l-Glutamic Acid Fermentation with Molasses

Conditions suitable for the cell wall lysis of a l-glutamate-producing bacterium, Microbacterium ammoniaphilum, by egg white lysozyme were studied, in order to make clear the correlation of the fatty acid composition of the cellular fractions and the extracellular accumulation of l-glutamate,

The cell wall of a phage-resistant strain was recognized to be almost completely lyzed by the lysozyme.

Using this result, the relationship between the fatty acid composition of each fraction and extracellular accumulation of l-glutamate was investigated, and the following thesis was proposed: The extracellular accumulation of l-glutamate in large quantity took place when the molar ratio of saturated/unsaturated fatty acid in the cell membrane fraction was above 1.     

Glutamate Metabolism in a Glutamate-producing Bacterium,Brevibacterium flavum

Brevibacterium flavum No. 2247 was found to grow with l-glutamate as the sole carbon and nitrogen source on an agar-plate medium when high concentrations of l-glutamate, FeSO₄ and biotin were added to the medium. It grew on l-glutamate in liquid medium only when yeast extract or high concentrations of FeSO₄ and glucose or organic acids of the tricarboxylic acid cycle were added to the medium. The growth on l-glutamate in liquid medium was also stimulated by high concentrations of l-glutamate, biotin and MgSO₄, and inhibited by a high concentration of (NH₄)₂SO₄.

Aspartate aminotransferase (TA)- and α-ketoglutarate dehydrogenase (KD)-defective mutants did not grow on l-glutamate, and glutamate-utilizing revertants derived from these mutants recovered TA and KD activity, respectively, whereas glutamate dehydrogenase (GD)-defective mutants grew on l-glutamate. Washed cells of strain No. 2247 grown on glutamate decomposed the amino acid, whereas those grown on glucose did not. The degradation was observed only under aerobic conditions. The former cells showed higher KD, succinate dehydrogenase and fumarase activities than the latter cells. Of 75 mutants which did not grow on glutamate but grew on succinate, three strains lacked KD but showed the same glutamate productivity as the parent strain. Four other strains with normal KD levels showed higher glutamate productivity than the parent.     

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.     

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.     

Synthesis of γ-Glutamylpeptides by γ-Glutamylcysteine Synthetase from Proteus mirabilis

Syntheses of various γ-glutamylpeptides were examined taking use of the highly purified γ-glutamylcysteine synthetase from Proteus mirabilis. The accumulation of each peptide was measured after long time incubation, and good formation was observed in the synthesis of peptides of following amino acids, l-cysteine, l-α-aminobutyrate, l-serine, l-homoserine, glycine, l-alanine, l-norvaline, l-lysine, l-threonine, taurine and l-valine. Peptide syntheses were confirmed by analyses of the component amino acids, after hydrolysis of the peptides.

The structure of the glutamylpeptides, especially the peptide-linkage at the γ-carbonyl residue of l-glutamate, was determined by mass spectrometry of the N-trifluoroacetyl methylester derivatives of the glutamylpeptides. Enzymatic synthesis of γ-glutamyl-l-α-aminobutyrate was also confirmed by PMR spectrometry in the comparison with chemically synthesized compound.     

Regulation of l-Glutamate Biosynthesis by Copper Ions

A specific regulatory effect of copper ions on the microbiological synthesis of l-glutamate from acetate was found. The minimal concentration of copper ions necessary for the maximal production of l-glutamate was about 0.025 µg/ml at which the yield of l-glutamate was four times greater than that in the absence of copper ions. This effect of copper was demonstrated only when acetate was the substrate; it was not observed when the substrate was glucose ethanol, lactate or n-paraffin.

The physiological features of the l-glutamate production from acetate were examined in the presence or absence of copper ions. The most striking features of the culture without added copper ions were the increase in Q_O2 and NADH oxidase and the marked reduction of succinate oxidase accompanied with the reduction of l-glutamate formation. In addition, the regulation of l-glutamate synthesis by copper ions proved to have no relation to the wellknown regulatory factor, cell permeability. These facts suggest that the l-glutamate biosynthesis from acetate is regulated through unknown factors related to the respiratory activities.     

l-Glutamic Acid Fermentation with Molasses

When an l-Glutamic acid (l-GA)-forming bacterium, Microbacterium ammoniaphilum, was cultured in the molasses medium with the addition of penicillin to accumulate large quantity of l-GA extracellularly, no significant differences were observed in the phospholipid quantity and the fatty acid composition which were found between the l-GA-accumulating cells grown either in the molasses medium with addition of polyoxyethylene fatty acid ester (POEFE) or in the glucose medium with the addition of biotin.

Moreover, it was shown that, in the molasses-POEFE system, the amount of l-GA accumulated was nearly constant, independent of the extracellular osmotic pressure caused by the presence of NaNO₃ or β-alanine, while, in the molasses-penicillin system, the amount varied inversely to the osmotic pressure.

From these results, it is assumed that either chemical or mechanical process can eliminate the permeability barrier in the cell membrane, thus allowing the extracellular accumulation by l-GA-forming bacteria.     

Biochemical Effects of Fatty Acid and its Derivatives on l-Glutamic Acid Fermentation

Brev. lactofermentum rapidly took up biotin from culture medium and stored it in the cells. The saturation level of the stored biotin (3.8 × 10⁴ molecules/cell) exceeded the level required for the maximum growth by ten times, and the minimum level (1.3 × 10³ molecules/cell) was the most adequate to the accumulation of l-glutamic acid. The stored cellular biotin over the minimum level was metabolically available in the subsequent culture lacking in supplemented biotin. The cellular biotin was gradually reduced to the minimum level with the multiplication of the cells, and them the accumulation of l-glutamic acid was observed. This relation between the level of cellular biotin and the accumulation of l-glutamic acid was impaired by the addition of Tween 60 or some saturated fatty acid. In the presence of biotin and Tween 60 the biotin-saturated cells turned into cells capable of accumulating l-glutamic acid keeping the maximum level; and in the same medium the cells having the minimum amount of biotin took up biotin and then were saturated with it, and yet the cells preserved the acid-accumulating property. It was confirmed with the use of bioautographic technique and avidin test that the biotin released from the cells by acid hydrolysis was identical with authentic d-biotin.     

Asymmetric synthesis of l-6-hydroxynorleucine from 2-keto-6-hydroxyhexanoic acid using a branched-chain aminotransferase

Abstract

l-6-Hydroxynorleucine was synthesized from 2-keto-6-hydroxyhexanoic acid using branched-chain aminotransferase from Escherichia coli with l-glutamate as an amino donor. Since the branched-chain aminotransferase was severely inhibited by 2-ketoglutarate, the branched-chain aminotransferase reaction was coupled with aspartate aminotransferase and pyruvate decarboxylase. Aspartate aminotransferase converted the inhibitory 2-ketoglutarate back to l-glutamate by using l-aspartate as an amino donor. On the other hand, pyruvate decarboxylase further shifted the reaction equilibrium towards l-6-hydroxynorleucine through decarboxylation of pyruvate to acetaldehyde. The concerted action of the three enzymes significantly enhanced the yield compared to that of branched-chain aminotransferase alone. In the coupled reaction, 90.2 mM l-6-hydroxynorleucine (> 99% ee) was produced from 100 mM 2-keto-6-hydroxyhexanoic acid, whereas in a single branched-chain aminotransferase reaction only 22.5 mM l-6-hydroxynorleucine (> 99% ee) was produced.     

Utilization of Hydrocarbons by Microorganisms

As already reported, Corynebacterium hydrocarboclastus S10B1 was able to accumulate a good deal of l-glutamate in a thiamine-deficient medium at the sole expense of n-alkanes, but unable to form l-glutamate in a thiamine-sufficient medium though an abundant cell growth was observed.

α-Ketoglutaric acid and dl-alanine were found to be produced in the same thiamine-deficient medium in which l-glutamate was accumulated. Both products formed from n-tetradecane by this organism were isolated from culture broth, purified and identified. The optimum concentration of thiamine in the culture medium was 3 to 5 µg per liter for their production. The maximum yields of α-ketoglutaric acid and dl-alanine reached 16 g and 1.5 g per liter in the calcium carbonate-added medium, respectively. However, the addition of more than 30 μg per liter of thiamine extremely repressed their accumulation.     

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.     

Distribution of 7-Keto-8-aminopelargonic Acid Synthetase in Bacteria and the Control Mechanism of the Enzyme Activity

The 7-keto-8-aminopelargonic acid (KAPA) synthetase activities of cell-free extracts from various bacteria were investigated. The experiments on the substrate specificity of KAPA synthetase, using crude cell-free extracts from bacteria having high enzyme activity, showed that l-serine and pyruvic acid could replace l-alanine, but that, when the enzyme was partially purified, these compounds were not effective. Many kinds of amino acids such as l-cysteine, l-serine, d-alanine, glycine, d-histidine, and l-histidine, inhibited the enzyme activity. This inhibition was found to be competitive with l-alanine. Pyridoxal 5′-phosphate, which is a cofactor of the enzyme, also inhibited the enzyme activity at high concentrations. The repression of KAPA synthetase by biotin occurred in Bacillus subtilis and B. sphaericus but not in Micrococcus roseus and Pseudomonas fluorescens, even at a concentration of 1000 mµg per ml of biotin.     

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp

Branched chain amino acid aminotransferase was partially purified from Pseudomonas sp. by ammonium sulfate fractionation, aminohexyl-agarose and Bio-Gel A-0.5 m column chromatography.

This enzyme showed different substrate specificity from those of other origins, namely lower reactivity for l-isoleucine and higher reactivity for l-methionine.

Km values at pH 8.0 were calculated to be 0.3 mm for l-leucine, 0.3 mm for α-ketoglutarate, 1.1 mm for α-ketoisocaproate and 3.2 mm for l-glutamate.

This enzyme was activated with β-mercaptoethanol, and this activated enzyme had different kinetic properties from unactivated enzyme, namely, Km values at pH 8.0 were calculated to be 1.2 mm for l-leucine, 0.3 mm for α-ketoglutarate.

Isocaproic acid which is the substrate analog of l-leucine was competitive inhibitor for pyridoxal form of unactivated and activated enzymes, and inhibitor constants were estimated to be 6 mm and 14 mm, respectively.     

Separation of l-Leucine-pyruvate and l-Leucine-α-ketoglutarate Transaminases in Acetobacter suboxydans and Identification of Their Reaction Products

l-Leucine-pyruvate and l-leucine-α-ketoglutarate(α-KGA) transaminases were separated by DEAE-cellulose column chromatography and partially purified to 200- and 50-fold, respectively, from the cell-free extract of Acetobacter suboxydans (Gluconobacter suboxydans IFO 3172). The optimum pH range of the former was 5.0~5.5 and that of the latter was 8.5~9.0. l-Leucine, l-citrulline, and l-methionine were the most effective amino donors for the l-leucine-pyruvate transaminase. Basic amino acids as well as aromatic amino acids were able to be amino donors for the transamination with pyruvate. α-KGA was effective as an amino acceptor for this enzyme. The l-leucine-α-KGA transaminase had the typical properties of the branched-chain amino acid transaminase in its substrate specificity.

The reaction products of the transaminations were identified. l-Alanine was formed from pyruvate and l-glutamate from α-KGA. α-Keto acids formed from various amino acids by the l-leucine-pyruvate transaminase were also identified.     

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.     

Production of l-Lysine by Fluoropyruvate-sensitive Mutants of Brevibacterium lactofermentum

Better producers of l-lysine were obtained by derivation of fluoropyruvate(FP)-sensitive mutants from Brevibacterium lactofermentum AJ3990. The coexistence of FP and excess biotin synergistically stimulated l-lysine formation by washed cells. FP inhibited 50% of growth and pyruvate dehydrogenase (PDH) activity of AJ3990 at 0.04 mm and 1 mm, respectively. Therefore, the synergistic effect of FP and excess biotin seems to be due to the optimization of the PDH/pyruvate carboxylase activity ratio in l-lysine biosynthesis. This was confirmed by the derivation of FP-sensitive mutants which have the optimal level of PDH activity for l-lysine production. The best producer, AJ11204, had about 27% PDH activity as compared with the parental strain and accumulated 70 g of l-lysine per liter with a conversion yield of 50% from glucose in the presence of excess biotin.     

Properties and Continuous Use of Microbial Fumarase Entrapped in Fibres

A new flavoprotein enzyme, l-glutamate oxidase, was purified to homogeneity from an aqueous extract of a wheat bran culture of Streptomyces sp. X-l 19–6. It showed absorption maxima at 273, 385 and 465 nm and a shoulder around 490 nm, and contained 2 mol of FAD per mol of enzyme. The enzyme had a molecular weight of approximately 140,000 and consisted of three sizes of subunits with molecular weights of 44,000, 16,000 and 9,000. Balance studies showed that 1 mol of l-glutamate was converted to 1 mol of α-ketoglutarate, ammonia and hydrogen peroxide with the consumption of 1 mol of oxygen. In addition to l-glutamate, l-aspartate was oxidized by the enzyme but only to an extent of 0.6% at pH 7.4; the Michaelis constants were as follows: 0.21 mM for l-glutamate and 29 mM for l-aspartate. The isoelectric point was pH 6.2, and the enzyme activity was optimal between pH 7.0 and 8.0. When the enzyme was heated at pH 5.5 for 15 min, the remaining activity was 100% of the original activity level at 65°C, 87% at 75°C and 47% at 85°C.     

Cultural Conditions for l-Leucine Production by Strain No. 218, a Mutant of Brevibacterium lactofermentum 2256

The excellent l-leucine producing mutant No. 218, derived from a biotin requiring glutamic acid producing strain, is methionine and isoleucine auxotrophic. A suboptimum growth condition made by adding a limiting amount of isoleucine was necessary for the maximum production of l-leucine. On the other hand, methionine was indifferent to the productivity if sufficiently supplied for growth.

Biotin of more than 50 μg/liter caused the accumulation of l-leucine; less than 50 μg/liter, however, gave a drastic change in accumulation pattern from l-leucine to l-glutamic acid. Strain No. 218 produced 28 mg/ml of l-leucine after 72 hr cultivation when 13 % glucose was supplied as a carbon source, thus giving the yield of 21.6%.

Effects on l-leucine production of concentrations of inorganic salts, pH, temperature and aeration were also investigated.     

Production and Purification of Alkaline Protease Inhibitors of Streptomyces sp.

N-Acetyl-d-glutamate deacetylase and N-acetyl-d-aspartate deacetylase were found in cell extracts from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. N-Acetyl-d-glutamate deacetylase was produced inducibly by N-acetyl-d-glutamate and was highly specific to N-acetyl-d-glutamate. N-Acetyl-d-aspartate deacetylase was produced inducibly by N-acetyl-d-aspartate and was highly specific to N-acetyl-d-aspartate.     

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.