Effect of Substrate Concentration on Plastein Productivity and Some Rheological Properties of the Products

A bacterial arginase was purified to homogeneity from a strain of Bacillus brevis. The native enzyme, with an estimated MW of 143,000, migrated on SDS-PAGE as a single polypeptide of estimated MW of 33,000. The enzyme, highly specific to l-arginine, showed the maximum activity at pH 11.0 in the presence of Mn²⁺ ions and the pI was 4.8 by isoelectric focusing. The enzyme activity was increased significantly by the addition of Mn²⁺, Ni²⁺, or Co²⁺ ions, and inhibited potently by chemicals such as HgCl₂, N-bromosuccinimide, or glutathione. The K_ms for l-arginine and l-canavanine were 0.69 and 22.2 mm, respectively. The enzyme was inhibited competitively by γ-guanidinobutyric acid, and non-competitively by l-lysine, l-ornithine, creatine, blasticidin S, and edeine B₁ Analysis of the N-terminal amino acid sequence of the purified bacterial enzyme found 33–36% homologies with the Agrobacterium, yeast, rat, and human enzymes.     

Inhibition of l-Alanine Adding Enzyme by Glycine

l-Alanine adding enzymes from Bacillus subtilis and Bacillus cereus which catalyzed l-alanine incorporation into UDPMurNAc were partially purified and the properties of the enzymes were examined. The enzyme from B. subtilis was markedly stimulated by reducing agents including 2-mercaptoethanol, dithiothreitol, glutathione and cysteine. Mn²⁺ and Mg²⁺ activated l-alanine adding activity and their optimal concentrations were 2 to 5 mm and 10 mm, respectively. The optimum pH was 9.5 and the Km for l-alanine was 1.8×10^?4m. l-Alanine adding reaction was strongly inhibited by p-chloromercuribenzoate and N-ethyl-maleimide. Among glycine, l- and d-amino acids and glycine derivatives, glycine was the most effective inhibitor of the l-alanine adding reaction. The enzyme from B. cereus was more resistant to glycine than that from B. subtilis. Glycine was incorporated into UDPMurNAc in place of l-alanine, and the Ki for glycine was 4.2×l0^?3m with the enzyme from B. subtilis. From these data, the growth inhibition of bacteria by glycine is discussed.     

DAHP Synthetase and Its Control in Corynebacterium glutamicum

Properties of 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthetase from Corynebacterium glutamicum were examined using the cell free extract. The optimum pH for the reaction was broad ranging from 5.5 to 7.0 and the optimum temperature was 37°C. Co²⁺ inhibited the enzyme activity at 20°C, whereas Co²⁺ apparently stimulated the enzyme activity at 37°C because the ion protected the enzyme from inactivation at 37°C. Co²⁺ reversed the inhibition of the enzyme activity by EDTA. The activity of DAHP synthetase was feedback inhibited only weakly by l-phenylalanine, l-tyrosine or l-tryptophan alone, but was strongly inhibited synergistically by l-phenylalanine and l-tyrosine. l-Tryptophan enhanced the inhibition by the pair of l-tyrosine and l-phenylalanine. Maximal inhibition was near 90 % in the simultaneous presence of the three amino acids. Sensitivity of the enzyme to the inhibitors was lost during the purification process of the enzyme or during the reaction at 37°C. Especially sensitivity to l-tryptophan was easily lost. Co²⁺ protected the enzyme from the desensitization. Mutants resistant to p-fluorophenylalanine plus l-tyrosine (or 3-aminotyrosine) had DAHP synthetase which was released from the feedback inhibition by the three amino acids. The formation of the enzyme was not affected by aromatic amino acids.     

The effects on plasma L-arginine levels of combined oral L-citrulline and L-arginine supplementation in healthy males

We investigated the effects of combining 1 g of l-citrulline and 1 g of l-arginine as oral supplementation on plasma l-arginine levels in healthy males. Oral l-citrulline plus l-arginine supplementation more efficiently increased plasma l-arginine levels than 2 g of l-citrulline or l-arginine, suggesting that oral l-citrulline and l-arginine increase plasma l-arginine levels more effectively in humans when combined.     

Fermentative Production of l-Arginine

Most of the bacteria, which were examined for the sensitivity to l-arginine analogs (l-canavanine, l-homoarginine, d-arginine and arginine hydroxamate), were insensitive to the analogs at a concentration of 8 mg/ml. Corynebacterium glutamicum DSS-8 isolated as d-serine-sensitive mutant from an isoleucine auxotroph KY 10150, was found to be sensitive to d-arginine and arginine hydroxamate. Furthermore, DSS-8 produced l-arginine in a cultural medium. l-Arginine analog-resistant mutants were derived from DSS-8 by N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment. Most of them were found to produce a large amount of l-arginine. An isoleucine revertant from one of these mutants produced 19.6 mg/ml of l-arginine in the medium containing 15% (as sugar) of molasses.

The mechanism of the sensitivity to l-arginine analogs and that of the production of l-arginine in the d-serine-sensitive mutant, DSS-8, were investigated. DSS-8 seems to be a mutant having increased permeability to d- and l-arginine.     

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.     

Isolation and Identification of O-Acetyl and 12-Hydroxyradiclonic Acids

An N-acetylglutamate-acetylornithine acetyltransferase-deficient arginine-requiring mutant AA–1, was derived from an l-arginine producer of Corynebacterium glutamicum. It accumulated a large amount (30 mg per ml) of l-glutamic acid and a small amount (1.2 mg per ml) of N^α-acetylornithine, an intermediate of arginine biosynthesis, in the culture medium.

The production of N^α-acetylornithine by AA–1 was not affected by the concentration of l-arginine in the medium, whereas that of l-glutamic acid was inhibited by a high concentration of l-arginine in the medium containing excess biotin.     

Properties of Tyrosinase from Pseudomonas melanogenum

The properties of the tyrosinase from Pseudomonas melanogenum was investigated with the crude enzyme preparation. Optimum temperature and pH of the enzyme were 23°C and 6.8, respectively. l-Tyrosine, d-tyrosine, m-tyrosine, N-acetyl-l-tyrosine and l-DOPA were utilized as a substrate by the enzyme. The value for Km obtained were as follows: l-tyrosine 6.90 × 10^?4 m, d-tyrosine 1.43 ×10^?3 m and l-DOPA 9.90 × 10^?4 m. The enzyme was inhibited by chelating agents of Cu²⁺ l-cysteine, l-homocysteine, thiourea and diethyl-dithiocarbamate and the inhibition was completely reversed by the addition of excess Cu²⁺ From these results it is concluded that the enzyme is a copper-containing oxidase.     

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.     

Fermentative Production of l-Glutamine by Sulfaguanidine Resistant Mutants Derived from l-Glutamate Producing Bacteria

Excellent l-glutamine producers were screened for among sulfaguanidine resistant mutants derived from the wild type l-glutamic acid-producing bacteria, Brevibacterium flavum, Brevibacterium lac to fermentum, Corynebacterium glutamicum and Microbacterium ammoniaphilum.

The best strain, No. 1~60, was a sulfaguanidine resistant mutant derived from B. flavum 2247 by mutation. Strain No. 1~60 accumulated 41.0 mg/ml of l-glutamine after 48 hr of cultivation from 10% glucose as a carbon source. This yield was the highest among those so far reported.

The addition of Mn^{2 +} (2 ppm) to the standard medium for B. flavum 2247 decreased the l- glutamine production and increased the l-glutamic acid excretion markedly. On the contrary, strain 1 —60 was not affected the Mn²⁺ (2 ppm) addition at all.

Glutamate kinase activity and the intracellular content of ATP in sulfaguanidine resistant mutant No. 1~60 were higher than those in the parent strain, B. flavum 2247.

It was confirmed that the increase in glutamate kinase and the increase in internal ATP, which were important for the l-glutamine synthesis, were very effective for the improvement of l-glutamine-producing mutants.     

Enzymatic Conversion of Dethiobiotin to Biotin in Cell-free Extracts of a Bacillus sphaericus bioB Transformant

The activity of biotin synthase, responsible for biotin synthesis from dethiobiotin, was demonstrated in a completely defined reaction mixture with cell-free extracts of a Bacillus sphaericus bioB transformant. Among the sulfur compounds tested, only S-adenosyl-l-methionine was active, while l-methionine and l-cysteine had no significant effect. Protein concentrations higher than 15mg/ml in the reaction mixture were needed to detect biotin synthase activity. When dialyzed cell-free extracts were used for the reaction, NADH, NADPH, or FAD among the well-known cofactors tested enhanced the activity, and Fe²⁺, Mn²⁺, and Ca²⁺ among the metal ions tested also had some effects.     

Studies on d-Glucose-isomerizing Enzyme from Bacillus coagulans,Strain HN-68

A functional role of Co²⁺ and Mn²⁺ in the d-glucose- and d-xylose-isomerizing reactions by d-glucose-isomerizing enzyme obtained from the cells of Bacillus coagulans, strain HN–68 was investigated. (1) The enzyme required Co²⁺ and Mn²⁺ for d-glucose- and d-xylose-isomerizing activities, respectively. (2) The enzyme which bound the metal, Co²⁺- or Mn²⁺-enzyme, was active form. Co²⁺ was bound to the enzyme in a molar ratio of 4:1. (3) The rate of activation by metal ion varied with incubation pH. (4) The binding of substrate to the enzyme was completely independent in the presence of metal ions. (5) However, it seemed unlikely that the Co²⁺ and Mn²⁺ acted as catalyzer on the reaction. (6) The binding sites for Co²⁺ and Mn²⁺ were different from each other. (7) The experimental data obtained might be successfully explained in terms of the suitable conformational changes for d-glucose and d-xylose isomerization, which were induced in the catalytic sites of the enzyme by binding Co²⁺ and Mn²⁺, respectively.     

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.     

Bacterial Racemization of α-Amino-ε-caprolactam

1. Several bacteria were isolated from soil which grew on both d- and l-aminolactam and whose cells had an activity to racemize them. They were identified as Achromobacter obae nov. sp., Achr. cycloclastes, Alcaligenes faecalis and Flavobacterium arborescens.

2. Racemization of d- and l-aminolactam was investigated using the lyophilized cells of Achr. obae nov. sp. The optimum pH value of the reaction was about 8.0. The racemizing activity was completely inhibited by 10^?4 m hydroxylamine, and the inhibition was removed by 10^?4 m pyridoxal phosphate. Five percent d- and l-aminolactam solutions were completely racemized with a concomitant slight formation of l-lysine.     

N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

Production of l-Leucine by a Mutant of Brevibacterium lactofermentum 2256

A potent l-leucine producer was screened among mutants of glutamic acid producing bacteria. This strain, No. 218, is one of 2-thiazolealanine resistant mutants derived from a methionine isoleucine double auxotroph of Brevibacterium lactofermentum 2256 by nitroso-guanidine.

Strain No. 218 produced 19 mg/ml of l-leucine after 72 hr cultivation when 8 % glucose and 4 % ammonium sulfate were supplied as a carbon and a nitrogen source, respectively, thus giving the yield of 23.1 % from glucose.

The addition of Fe²⁺ and Mn²⁺ in combination gave much more productivity than that of Fe²⁺ or Mn²⁺ alone.

Effects of amino acids, nucleic acids, vitamins, and the other nutrients on l-leucine production were investigated.

The fermentation product was isolated and purified from the culture, and identified as l-leucine.     

D-Glucosaminate Aldolase Activity of D-Glucosaminate Dehydratase from Pseudomonas fluorescens and Its Requirement for Mn2+ Ion

When D-glucosaminate dehydratase (GADH) was incubated with D-glucosaminate (GlcNA) in veronal buffer (VB; 0.01 M, pH 8.0), GlcNA was converted stoichiometrically to glyceraldehyde, pyruvate, and ammonia (aldolase reaction A). This reaction occurred in addition to the dehydratase reaction (conversion of GlcNA to 2-keto-3-deoxy-o-gluconate and ammonia: α,β-elimination reaction, B). The ratio of the activities (A:B) was about 1:4. However, in potassium phosphate buffer (KPB; 0.04 M, pH 8.0), the aldolase reaction was inhibited to 3–4% of that in VB, and also inhibited by various derivatives of glycerol, in particular, glycerol-3-phosphate (glycerol-3-P) and glyceraldehyde-3-phosphate (glyceraldehyde-3-P) in VB. The native enzyme was inhibited by incubation with 0.1 M EDTA, and the activity was restored by incubation of the EDTA-treated enzyme with (Mn²⁺ + pyridoxal 5′-phosphate (PLP)). When the EDTA-treated enzyme was incubated with (Mn²⁺ + PLP + glycerol-3-P), the activity of reaction B increased to 131% but that of reaction A decreased to 21%. These results suggested that Mn²⁺, PLP, and the phosphate group of glycerol-3-P are involved in formation of the active enzyme. In the case of the aldolase reaction, Mn²⁺ ion, which might be essential for the reaction, is chelated by the phosphate group of glycerol-3-P with resultant inhibition of the aldolase reaction.     

Polymyxin Acylase: An Enzyme Causing Intramolecular N2⇄N6 Acyl Transfer in N-Monooctanoyl-l-Lysine

Polymyxin acylase from Pseudomonas sp. M-6-3 can deacylate not only polymyxin antibiotics, but also A-fatty acyl-peptides and N-fatty acyl-amino acids. We found that this enzyme causes intramolecular N²?N⁶ acyl transfer in monooctanoyl-l-lysine; when N²-octanoyl-l-lysine is the substrate, N⁶-octanoyl- l-lysine is produced at pH 10.5, but when N⁶-octanoyl- l-lysine is the substrate, N²-octanoyl- l-lysine is produced at pH 8.0. In these reactions, the deacylation proceeded gradually at the final stage and eventually, both N²-octanoyl- l-lysine and N⁶-octanoyl- l-lysine were hydrolyzed to l-lysine and octanoic acid. Furthermore, this enzyme showed intermolecular acyltrans- ferase activity, transferring several N-octanoyl- dl-amino acids to N-octanoyl-hydroxylamine. This acyltransfer ability of polymyxin acylase offers a new method of enzymic N-acylation of compounds containing amino components.     

Purification and Properties of d-Xylose Isomerase from Alkalophilic Bacillus No. KX-6

An alkalophilic Bacillus No. KX-6 isolated from soil produced a d-xylose isomerase in alkaline media. The striking characteristic of this bacterium was its especially good growth in alkaline media. The d-xylose isomerase of this bacterium was purified by ammonium sulfate fractionation, DEAE-Sepharose ion exchange column chromatography and G-200 gel Alteration. The molecular weight and sedimentation constant were approximately 120,000 and 9.35 S, respectively. The enzyme was most active at pH 7~10 and was stable at pH 6.0 to 11.0. Enzyme activity was stimulated by cobalt ion but inhibited by Hg^{2 +}, Ag^{2 +}, and Cu^{2 +}. Substrate specificity studies showed that this enzyme was active on d-xylose, d-glucose, d-ribose, and d-arabinose. The smaller Km value and larger V_max value for d-xylose indicated that this enzyme is essentially d-xylose isomerase.     

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.