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.     

Microbial Production of l-Threonine

The growth of Brevibacterium flavum No. 2247 was inhibited over 90% at a concentration above 1 mg/ml of α-amino-β-hydroxyvaleric acid, a threonine analogue, and the inhibition was reversed by the addition of l-threonine, and to lesser extent by l-leucine, l-isoleucine, l-valine and l-homoserine. l-Methionine stimulated the inhibition. Several mutants resistant to the analogue produced l-threonine in the growing cultures. The percentage of l-threonine producer in the resistant mutants depended on the concentration of the analogue, to which they were resistant. The best producer, strain B-183, was isolated from resistant strains selected on a medium containing 5 mg/ml of the analogue. Mutants resistant to 8 mg/ml of the analogue was derived from strain B-183 by the treatment with mutagen, N-methyl-N’-nitro-N-nitrosoguanidine. Among the mutants obtained, strain BB-82 produced 13.5 g/liter of l-threonine, 30% more than did the parental strain. Among the resistant mutants obtained from Corynebacterium acetoacidophilum No. 410, strain C-553 produced 6.1 g/liter of l-threonine. Several amino acids other than l-threonine were also accumulated, and these accumulations of amino acids were discussed from the view of regulation mechanism of l-threonine biosynthesis.     

Taurine Levels in Some Tissues of Yellowtail (Seriola quinqueradiata) and Mackerel (Scomber japonicus)

The mechanism of stereospecific production of l-amino acids from the corresponding 5-substituted hydantoins by Bacillus brevis AJ-12299 was studied. The enzymes involved in the reaction were partially purified by DEAE-Toyopearl 650M column chromatography and their properties were investigated. The conversion of dl-5-substituted hydantoins to the corresponding l-amino acids consisted of the following two successive reactions. The first step was the ring-opening hydrolysis to N-carbamoyl amino acids catalyzed by an ATP dependent l-5-substituted hydantoin hydrolase. This reaction was stereospecific and the N-carbamoyl amino acid produced was exclusively the l-form. N-Carbamoyl-l-amino acid was also produced from the d-form of 5-substituted hydantoin, which suggests that spontaneous racemization occurred in the reaction mixture. In the second step, N-carbamoyl-l-amino acid was hydrolyzed to l-amino acid by an N-carbamoyl-l-amino acid hydrolase, which was also an l-specific enzyme. The ATP dependency of the l-5-substituted hydantoin hydrolase was supposed to be the limiting factor in the production of l-amino acids from the corresponding 5-substituted hydantoins by this bacterium.     

Elimination,Replacement and Isomerization Reactions by Intact Cells Containing Tyrosine Phenol Lyase

Tyrosine phenol lyase catalyzes a series of α,β-elimination, β-replacement and racemization reactions. These reactions were studied with intact cells of Erwinia herbicola ATCC 21434 containing tyrosine phenol lyase.

Various aromatic amino acids were synthesized from l-serine and phenol, pyrocatechol, resorcinol or pyrogallol by the replacement reaction using the intact cells. l(d)-Tyrosine, 3,4-dihydroxyphenyl-l(d)-alanine (l(d)-dopa), l(d)-serine, l-cysteine, l-cystine and S-methyl-l-cysteine were degraded to pyruvate and ammonia by the elimination reaction. These amino acids could be used as substrate, together with phenol or pyrocatechol, to synthesize l-tyrosine or l-dopa via the replacement reaction by intact cells. l-Serine and d-serine were the best amino acid substrates for the synthesis of l-tyrosine or l-dopa. l-Tyrosine and l-dopa synthesized from d-serine and phenol or pyrocatechol were confirmed to be entirely l-form after isolation and identification of these products. The isomerization of d-tyrosine to l-tyrosine was also catalyzed by intact cells.

Thus, l-tyrosine or l-dopa could be synthesized from dl-serine and phenol or pyrocatechol by intact cells of Erwinia herbicola containing tyrosine phenol lyase.     

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 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.     

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.     

Screening of 3,4-Disubstituted Phenyl-L-alanine-producible Microorganisms

Microbial transaminase which catalyzes the reaction between 3,4-disubstituted phenyl pyruvate and certain amino acids to produce 3,4-disubstituted phenyl-L-alanine was investigated. Wide distribution of this enzyme among the various kinds of microorganisms was confirmed.

3,4-Dihydroxyphenyl-L-alanine (L-Dopa) or 3,4-dimethoxyphenyl-L-alanine was isolated from corresponding keto acids using three strains of selected microorganisms.     

Studies on the Reaction Conditions of DOPA Production with Alcaligenes faecalis.

The effects of several factors on the enzymatic production of 3,4-dihydroxyphenyl-l-alanine (DOPA) and 3,4-dimethoxyphenyl-l-alanine (DMPA) by transamination reaction were investigated using wet cells of Alcaligenes faecalis IAM 1015. In addition, some experiments for the cultural conditions for transaminase production were performed. DOPA and DMPA were obtained in 80 and 90% yields, respectively, using the mixture of l-glutamate and l-aspartate as amino donors. Accumulation of DMPA in the culture under the growing state of the bacteria was also confirmed.     

Multistep enzyme catalysed deracemisation of 2-naphthyl alanine

Kinetic parameters of d-amino acid oxidase from R. gracilis (DAAO) towards d-2-naphthyl alanine (d-2-NAla) and of l-aspartate amino transferase (l-AAT) from Escherichia coli towards 2-naphthyl pyruvate (2-NPA) were measured. The two enzymes were then combined in a one-pot reaction in which DAAO was used to generate 2-NPA which was the substrate of l-AAT in the presence of cysteine sulphinic acid (CSA) as an amino donor. The combined reactions afforded enantiomerically pure l-2-NAla in almost quantitative yield. The extremely low water solubility of 2-NAla can be partially overcome by running the biotransformation in suspension with higher formal concentration. In these conditions multiple enzyme additions are required.     

The Biosynthetic Control in Aromatic Amino Acid Producing Mutants of Corynebacterium glutamicum

Regulatory properties of the enzymes involved in aromatic amino acid biosynthesis in the mutant of Corynebacterium glutamicum which produces a large amount of aromatic amino acids were examined. A phenylalanine auxotrophic l-tyrosine producer, pr-20, had a 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthetase released from the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a two-fold derepressed chorismate mutase. A pair of l-phenylalanine and l-tyrosine still strongly inhibited the chorismate mutase activity, though the enzyme was partially released from the inhibition by l-phenylalanine alone. A tyrosine auxotrophic l-phenylalanine producer, PFP-19-31, had a DAHP synthetase sensitive to the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a prephenate dehydratase and a chorismate mutase both partially released from the feedback inhibition by l-phenylalanine. The mutant produced a large amount of prephenate as well as l-phenylalanine. A phenylalanine and tyrosine double auxotrophic l-tryptophan producer, Px-115-97, had an anthranilate synthetase partially released from the feedback inhibition by l-tryptophan and had a DAHP synthetase sensitive to the feedback inhibition. These data explained the mechanism of the production of aromatic amino acids by these mutants and supported the in vivo functioning of the control mechanisms of aromatic amino acid biosynthesis in C. glutamicum previously elucidated in vitro experiments.     

Synthesis of γ-Glutamyl Peptides Catalyzed by Transamidase from Bacillus natto

Crude ammonium sulfate fraction of a cell free extract from Bacillus natto contained an enzyme (or enzymes) which catalyzed the transamidation reaction specific for glutamine. Both l- and d-isomers of glutamine were active as substrate. On incubation of l- or d-glutamine with the enzyme preparation, two peptides consisting of glutamic acid and glutamine were formed. The main component of the peptides was readily isolated by ion-exchange chromatography and identified as γ-glutamylglutamine by paper chromatography and by paper electrophoresis using authentic peptides. The optical configuration of the amino acid residues in the dipeptide was determined by digestion of the acid hydrolyzate with l-glutamic acid decarboxylase, and the result showed that the dipeptide obtained from l-glutamine was a l-l isomer, while the dipeptide from d-glutamine was a d-d isomer.     

Partial Purification and Some Properties of 7-Keto-8-aminopelargonic Acid Synthetase,an Enzyme Involved in Biotin Biosynthesis

7-Keto-8-aminopelargonic acid synthetase (KAPA synthetase) which catalyzes the formation of KAPA from pimelyl CoA and l-alanine, and is involved in biotin biosynthesis, was partially purified from a cell-free extract of Bacillus sphaericus by a procedure involving ammonium sulfate fraction ation, protamine treatment, and DEAE-cellulose column chromatography. The reaction product was bioautographically confirmed to be KAPA. Some properties of the enzyme were also investigated. Among the amino acids, only l-alanine was active as a substrate, condensing with pimelyl CoA, The reaction required pyridoxal phosphate but the other vitamin B₆ compounds were inert. Typical inhibitors of pyridoxal phosphate enzymes showed marked inhibition to the reaction. Various amino acids such as l-cysteine, glycine, d-alanine, l-serine, l-histidine, and d-histidine were also found to be inhibitory.     

Synthesis of l-Tryptophan from Pyruvate,Ammonia and Indole

The tryptophanase activity which synthesizes l-tryptophan from pyruvate, ammonia and indole, was found to be widely distributed in cells of bacteria belonging to Enterobacteriaceae, such genera as Escherichia, Kluyvera, Enterobacter, Erwinia and Proteus. With the cells of Proteus rettgeri, equilibrium of the elimination reaction of l-tryptophan in the presence of high concentration of ammonia was studied. It was found that the equilibrium inclines toward the synthetic state.

When 5-hydroxy- and 5-methyl-indole were substituted for indole, 5-hydroxy- and 5-methyl-l-tryptophan, respectively, were synthesized. The synthesis of l-tryptophan was also observed with indole and various amino acids, S-methyl-l-cysteine, S-ethyl-l-cysteine, l-cysteine, 5-fluoro-dl-tryptophan, or oxalacetic acid.     

Distribution of Tyrosine Phenol Lyase in Microorganisms

The distribution of tyrosine phenol lyase activity in microorganisms was studied with intact cells in a synthetic reaction mixture containing l-serine and phenol or pyrocatechol. This activity was found in various bacteria, most of which belonged to the Enterobacteriaceae; especially to the genera Escherichia, Proteus and Erwinia. Cells of Erwinia herbicola ATCC 21434 were selected as a promising source of enzyme.

Intact cells of Erwinia herbicola ATCC 21434 prepared from a broth cultured for 24 hr contained markedly high enzymic activity and catalyzed the synthetic reaction of l-tyrosine or 3,4-dihydroxyphenyl-l-alanine (l-dopa) from l-serine and phenol or pyrocatechol in significantly high yields.

Results of the isolation and identification of the products showed that the amino acid synthesized by this enzymatic method was identical with l-tyrosine or l-dopa.     

Effects of Methionine,Cystine and Taurine on Plasma Cholesterol Level in Rats Fed a High Cholesterol Diet

l-Methionine γ-lyase (EC 4.4.1.11) catalyzes α,β-elimination of l-2-amino-3-(N-methylamino)propionic acid and l-2-amino-3-(N-hydroxyethylamino)propionic acid to yield pyruvate, ammonia, and the corresponding amines. These amino acids also undergo the enzymatic β-replacement reaction with thiols to produce the corresponding S-substituted cysteines. Thus, l-methionine γ-lyase cleaves a C-N bond in addition to C-S, C-Se, and C-O bonds at the β position of amino acids by elimination and replacement reactions. A linear relationship between the reactivity, (log(V_max/Km) and the pKa value of the conjugated acid of the leaving group has been found for Se-methyl-l-selenocysteine, S-methyl-l-cysteine, and O-methyl-l-serine. However, l-2-amino-3-(N-methylamino)propionic acid has shown lower reactivity than that expected from the pKa value of methylammonium ions.     

Syntheses of Several β-l-Rhamnopyranosides

To investigate the substrate specificity of β-l-rhamnosidase, the following β-l-rhamnopyranosides were synthesized: 1-(β-l-rhamnopyranosyl)-dl-glycerol (1), methyl β-l-rhamnopyranoside (2), methyl 2-O-(β-l-rhamnopyranosyl)-β-d-glucopyranoside (3) and methyl 2-O-β(β-l-rhamnopyranosyl)-α-l-arabinopyranoside (4). The synthesis of 3 was performed using l-quinovose with neighboring group participation, which lead stereoselectively to the β-l-quinovoside. The 2-OH of the l-quinovo-unit was selectively deblocked, oxidized to the keto group, and then stereoselectively reduced, whereby 3 was produced.     

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.     

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.     

Genetic Changes of Regulatory Mechanisms Occurred in Leucine and Valine Producing Mutants Derived from Brevibacterium lactofermentum 2256

The regulatory mechanisms in branched-chain amino acid synthesis were compared between 2-thiazolealanine (2-TA) resistant l-leucine and l-valine producing mutants and the 2-TA sensitive original strains of Brevibacterium lactofermentum 2256.

In the original strains, sensitive to 2-TA, α-isopropylmalate (IPM) synthetase, the initial enzyme specific for l-leucine synthesis, is sensitive to feedback inhibition and to repression by l-leucine, and α-acetohydroxy acid (AHA) synthetase, the common initial enzyme for synthesis of l-isoleucine, l-valine as well as l-leucine, is sensitive to feedback inhibition by each one of these amino acids, and to repression by them all. In strain No. 218, a typical l-leucine producer resistant to 2-TA, IPM synthetase was found to be markedly desensitized and derepressed, and AHA synthetase remained unaltered. On the contrary, in strain No. 333, l-valine producer resistant to 2-TA, AHA synthetase was found to be desensitized and partially derepressed, and IPM synthetase remained unaltered.

The genetic alteration of these regulatory mechanisms was discussed in connection with the accumulation pattern of amino acids.