Crystallization and Properties of N-Benzoylglycine Amidohydrolase from Pseudomonas putida

N-Benzoylgiycine amidohydrolase (hippurate hydrolase EC 3.5.1.32), which catalyzes the hydrolysis of hippuric acid to benzoic acid and glycine, was found in a cell-free extract of Pseudomonas putida C692-3 grown on a medium containing hippuric acid. The enzyme was purified from the extract by ammonium sulfate fractionation and column chromatographies on DEAE-cellulose, DEAE-Sephadex A-50, hydroxyapatite, and Sepharose CL-6B. The enzyme was finally crystallized. The crystalline enzyme was almost homogeneous on electrophoresis. The enzyme had a molecular weight of about 170,000 and consisted of four subunits identical in molecular weight (approximately 42,000). The enzyme hydrolyzed N-benzoylglycine most rapidly, and N-benzoyl-l-alanine and N-benzoyl-l-aminobutyric acid. The Km value for these substrates were 0.72 mm, 0.87 mm, and 0.87mm, respectively. The optimum pH of the enzyme reaction was 7.0 to 8.0 and the enzyme was stable from pH 6.0 to 8.0.     

New Nematicidal Metabolites from a Fungus,Irpex lacteus

N-Benzoyl-l-alanine amidohydrolase was purified from a cell-free extract of Corynebacterium equi H-7 which was grown in a medium containing hippuric acid as the sole carbon source. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The molecular weight was 230,000 and the enzyme consisted of six subunits, identical in molecular weight (approximately 40,000). The isoelectric point of the enzyme was pH 4.6. The optimum pH of the enzyme reaction was 8.0 and the enzyme was stable from pH 7.0 to 8.0. The enzyme hydrolyzed N-benzoyl-l-alanine, N-benzoylglycine, and N-benzoyl-l-aminobutyric acid. The Km values for these substrates were 4.3 mm, 6.7 mm, and 4.3 mm, respectively. The enzyme was activated by Co²⁺.     

Purification and Characterization of l-Aminoacylase from Alcaligenes denitrificans DA181

The l-aminoacylase produced intracellularly by Alcaligenes denitrificans DA181 was puritied to homogeneity. This enzyme had an apparent molecular weight of 80,000, and was composed of two subunits of identical molecular weight. Its isoelectric point was pH 5.1. The optimal reaction temperature and pH were 65°C and 8.0, respectively. This enzyme showed specificity toward N-acetyl-derivative of hydrophobic l-amino acids with N-acetyl-l-valine as the favored substrate, followed by N-acetyl-l-alanine.     

Biosynthetic Threonine Deaminase from Proteus morganii

Biosynthetic threonine deaminase was purified to an apparent homogeneous state from the cell extract of Proteus morganii, with an overall yield of 7.5%. The enzyme had a s⁰_20,w of 10.0 S, and the molecular weight was calculated to be approximately, 228,000. The molecular weight of a subunit of the enzyme was estimated to be 58,000 by sodium dodecyl sulfate gel electrophoresis. The enzyme seemed to have a tetrameric structure consisting of identical subunits. The enzyme had a marked yellow color with an absorption maximum at 415 nm and contained 2 mol of pyridoxal 5′-phosphate per mol. The threonine deaminase catalyzed the deamination of l-threonine, l-serine, l-cysteine and β-chloro-l-alanine. Km values for l-threonine and l-serine were 3.2 and 7.1 mm, respectively. The enzyme was not activated by AMP, ADP and ATP, but was inhibited by l-isoleucine. The Ki for l-isoleucine was 1.17 mm, and the inhibition was not recovered by l-valine. Treatment with mercuric chloride effectively protected the enzyme from inhibition by l-isoleucine.     

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.     

Properties of Crystalline ω-Amino Acid : Pyruvate Aminotransferase of Pseudomonas sp. F–126

ω-Amino acid: pyruvate aminotransferase, purified to homogeneity and crystallized from a Pseudomonas sp. F–126, has a molecular weight of 172,000 or 167,000±3000 as determined by the gel-filtration or sedimentation equilibrium method, respectively. The enzyme catalyzes the transamination between various ω-amino acids or amines and pyruvate which is the exclusive amino acceptor. α-Amino acids except l-α-alanine are inert as amino donor. The Michaelis constants are 3.3 mm for β-alanine, 19 mm for 2-aminoethane sulfonate and 3.3 mm for pyruvate. The enzyme has a maximum activity in the pH range of 8.5~10.5. The enzyme is stable at pH 8.0~10.0 and at up to 65°C at pH 8.0. Carbonyl reagents strongly inhibit the enzyme activity. Pyridoxal 5′-phosphate and pyridoxamine 5′-phosphate reactivate the enzyme inactivated by carbonyl reagents. The inhibition constants were determined to be 0.73 mm for d-penicillamine and 0.58 mm for d-cycloserine. Thiol reagents, chelating agents and l-α-amino acids showed no effect on the enzyme activity.     

Leucine Dehydrogenase from Corynebacterium pseudodiphtheriticum: Purification and Characterization

Leucine dehydrogenase [EC 1.4.1.9] was purified to homogeneity from Corynebacterium pseudodiphtheriticum ICR 2210. The enzyme consisted of a single polypeptide with a molecular weight of about 34,000. Stepwise Edman degradation provided the N-terminal sequence of the first 24 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 2 amino acids. Although the enzyme catalyzed the reversible deamination of various branched-chain l-amino acids, l-valine was the best substrate for oxidative deamination at pH 10.9 and the saturated concentration. The enzyme, however, had higher reactivity for l-leucine, and the k_cat/Km value for l-leucine was higher than that for l-valine. The enzyme required NAD⁺ as a natural coenzyme. The NAD⁺ analogs 3-acetylpyridine-NAD⁺ and deamino-NAD⁺ were much better coenzymes than NAD ⁺. The enzyme activity was significantly reduced by sulfhydryl reagents and pyridoxal 5′-phosphate. d-Enantiomers of the substrate amino acids competitively inhibited the oxidation of l-valine.     

Mechanism of Asymmetric Production of d-Amino Acids from the Corresponding Hydantoins by Pseudomonas sp.

The mechanism of asymmetric production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. AJ-11220 was examined by investigating the properties of the enzymes involved in the hydrolysis of dl-5-substituted hydantoins. The enzymatic production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. AJ-11220 involved the following two successive reactions; the d-isomer specific hydrolysis, i.e., the ring opening of d-5-substituted hydantoins to d-form N-carbamyl amino acids by an enzyme, d-hydantoin hydrolase (d-HYD hydrolase), followed by the d-isomer specific hydrolysis, i.e., the cleavage of N-carbamyl-d-amino acids to d-amino acids by an enzyme, N-carbamyl-d-amino acid hydrolase (d-NCA hydrolase).

l-5-Substituted hydantoins not hydrolyzed by d-HYD hydrolase were converted to d-form 5- substituted hydantoins through spontaneous racemization under the enzymatic reaction conditions.

It was proposed that almost all of the dl-5-substituted hydantoins were stoichiometrically and directly converted to the corresponding d-amino acids through the successive reactions of d-HYD hydrolase and d-NCA hydrolase in parrallel with the spontaneous racemization of l-5-substituted hydantoins to those of dl-form.     

Production,Purification, and Characterization of D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

The best inducers for D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) were a poor substrate, N-acetyl-;-methyl-D-leucine, and an inhibitor, N-acetyl-D-alloisoleucine. The enzyme has been homogeneously purified. The molecular weight of the native enzyme was estimated to be 58,000 by gel filtration. A subunit molecular weight of 52,000 was measured by SD8–PAGE, indicating that the native protein is a monomer. The isoelectric point was 5.2. The enzyme was specific to the D-isomer and hydrolyzed N-acetyl derivatives of D-leucine, D-phenylalanine, D-norleucine, D-methionine, and D-valine, and also N-formyl, N-butyryl, and N-propionyl derivatives of D-leucine. The K_m for N-acetyl-D-leucine was 9.8mM. The optimum pH and temperature were 7.0 and 50°C, respectively. The stabilities of pH and temperature were 8.1 and 40°C. D-Aminoacylases from three species of the genus Alcaligenes differ in inducer and substrate specificities, but are similar with respect to molecular weight and N-terminal amino acid sequence.     

Substrate Specificity of Alkaline Proteinases from Aspergillus sydowi and Aspergillus sulphureus

l-Fucose (l-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1143 and purified about 380-fold with a yield of 23 %. The purification procedures were: treatment with polyethyleneimine, ammonium sulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of about 34,000. The optimum pH was at 9 — 10.5 and the isoelectric point was at pH 5.1. l-Fucose and l-galactose were effective substrates for the enzyme reaction, but d-arabinose was not so much. The anomeric requirement of the enzyme to l-fucose was the β-pyranose form, and the reaction product from l-fucose was l-fucono- lactone. The hydrogen acceptor for the enzyme reaction wasNADP⁺, and NAD ⁺ could be substituted for it to a very small degree. Km values were 1.9mm, 19mm, 0.016mm, and 5.6mm for l-fucose, l- galactose, NADP⁺, and NAD⁺, respectively. The enzyme activity was strongly inhibited by Hg^{2 +}, Cd^{2 +}, and PCMB, but metal-chelating reagents had almost no effect. In a preliminary experiment, it was indicated that the enzyme may be usable for the measurement of l-fucose.     

Purification and Crystallization of d-Arabinose (l-Fucose) Isomerase from Aerobacter aerogenes by Polyethylene Glycol

d-Arabinose(l-fucose) isomerase (d-arabinose ketol-isomerase, EC 5.3.1.3) was purified from the extracts of d-arabinose-grown cells of Aerobacter aerogenes, strain M-7 by the procedure of repeated fractional precipitation with polyethylene glycol 6000 and isolating the crystalline state. The crystalline enzyme was homogeneous in ultracentrifugal analysis and polyacrylamide gel electrophoresis. Sedimentation constant obtained was 15.4s and the molecular weight was estimated as being approximately 2.5 × 10⁵ by gel filtration on Sephadex G-200.

Optimum pH for isomerization of d-arabinose and of l-fucose was identical at pH 9.3, and the Michaelis constants were 51 mm for l-fucose and 160 mm for d-arabinose. Both of these activities decreased at the same rate with thermal inactivation at 45 and 50°C. All four pentitols inhibited two pentose isomerase activities competitively with same Ki values: 1.3–1.5 mm for d-arabitol, 2.2–2.7 mm for ribitol, 2.9–3.2 mm for l-arabitol, and 10–10.5 mm for xylitol. It is confirmed that the single enzyme is responsible for the isomerization of d-arabinose and l-fucose.     

S-Carboxymethylcysteine Synthase from Escherichia coli

An enzyme that catalyzes the synthesis of S-carboxymethyl- l-cysteine from 3-chloro- l-alanine (3-Cl-Ala) and thioglycolic acid was found in Escherichia coli W3110 and was designated as S- carboxymethyl-l-cysteine synthase. It was purified from the cell-free extract to electrophoretic homogeneity and was crystallized. The enzyme has a molecular weight of 84,000 and gave one band corresponding to a molecular weight of 37,000 on SDS-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the β-replacement reactions between 3-CI-AIa and various thiol compounds. The apparent Km values for 3-Cl-Ala and thioglycolic acid were 40 mM and 15.4 mM. The enzyme showed very low activity as to the α,β-elimination reaction with 3-Cl-Ala and l-serine. It was not inactivated on the incubation with 3-Cl-Ala. The absorption spectrum of the enzyme shows a maximum at 412 nm, indicating that it contains pyridoxal phosphate as a cofactor. The N-terminal amino acid sequence was determined and the corresponding sequence was detected in the protein sequence data bank, but no homogeneous sequence was found.     

An Aminopeptidase of Aspergillus sojae

An aminopeptidase was purified from Aspergillus sojae X–816. The molecular weight of the enzyme was estimated to be 220,000. The isoelectric point was at pH 5.3. The optimum pH for l-leucylglycylglycine was 7.5. The enzyme was stable up to 37°C against temperature treatment for 15 min. Some chelating agents inhibited the enzyme activity. The Km value for l-leucylglycylglycine at pH 7.5 and 37°C was 45 mm. The Km value for l-leucyl-β-naphthylamide at pH 7.0 and 37°C was 2.2 mm.     

Production of Xylanase by Streptomyces sp., Using Non-metabolizable Inducer

meso-Diaminopimelate dehydrogenase (EC 1.4.1.16) was purified to homogeneity from Corynebacterium glutamicum ATCC 13032. The enzyme had a molecular weight of about 70,000 and consisted of two subunits identical in molecular weight. The enzyme was highly specific for meso-2,6-diaminopimelate. The pH optima for deamination and amination were about 9.8 and 7.9, respectively. The Michaelis constants were 3.1mm for meso-2,6-diaminopimelate, 0.12mm for NADP⁺, 0.28 mm for l-2-amino-6-ketopimelate, 36 mm for ammonia, and 0.13 mm for NADPH. d and l isomers of 2,6-diaminopimelate competitively inhibited the oxidative deamination of meso-2,6-diaminopimelate. The enzyme was distributed in a wider range of bacterial species than reported previously [Misono et al., J. Bacteriol., 137, 22 (1979)] when assayed by a sensitive formazan formation method.     

Changes of α-Glycerophosphate Dehydrogenase Activity in Fatty Liver of Rats by Amino Acid Imbalance

An aminopeptidase was purified from an aqueous extract of mullet roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose and Sephadex G-200. The molecular weight of the enzyme was 184,000 by gel filtration, and the enzyme appeared to consist of two homogenous subunits. The optimal pH and optimal temperature for activity were 7.4 and 45°C, respectively. Puromycin, p-chloromercuribenzoic acid, and o-phenanthroline inhibited the enzyme n on-competitively (their Ki = 1.34 μm, 0.113mm and 0.145 mm, respectively), while 2-mercaptoethylamine was competitive (Ki = 0.056 mm). The enzyme was also inhibited by l-amino acids, in particular glutamic acid. The enzyme could hydrolyze a variety of α-aminoacyl β-naphthylamides and was most active on l-alanyl-β-naphthylamide. Judging from these properties, the mullet roe aminopeptidase resembles soluble alanyl amino-peptidase [EC 3.4.11.14].     

Regulatory Properties of Chorismate Mutase from Corynebacterium glutamicum

Regulatory properties of chorismate mutase from Corynebacterium glutamicum were studied using the dialyzed cell-free extract. The enzyme activity was strongly feedback inhibited by l-phenylalanine (90% inhibition at 0.1~1 mm) and almost completely by a pair of l-tyrosine and l-phenylalanine (each at 0.1~1 mm). The enzyme from phenylalanine auxotrophs was scarcely inhibited by l-tyrosine alone but the enzyme from a wild-type strain or a tyrosine auxotroph was weakly inhibited by l-tyrosine alone (40~50% inhibition, l-tyrosine at 1 mm). The enzyme activity was stimulated by l-tryptophan and the inhibition by l-phenylalanine alone or in the simultaneous presence of l-tyrosine was reversed by l-tryptophan. The Km value of the reaction for chorismate was 2.9 } 10^?3 m. Formation of chorismate mutase was repressed by l-phenylalanine. A phenylalanine auxotrophic l-tyrosine producer, C. glutamicum 98–Tx–71, which is resistant to 3-amino-tyrosine, p-aminophenylanaine, p-fluorophenylalanine and tyrosine hydroxamate had chorismate mutase derepressed to two-fold level of the parent KY 10233. The enzyme in C. glutamicum seems to have two physiological roles; one is the control of the metabolic flow to l-phenylalanine and l-tyrosine biosynthesis and the other is the balanced partition of chorismate between l-phenylalanine-l-tyrosine biosynthesis and l-tryptophan biosynthesis.     

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.     

Properties of α-Amino-ε-caprolactam Racemase from Achromobacter obae

α-Amino-ε-caprolactam racemase, which occurs in the cytoplasmic fraction of Achromobacter obae, has been purified to homogeneity. It has a monomeric structure with a molecular weight of approximately 50,000. The absorption spectrum of the enzyme exhibits maxima at 280 and 412 nm at pH 7.3, and is independent of pH from 6.0 to 8.0. One mole of pyridoxal 5′-phosphate is bound per mol of the enzyme. Incubation of the enzyme with hydroxylamine resulted in the formation of the apoenzyme. d- and l-α-Amino-ε-caprolactams are the only substrates. The maximum activity is found at pH 8.8 for both the isomers. Michaelis constants are as follows: 8 mm for d-α-amino-ε-caprolactam, 6mm for l-α-amino-ε-caprolactam and 2.1 × 10^?7 m for pyridoxal 5′-phosphate. The enzyme is inhibited significantly by CuSO₄, HgCl₂, thiol reagents such as N-ethylmaleimide and p-chloromercuribenzoate, and carbonyl reagents (e.g., phenylhydrazine and hydroxylamine). α-Amino-ε-caprolactam racemase catalyzes the α-proton exchange of the substrate with deuteron during racemization in deuterium oxide.     

Biological Activities of Sex Hormone Produced by Tremella mesenteriea

d-Aminoacylase was found to be produced not only by S. olivaceus 62–3 isolated from soil but also by three strains of type culture of Streptomyces species. All four of these strains produced d-aminoacylase intracellularly only when an inducer was added to the culture medium. d-Amino acids or N-acetyl-d-amino acids were effective as inducers.

As S. tuirus showed the highest d-aminoacylase activity, the enzyme extract of this strain was subjected to further investigation to determine the optimal conditions for optical resolution of N-acetyl-dl-phenylglycine. Almost all contaminating l-aminoacylase in the enzyme extract could be eliminated by DEAE-Sephadex adsorption. d-Phenylglycine of 99.9% optical purity was obtained after complete hydrolysis of d-isomer with the use of d-aminoacylase solution.     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.