial Production of l-Glutamic Acid by Glycerol Auxotrophs

To establish a novel process for the production of l-glutamic acid from n-paraffins, a glycerol auxotroph GL-21, a new type mutant, was successfully obtained from Corynebacterium alkanolyticum No. 314 by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. This auxotroph required glycerol for its growth regardless of the carbon source used.

At 72 hr, this mutant GL-21 produced about 40 mg/ml of l-glutamic acid from n-paraffins in the culture broth at 0.01 per cent addition of glycerol in the absence of penicillin.

A thiamine auxotroph, a biotin auxotroph and an oleic acid auxotroph were also obtained by a similar technique, but these auxotrophs were found to be inapplicable for the production of l-glutamic acid from n-paraffins.     

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 the Behaviors of Impurities on the Crystallization of l-Glutamic Acid

The behaviors of impurities such as amino acids and inorganic salts at the time of crystallization of l-glutamic acid were investigated; and it was concluded that amino acids which co-existed in the solution of l-glutamic acid followed the crystals of l-glutamic acid persistently, and the contamination mechanism would not be clarified by the adherence of mother liquor or the formation of liquid foams in the crystals, or by the mixed crystal formation, but by a physical adsorption on the crystal surfaces.     

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.