On the activation mechanism of L-aspartate beta-decarboxylase from Pseudomonas dacunhae by alpha-ketoglutarate.
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Biomedical subjects
Publications and source records attributed to I Chibata.
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A number of organisms were screened for their ability to produce l-proline. Kurthia catenaforma, which we recently isolated, was selected. A serine-requiring mutant, strain 45, produced about 1.5 times the amount of this amino acid that the parent strain did. In investigations of various media, it was found that approximately 30 ml of l-proline per ml was produced in shaken culture at 30 C in a medium containing glucose, urea, corn steep liquor, casein hydrolysate, l-aspartic acid, and inorganic salts. To study the effect of l-aspartic acid on the production of l-proline, various amino or organic acids were substituted for l-aspartic acid, and the changes during fermentation were investigated. l-Aspartic acid was not replaced by the compounds tested, and this acid appeared to increase growth during the later stages of fermentation with a concurrent increase in the production of l-proline.
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To establish an advantageous method for the production of l-alanine, a procedure was studied for converting l-aspartic acid to l-alanine by microbial l-aspartic beta-decarboxylase. A number of organisms were screened to test their ability to form and accumulate alanine from aspartic acid. Pseudomonas dacunhae was selected as the most advantageous organism. With this organism, enzyme activity as high as 3,910 muliters of CO(2) per hr per ml of medium could be produced by shaking the culture at 30 C in the medium containing ammonium fumarate, sodium fumarate, corn steep liquor, peptone, and inorganic salts. For the enzymatic conversion of l-aspartic acid to l-alanine, the culture broth was employed as the enzyme source. A large amount of l-aspartic acid (as much as 40% of the broth) was converted stoichiometrically to alanine in 72 hr at 37 C. Furthermore, appropriate addition of a surface-active agent to the reaction mixture was found to be highly effective in shortening the time required for the conversion. Accumulated l-alanine was readily isolated in pure form by ordinary procedures with ion-exchange resins. Yields of isolated l-alanine of over 90% from l-aspartic acid were easily attainable.
The use of four stereoisomers of isoleucine by Lactobacillus fermenti strain 36 was studied in detail. All four isoleucine isomers were used for growth in the presence of vitamin B(6) compounds, but only l-isoleucine was active in the absence of these vitamins. Of the vitamin B(6) compounds, pyridoxal and pyridoxamine were equally more effective than pyridoxine for the utilization of these isomers. Lowering the initial pH, decreasing the amounts of leucine and valine, and adapting the organism to d-alloisoleucine medium accelerated the use of isoleucine isomers. Thus, the conditions were established under which respective isomers gave the same growth response, and these findings were applied to the separate microbiological assay of l-isoleucine and total isoleucine isomers.
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Studies were conducted on the participation of specific amino acids in the alterations of their levels in plasma and muscle of rats after short term intravenous infusion. Sufficient amounts of amino acids and glucose were used as a basal solution, and the levels in plasma and muscle were determined 30 min after the end of infusion. When an infusion solution devoid of one of the essential amino acids from the basal solution, -Leu, -Ile, -Lys or -Thr, was administered, the plasma and muscle levels of the deleted amino acids decreased in different degrees. With infusion of the deficient solutions except for the -Leu, no significant changes were observed in amino acids other than those deleted, although occasional changes were noted. On the other hand, the infusion of the -Leu resulted in significant increase of isoleucine and valine levels, and a moderate increase of many other amino acids both in plasma and in muscle. In contrast, when leucine was administered singly in an amount equivalent to that in the basal solution, isoleucine and valine decreased significantly. Most of the other amino acids also decreased markedly after the infusion of leucine alone. These results suggest that, in intravenous infusion, leucine plays a specific role on amino acid levels in plasma and muscle of rats.
Nutritional effects of intravenous infusion of an amino acid (AA) mixture enriched with the branched chain AA were previously evaluated at a daily level of 45 kcal and 200 mg N using male rats weighing approximately 200 g. The present study was conducted with male rats weighing approximately 200 g to evaluate the nutritional effects of 1) an AA infusion solution at further increased energy level, and 2) an AA solution devoid of aspartic and glutamic acids. By increasing daily energy input from 45 to 55 kcal/rat, the body weight gain of rat was markedly increased and more positive nitrogen balance was observed. Glucose, albumin, and free AA levels were unchanged in plasma of rats after the infusion period, while plasma urea level was somewhat lowered. Organ weights and liver lipid content were also unchanged. The administration of an infusion solution devoid of aspartic and glutamic acids resulted in little alteration in the amounts of urinary excretion and plasma levels of these acidic AA. Furthermore, other parameters measured showed no significant effect of the deletion of the AA. These results indicate that no advantage is expected in the use of acidic AA for parenteral nutrition.
Two experiments were conducted with male rats weighing 170 to 190 grams. In experiment 1, some nutritional parameters were determined in tumor-bearing (TB) (Walker 256 carcinosarcoma) rats fed a 23.6% casein diet for 4 weeks after the tumor inoculation. Cumulative weight gain and food intake were less in TB rats than in nontumor-bearing (NTB) rats. At 3 and 4 weeks after the tumor inoculation, plasma histidine, alanine, and glycine levels were higher in TB rats than in NTB animals. The arginine level was lower in the plasma of TB rats at 4 weeks after the inoculation. The significance of decrease in plasma arginine with regard to tumor growth is discussed. In experiment 2, the effects of total parenteral nutrition (TPN) on TB rats were evaluated as compared with those of 5% glucose (Glc) solution. Body weights of TPN rats were maintained and their nitrogen (N) balances were positive during a 7-day experimental period, while 5% Glc animals showed severe body weight loss and apparent negative N balance. After the end of infusion, the plasma urea level of the TPN group was within normal range, whereas that of 5% Glc group showed a markedly high value. The plasma albumin level was higher in the TPN group. Liver and spleen weights were increased in TPN rats. Absolute tumor weight was somewhat greater in TPN rats than in 5% Glc rats, but the difference in tumor weight:body weight ratios became more slight. These results indicate that TPN was effective for maintaining the nutritional status of TB host without significant acceleration in tumor growth.