Production and utilization of amino acids.
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Amino Acids↗
Biomedical subjects
I Chibata
Publications and source records attributed to I Chibata.
Continuous production of NADP by immobilized Achromobacter aceris cells.
Several microorganisms having higher nicotinamide adenine dinucleotide kinase (NAD kinase, EC 2.7.1.23) activity were immobilized into polyacrylamide gel lattices. The enzyme activity field by immobilization was highest in Achromobacter aceris AKU 0120. By the incubation of the immobilized A. aceris cells at pH 4.0, the NAD kinase activity increased and the adenosine triphosphate (ATP)-degradation activity disappeared completely. Enzymatic properties of the immobilized A. aceris cells were investigated and compared with those of intact cells. The optimal pH and the optimal temperature of immobilized cells were the same as those of intact cells. Immobilized cell NAD kinase was more stable than that of intact cells. The operational half-life of immobilized cells was 20 days when the substrate solution was passed through a column packed with immobilized cells at a flow rate which gives a space velocity (SV) of 0.1 hr-1 at 37 degrees C. On the other hand, the half-life of the intact cells was only 6 hr.
Adenosine Triphosphate↗
Construction of an L-arginine-producing mutant in Serratia marcescens. Use of the wide substrate specificity of acetylornithinase.
L-Arginine biosynthesis in Serratia marcescens Sr41 was found to be controlled by (a) feedback inhibition of N-acetylglutamate synthetase and (b) repression of some L-arginine biosynthetic enzymes, and an L-arginine-degrading system was found to exist. Accordingly, an L-arginine-producing mutant (aru argR argA) of S. marcescens Sr41 was constructed as follows. A mutant incapable of L-arginine utilization (aru) was obtained from the wild strain. Subsequently, from the lysine auxotroph (lysA) of aru mutant, a mutant having derepressed L-arginine biosynthetic enzymes (argR) was isolated by screening for colonies that could utilize Nalpha-acetyl-L-lysine in the presence of L-arginine. This selection was based on the finding that acetylornithinase of S. marcescens hydrolyzed Nalpha-acetyl-L-lysine. On the other hand, to obtain a mutant with feedback-resistant N-acetylglutamate synthetase (argA), the proAB argD argR triple mutant was isolated from the indirectly suppressed revertant (proAB argD) of the proline auxotroph (proAB). Next, the argA mutant was isolated from the triple mutant by selection for resistance to 3,4-dehydro-DL-proline in the presence of L-arginine. The argA mutation was introduced into the aru lysA argR strain by PS20-mediated cotransduction with lysA+. The aru argR argA lysA+ transductant produced 25 mg/ml of L-arginine in the medium.
Acetyl Coenzyme A↗
Construction of a urocanic acid-producing strain of Serratia marcescens by transduction.
In Serratia marcescens, the mutation responsible for triazolealanine (TRA) resistance was transferred from a TRA-resistant mutant to a urocanase-less mutant by PS20-mediated transduction. The two crosses were performed using as donors two TRA-resistant mutants, whose phenotypes included increased levels of histidine-biosynthetic enzymes and feedback-insensitive phosphoribosyltransferase. In one cross, TRA-resistant transductants were urocanase-less mutants having only increased levels of the enzymes and barely detectable levels of urocanic acid. In the other cross, the transductants were urocanase-less mutants having both phenotypes of the donor, and most produced high concentrations (10.5 mg/ml) of urocanic acid.
Alanine↗
Threonine production by regulatory mutants of Serratia marcescens.
beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.
Aspartate Kinase↗
Threonine degradation by Serratia marcescens.
The wild strain of Serratia marcescens rapidly degraded threonine and formed aminoacetone in a medium containing glucose and urea. Extracts of this strain showed high threonine dehydrogenase and "biosynthetic" threonine deaminase activities, but no threonine aldolase activity. Threonine dehydrogenase-deficient strain Mu-910 was selected among mutants unable to grow on threonine as the carbon source. This strain did not form aminoacetone from threonine, but it slowly degraded threonine. Strain D-60, deficient in both threonine dehydrogenase and threonine deaminase, was derived from strain Mu-910 and barely degraded threonine. A glycine-requiring strain derived from the wild strain grew in minimal medium containing threonine as the glycine source, whereas a glycine-requiring strain derived from strain Mu-910 did not grow. This indicates that threonine dehydrogenase participates in glycine formation from threonine (via alpha-amino-beta-ketobutyrate) as well as in threonine degradation to aminoacetone.
Alcohol Oxidoreductases↗
Effect of nitrogen source on nutritional management after small bowel resection in rats.
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Amino Acids↗
Reason for higher stability of aspartase activity of immobilized Escherichia coli cells.
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Ammonia-Lyases↗
Transformations of organic compounds by immobilized microbial cells.
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Arginine↗
Pathway for isoleucine formation form pyruvate by leucine biosynthetic enzymes in leucine-accumulating isoleucine revertants of Serratia marcescens.
Leaky revertants isolated from isoleucine auxotrophs of Serratia marcescens mutant resistant to alpha-aminobutyric acid were previously reported to accumulate leucine in the medium, due to the absence of both feedback inhibition and repression of leucine biosynthesis. Growth of the revertant was accelerated by pyruvate, D(-)-citramalate, citraconate, and alpha-ketobutyrate, but not by threonine. Extracts of the revertant exhibited high activities of pyruvate-dependent coenzyme A liberation from acetyl-coenzyme A, hydration of citraconate, and conversion of citraconate to alpha-ketobutyrate, but showed no threonine-deaminating activity. In the leucine-accumulating revertants the above three activities were not affected by leucine, but in the wild strain and other revertants accumulating no leucine all or one of these activities was controlled by leucine. A leucine auxotroph isolated from the leucine-accumulating revertant showed isoleucine auxotrophy as well. From these data, it is concluded that, in leucine-accumulating revertants, of S. marcescent, isoleucine, is synthesized from alpha-ketobutyrate via citramalate formed from pyruvate annd acetyl-coenzyme A by leucine biosynthetic enzymes, as a result of desensitization of alpha-isopropylmalate synthetase to feedback inhibition.
Acetyl Coenzyme A↗
Norleucine accumulation by a norleucine-resistant mutant of Serratia marcescens.
A norleucine-resistant mutant was derived from an isoleucine-valine auxotroph of a leucine accumulator of Serratia marcescens. The norleucine-resistant mutant could accumulate norleucine from norvaline in the medium without the addition of methionine, which antagonized norleucine. This mutant constitutively formed homoserine-O-transsuccinylase.
Acyltransferases↗
L-Histidine production by histidase-less regulatory mutants of Serratia marcescens constructed by transduction.
2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.
Drug Resistance, Microbial↗
Enhancement of isoleucine hydroxamate-mediated growth inhibition and improvement of isoleucine-producing strains of Serratia marcescens.
Growth inhibition by isoleucine hydroxamate in Serratia marcescens was significantly enhanced by adding valine plus leucine and by using glycerol as the carbon source. Isoleucine hydroxamate-resistant mutants were isolated under conditions in which growth inhibition was enhanced. One of the mutants, strain GIHVLr2179, lacked both feedback inhibition and repression of threonine deaminase. An alpha-aminobutyric acid-resistant mutant derived from strain GIHVLr2179, strain GIHVLAr2795, produced 12 mg of isoleucine per ml in the medium containing glucose and urea as carbon and nitrogen sources (a twofold increase over prior reports). This strain had increased activities of threonine deaminase, acetohydroxy acid synthase, aspartokinase, and homoserine dehydrogenase.
Acetolactate Synthase↗
Increase in arginine and citrulline production by 6-azauracil-resistant mutants of Bacillus subtilis.
In the arginine producer AHr-5, an L-arginine hydroxamate-resistant mutant of Bacillus subtilis, accumulation of N8-acetyl-L-ornithine increased as the level of L-arginine accumulation increased in the medium containing L-glutamic acid. Ornithine carbamoyltransferase of this strain was genetically derepressed. These results suggested that carbamoylphosphate might be deficient in vivo. With the intention to increase endogenous carbamoylphosphate, pyrimidine analogs inhibiting growth were selected and the mutants resistant to these compounds were derived from the AHr-5 mutant. Of the resistant mutants derived, the 6-azauracil-resistant mutant AAr-9 produced 28 mg of L-arginine per ml, which corresponded to more than twofold the amount produced by the parent strain. Derivation of an arginine-requiring mutant from the double-resistant mutant AAr-9 provides a new advantageous method for the production of L-citrulline. The increase in arginine and citrulline production is discussed.
Arginine↗
Norvaline accumulation by regulatory mutants of Serratia marcescens.
A gene coding for desensitized L-threonine dehydratase was transduced with phage PS20 into a leucine accumulator of Serratia marcescens Sr41. The transductant converted L-threonine to alpha-ketobutyrate, a precursor of both norvaline and isoleucine. An isoleucine-valine auxotroph of the transductant accumulated large amount of norvaline from L-threonine as well as from D-threonine.
Cell-Free System↗
Production of L-aspartic acid by microbial cells entrapped in polyacrylamide gels.
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Acrylic Resins↗
Production of L-amino acids by aminoacylase adsorbed on DEAE-Sephadex.
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Amidohydrolases↗
L-Norvaline and L-homoisoleucine formation by Serratia marcescens,.
Two unnatural amino acids were found as by-products in isoleucine fermentation from threonine by Serratia marcescens. These amino acids were identified as L-norvaline (2-aminopentanoic acid) and L-homoisoleucine (2-amino-4-methylhexanoic acid). Formation of L-norvaline and L-homoisoleucine was not observed when L-leucine was added to the medium. The leucine auxotroph derived from the isoleucine accumlator did not produce L-norvaline or L-homoisoleucine even during the accumulation of large amounts of isoleucine. It is suggested that L-norvaline and L-homoisoleucine formation is closely related to leucine biosynthesis.
Isoleucine↗