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.     

Studies on the Behaviors of Impurities on the Crystallization of L-Glutamie Acid

The influences of some factors at the both sides of the solid and liquid phases on the solubilities of L-glutamic acid in α and β-forms in water were examined.

As differences in the conditions of the liquid phase, the effects of co-existing inorganic neutral salts and amino acids on the solubilities were investigated; and as for the inorganic neutral salts applied here, salting-in effects were recognized, the degrees of which were in the following order, i.e., NH₄Cl, (NH₄)₂SO₄>KCl>NaCl. The effect in the β-form was larger than that of in the α-form. The influence of co-existing amino acids was very little if the amount was not so much, but the tendency to increase the solubility could be recognized.

As a difference in the condition of the solid phase, the purity of the crystals was picked up, and it was clarified that the crude crystal of L-glutamic acid in α-form gave a larger value of the solubility than the purified one.     

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 l-Glutamic Acid Fermentation

The authors have carried out a series of studies on l-glutamic acid fermentation with a strain of Brevibacterium divaricatum nov. sp. in the previous papers.

In this paper, some metabolism of l-glutamic acid and oxidative decomposition of several organic acids concerning the tricarboxylic acid cycle by the resting cells have been studied. The results suggest that l-glutamic acid is one of the final fermentative products of this bacterium, and the tricarboxylic acid cycle is working as a glutamic acid forming cycle.

The presence of glucokinase, phosphoglucoisomerase, phosphofructokinase, aldolase, DPN-linked glyceraldehyde-3-phosphate dehydrogenase, and TPN-linked glucose-6-phosphate dehydrogenase in cell-free extracts of this bacterium was also demonstrated.     

Studies on the Polymorphism of l-Glutamic Acid

The enthalpy differences between the α- and β-crystals of l-glutamic acid were measured and ΔH_α?β of ?75, ?95, 8 and 27±20cal./mol. were obtained at temperatures of 5, 15, 30 and 40°C, respectively. From this result, it was found that the α-form is the more stable form in solid phase at temperatures lower than 30°C and vice versa when higher than 30°C, though the α-form had been found to be the less stable form as the saturating body in the aqueous solution in all range of temperature.     

Studies on l-Glutamic Acid Fermentation

In the previous paper, most of the enzymes of the Embden-Meyerhof-Parnas pathway and glucose-6-phosphate dehydrogenase have been demonstrated to be present in cell-free extracts of Brevibacterium divaricatum, No. 1627. In this paper, the presence of condensing enzyme, aconitase, TPN-linked isocitric dehydrogenase, succinic dehydrogenase, fumarase, DPN-linked malic dehydrogenase, TPN-linked malic enzyme, oxalacetic carboxylase, isocitritase and malate synthetase in cell-free extracts of this bacterium was also demonstrated. From these results it was concluded that a strain of Brevibacterium divaricatum which has been found to contain all of the enzymes of the tricarboxylic acid cycle, would be capable of forming the key enzymes of the glyoxylate bypass as well. It suggests that the accumulation of α-ketoglutarate involves the glyoxylate bypass besides the tricarboxylic acid cycle in this bacterium.     

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 Polymorphism of l-Glutamic Acid

The β-crystal formation of l-glutamic acid in the seeded solution was investigated; and it was found that the growth rate of the seed crystals in a-axis direction was nearly as large as that of the α-crystal, but the growth rate in b- and c-axes was little recognized. The activation energy of the crystallization process of the β-crystal in a-axis direction was calculated from the growth rate constants determined at various temperatures, and 6~7 kcal/mol was obtained. On the assumption that the crystallization of β-crystal growth was controlled by the diffusional operation, the thickness of the laminar film was calculated from the growth rate constant and the estimated value of the diffusional constant. The calculated value of the thickness was much greater than the value reported by Nernst; therefore, the crystallization process should be controlled by the surface reaction. The co-existence of a small quantity of amino acids caused a great reduction in the growth rate of the β-crystal.     

Studies on l-Glutamic Acid Fermentation by Microbacterium ammoniaphilum

The present investigation is concerned with the effects of biotin and glucose on glutamic acid fermenation by Microbacterium ammoniaphilum. Both optimal amounts of biotin necessary for maximum growth and maximum accumulation of glutamic acid were determined under the conditoin of various concentration of glucose. As the glucose concentratin was increased, the amounts of biotin required for maximum growth also increased proportionally to the glucose concentrations. The optimal amounts of biotin for maximum accumulation of glutamic acid were smaller than those for maximum growth of cells at any glucose concentration. It is suggested that the process of glutamic acid accumulation is inevitably associated with the process of cell multiplication by both experiments of successive culture of cells grown under the dose of biotin sufficient and deficient for maximum growth.     

l-Glutamic Acid Fermentation

A study was made on the differences between Brevibacterium thiogenitalis No. 653 and its oleic acid-requiring mutant D-248 in some physiological characteristics.

The most important difference of the characteristics was found in their intracellular fatty acid contents. Namely, the cellular oleic acid content of D-248 was scarcely affected by biotin but limited by the oleic acid which was added to the medium.

On the other hand, various enzyme activities and rates of oxygen uptake for several organic acids were found to be slightly different between the two strains.

These observations suggest that oleic acid has an important role for the production of l-glutamic acid.

The effect of biotin and oleic acid on the cellular fatty acid contents, and the relation between the cellular fatty acid contents and the productivity of l-glutamic acid were investigated using Brevibacterium thiogenitalis No. 653 and its oleic acid-requiring mutant, D-248.

While the synthesis of palmitic acid in D-248 was stimulated by biotin and competitively reversed by oleic acid added to the culture medium, the level of cellular oleic acid was scarcely affected by biotin but regulated by oleic acid in the medium.

For the productivity of L-glutamic acid, the most important factor was the level of cellular oleic acid, and the effect of cellular palmitic acid was considerably weak. This relation was subjected to a figuration and able to be expressed on the whole as one exponential-like curve. An amount of over 70 per cent of cellular fatty acids was distributed in the phospholipid fraction and its fatty acid composition was almost the same as that of whole cells.     

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.     

l-Glutamic Acid Fermentation

It is well known that biotin has a marked effect on l-glutamic acid fermentation.

The authors have intended to find strains which are independent of the amounts of biotin in the culture medium. As a result, oleic acid-requiring mutants were obtained from a strain of Brevibacterium thiogenitalis which is an auxotroph for biotin. The growth of the mutant was remarkably stimulated by Tween 20, 40, 60, Ca ions and a small amount of corn steep liquor. And also, the mutant was found to have lost its requirement for biotin and showed growth response only to oleic acid or unsaturated fatty acids.

The effect of biotin, oleic acid and other unsaturated fatty acids on the production of l-glutamic acid was investigated by using an oleic acid-requiring mutant of Brevibacterium thiogenitalis No. 653. The results described in the present paper showed that the oleic acid-requiring mutant D-248 produced a large amount of l-glutamic acid in the excess biotin-contaming media, and that oleic acid seemed to be completely replaced by other unsaturated fatty acids such as palmitoleic acid and linoleic acid.     

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.     

l-Glutamic Acid Fermentation with Molasses

The relation between oleate and biotin to the extracellular accumulation of l-glutamate in Microbacterium ammoniaphilum was studied. And it was suggested that oleate was the essential constituent for the bacterial cell structure, and, at the same time, it participated in the cellular permeability of l-glutamate. On the other hand, biotin was recognized to play a role on the synthesis of cellular fatty acid, mainly oleate and palmitate. Through the discussion above mentioned, the reason was made clear that biotin was not necessary for the bacterial growth or the extracellular accumulation of l-glutamate, if oleate had been added.     

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.     

l-Glutamic Acid Fermentation with Molasses

An l-glutamic acid (l-GA)-forming bacterium. Microbacterium ammoniaphium was cultured in the molasses medium with or without poiyoxyethylene fatty acid esters to obtain l-GA-accumulating cells or non-accumulating cells, respectively.

Then protoplast-like bodies (PLB) were prepared from each group of cells by reacting them with egg white lysozyme.

l-GA-accumulating reaction by the PLB was carried out under high and low osmotic pressures.

From the results of the experiment, it was shown that the difference in the ability of l-GA accumulation between l-GA-accumulating cells and non-accumulating cells was attributed mainly to the difference in the nature of the cell membrane.

Further, the relationship between the molar ratio of saturated fatty acids/unsaturated fatty acids which was reported previously and the nature of the membrane was discussed.

The lipid composition of the cell membrane from Microbacterium ammoniaphilum was determined by thin-layer and column chromatographies to make clear the relation between the extracellular accumulation of l-glutamic acid and the lipid in the cell membrane. When polyoxyethylene fatty acid ester was added to the beet medium and a large amount of l-glutamic acid was accumulated, the increase of the saturated fatty acid (C₁₆, C₁₈) in the neutural lipid fraction and the decreases of the phospholipid fraction and the unsaturated fatty acid $({\text{C}}_{18}^{1=})$ in the neutral lipid fraction were recognized.     

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

It has been found that although Brevibacterium lactofermentum No. 2256 is incapable of accumulating l-glutamic acid in a biotin sufficient medium, it produces a large quantity of the acid in the presence of sucrose fatty acid ester. In a biotin deficient medium, however, the ester brought the unfavorable diminution of l-glutamic acid accumulation caused by the decrease of glucose consumption in an incubation period. The undesirable effects were practically lost when the ester was added to the culture medium after more than eight hours in the course of incubation. This fact suggests that the ester is concerned with the growth of microorganism. It is very interesting to elucidate the interrelation between sucrose fatty acid ester and biotin. For the maximum accumulation of l-glutamic acid corresponding increase in amount of the ester to the increasing concentration of biotin was necessary. The proportional relation did not extend to excedingly high levels of the two implicating factors. The further observations concerning the effects of the individual fatty acid esters such as sucrose stearate remain unsatisfactory.     

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

Tween 60 (polyoxyethylene sorbitan monostearate) has been found to be the most effective derivative of fatty acid in accumulating l-glutamic acid in biotin-sufficient medium. The effect was exceedingly subject to the influence of the addition time of the ester, and this was observed also on the growth curve of Brev. lactofermentum. Changes of the growth curve caused by the varied addition time of the ester corresponded to those by the concentration of biotin in the medium that did not contain Tween 60. The patterns of fermentation course in the two corresponding conditions, such as biotin 3 μg/l and biotin 20 μg/l-Tween 60 mg/ml, agreed closely with each other. It seemed that identical cells were grown on the conditions. The only difference between the cells was observed as to the contents of intracellular biotin. Although l-glutamic acid was not accumulated by biotin-sufficient cells, cells with sufficient biotin and capable of accumulating l-glutamic acid were obtained in the presence of Tween 60, in which case the ester neither prevented the cells from taking up biotin nor controlled the level of intracellular biotin.     

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.     

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

In the preceding paper on the interrelation between sucrose ester of fatty acid and biotin, the fatty acid being a mixture of C₁₀ to C₁₈ acid, it was described that carbon chain length of fatty acid has a great influence on the accumulation of l-glutamic acid. Fatty acids with C₁₂ to C₁₈ chain length, particularly myristic, palmitic and margaric acids were effective on the accumulation of l-glutamic acid in the culture medium containing sufficient biotin, whereas lower and higher length acids were ineffective. In the form of polyoxyethylene sorbitan or polyethylene glycol ester, C₁₆ and C₁₈ acids were remarkably effective. However, the ester of C₁₂ acid and polyoxyethylene ethers of C₁₂ to C₁₈ alcohols had little or no effect.