Substrate Specificity of 2-Ketogluc nate Reductase from Gluconobacti liquefaciens

The significant betaine aldehyde dehydrogenase activity was found in the cells of Pseudomonas aeruginosa A-16. The enzyme was inducibly formed and accumulated in the presence of choline, acetylcholine or betaine in the medium. The enzyme was purified approximately 620-fold with an overall recovery of 2.6% and proved to be homogeneous by ultracentrifugation. The molecular weight of the enzyme was determined as approximately 145,000 by gel filtration method. The enzyme had an isoelectric point around pH 5.1. The enzyme was quite specific for its substrate, betaine aldehyde. Both NADP and NAD functioned as coenzyme. The estimated values of Km at pH 7.4 and 25°C were 3.8 × 10^?4 m for betaine aldehyde, 8.9 × 10^?5 m for NADP and 2.2 × 10^?4 m for NAD.     

Studies on Isomerization of Sugars by Bacteria

An enzyme, which catalyzes the isomerization of d-glucose to d-fructose, has been found in a newly isolated bacterium which tentatively identified as Pacacolobacterum aerogenoides. The enzyme converts not only d-glucose but also d-mannose to d-fructose, and NAD and Mg⁺⁺ are required as cofactor for this isomerization. The properties of this enzyme were summarized as follows: (1) As a cofactor for the isomerization by this enzyme, NAD was absolutely necessary, whereas NADP, FMN and FAD were not. (2) The optimum pH was found to be at 7.5 and optinum temperature was at about 40°C. (3) The enzyme activity was markedly reduced by EDTA treatment and the reduced activity by EDTA was restored by the addition of Mg⁺⁺, Mn⁺⁺ or Co⁺⁺. (4) The enzyme activity was strongly inhibited by monoiodoacetate, p-chloromercuribenzoate, and Cu⁺⁺, however, the activity was recovered by adding cysteine or glutathione.     

Purification and Properties of 5-Ketogluconate Reductase from Gluconobacter liquefaciens

5-Ketogluconate reductase (5KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was partially purified about 120-fold by a procedure employing ammonium sulfate fractionation, and DEAE-cellulose-, hydroxylapatite- and DEAE-Sephadex A-50-column chromatographies. NADP was specifically required for the oxidative reaction of gluconic acid. The optimum pH for the oxidation of gluconic acid (GA) to 5-ketogluconic acid (5KGA) by the enzyme was 10.0 and for the reduction of 5KGA was 7.5. The optimum temperature of the enzyme was 50°C for both reactions of oxidation and reduction. The enzyme was considerably unstable and lost all of its activity within 3 days. The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ion, but remarkably stimulated by EDTA (1 × 10^?3m). Apparent Km values were 1.8 × 10^?2m for GA, 0.9 × 10^?3m for 5KGA, 1.6 × 10^?5 m for NADP, and 1.1 × 10^?5 m for NADPH₂.     

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.     

Amino Acid Metabolism in Microorganisms

3-Methylthiopropylamine (MTPA) formation from l-methionine in Streptomyces sp. K37 was studied in detail. The reaction was confirmed to be catalyzed by the decarboxylase of l-methionine. The properties of the enzyme were studied in detail using acetone dried cells or cell-free extract. The enzyme was specific for l-methionine. Pyridoxal phosphate stimulated the reaction and protected the enzyme against heat inactivation. The optimum pH for the reaction was 6.0~8.0 and the optimum temperature was about 40°C. Carbonyl reagents (10^?2~10^?3 m) inhibited the reaction completely, and silver nitrate and mercuric chloride (10^?3~10^?4 m) markedly inhibited the reaction. Km value for the reaction was 1.21 × 10^?5 m. l-Methionine assay using the decarboxylase was attempted and was found to be applicable to practical use.     

Phenol Ethers and Terpenes of Six Heterotropa Species Distributed in the Ryukyu Islands and Formosa

A new intracellular peptidase, which we call “d-peptidase S,” was purified from Nocardia orientalis IFO 12806 (ISP 5040). The purified enzyme was homogeneous on disc gel electrophoresis. The molecular weight and the isoelectric point were estimated to be 52,000 and 4.9, respectively. The optimum pH for the hydrolysis of d-leucyl-d-leucine was 8.0 to 8.1, and the optimum temperature was 36°C. The purified enzyme usually hydrolyzed the peptide bonds preceding the hydrophobic D-amino acids of dipeptides. Tri- and tetra-peptides extending to the amino terminus of such peptides were also hydrolyzed. Therefore, the enzyme is a carboxylpeptidase-like peptidase specific to d-amino acid peptides. The Km values for d-leucyl-d-leucine and l-leucyl-d-leucine were 0.21 × 10^-3 and 0.44 × 10^-3 m respectively. The activity was inhibited by several sulfhydryl reagents and two chelators, 8-hydroxyquinoline and o-phenanthroline.     

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.     

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.     

Metabolism of Aromatic Amino Acid in Microorganisms

Phenylalanine ammonia-lyase, which catalyzes the conversion of l-phenylalanine to trans-cinnamic acid and ammonia, has been partially purified from the cells of Rhodotorula. Some of the properties of this phenylalanine ammoyia-lyase were investigated. The enzyme was stable in phosphate buffer of pH over the range of 6.0 to 7.0 On heating, the enzyme was stable up to 50°C, but above 60°C, it was destroyed. The enzyme activity was strongly inhibited by p-chloromercuribenzoate at 10^?5 m and almost recovered by the addition of glutathione or mercaptoethanol at 10^?3 m. The present enzyme preparation of Rhodotorula also catalyzed the deamination of l-tyrosine to trans-p-coumaric acid. trans-p-Coumaric acid was isolated from the reaction mixture and identified by its absorption spectra. The rates of deamination showed optima at pH 9.0 and 9.5 for l-phenylalanine and l-tyrosine, respectively.     

Purification and Properties of 2-Ketogluconate Reductase from Gluconobacter liquefaciens.

2-Ketogluconate reductase (2KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was purified about 1000-fold by a procedure involving ammonium sulfate fractionation and column chromatographies using DEAE-cellulose, hydroxylapatite, and Sephadex gel The purified enzyme gave a single band on polyacrymamide gel electrophoresis. NADP was specifically required for the oxidation reaction of gluconic acid. Using gel filtration a molecular weight of about 110,000 was estimated for the enzyme. The pH optimum for the oxidation of gluconic acid (GA) to 2-ketogluconic acid (2KGA) by the enzyme was 10.5 and for the reduction of 2KGA was 6.5. The optimum temperature of the enzyme was 50 C for both reactions of oxidation and reduction. The enzyme was stable at pH between 5.0 and 11.0 and at temperature under 50°C, The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ions, but remarkably stimulated by manganese ions (1×10^?3 m). Km value of the enzyme for GA was 1.3×10^?2 m and for 2KGA was 6.6×10^?3 _m. Km values for NADP and NADPH₂ were 1.25×10^?5 and 1.52×10^?5 m respectively.     

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 Properties of NADP-Dependent Maltose Dehydrogenase Produced by Alkalophilic Corynebacterium sp. No. 93–1

NADP-dependent maltose dehydrogenase (NADP-MalDH) was completely purified from the cell free extract of alkalophilic Corynebacterium sp. No. 93–1. The molecular weight of the enzyme was estimated as 45,000~48,000. The enzyme did not have a subunit structure. The isoelectric point of the enzyme was estimated as pH 4.48. The pH optimum of the enzyme activity was pH 10.2, and it was stable at pH 6 to 8. The temperature optimum was 40°C, and the enzyme was slightly protected from heat inactivation by 1 mm NADP. The enzyme oxidized d-xylose, maltose and maltotriose, and the Km values for these substrates were 150mm, 250 mm and 270 mm, respectively. Maltotetraose and maltopentaose were suitable substrates. The Km value for NADP was 1.5 mm with 100mm maltose as substrate. The primary product of this reaction from maltose was estimated as maltono-δ-lactone, and it was hydrolyzed non-enzymatically to maltobionic acid. The enzyme was inhibited completely by PCMB, Ag⁺ and Hg²⁺.     

Preparation of Zeaxanthin from Berries of Boxthorn,Lycium chinensis

Purification and properties of a new alkaline protease of rat skeletal muscle have been reported. The purification procedure of the enzyme is as follows: skeletal muscle tissue was extracted successively with Hasselbach-Schneider solution, 5 m urea solution and 2% sodium deoxycholate solution. After then, the enzyme was extracted from the residue with 1.1 m potassium iodide solution. This enzyme solution was treated with n-butanol, and dialyzed against water. The enzyme precipitated during dialysis was collected and dissolved in 1.1 m potassium iodide solution. The enzyme solution was fractionated with acetone, and chromatographed on Sephadex G-200. The final preparation showed over 20,000 times of purity.

The optimum pH range of the enzyme activity is 9.5~10.5, and the maximum reaction rate occurs at 47~57°C. The enzyme is stable below 47°C at pH 7.3. At 37°C, the enzyme is stable during 30 min at least, in the pH range of 5.5~10.0. Below pH 5.0, it is relatively labile. Hg²⁺, Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, and Zn²⁺ scarcely affect the enzyme activity at the concentration of 1 mm. Ethylenediaminetetraacetate shows little effect on the activity at the concentration of 10 mm, and iodoacetamide, 2,4-dinitrophenol, p-chloromercuribenzoate show the similar effect at the concentration of 1 mm. Diisopropyl-flurophosphate inhibits the enzyme activity. From the results obtained, this enzyme is presumed to be responsible for the activity of autolytic breakdown of rat skeletal muscle proteins in the alkaline pH range.     

Glucose-6-phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase of Candida tropicalis Partial Purification and Some Properties

Glucose-6-phosphate (G6P) dehydrogenase and 6-phosphogluconate (6PG) dehydrogenase were partially purified about 53-fold and 47-fold, respectively, from the cell-free extract of glucose-grown Candida tropicalis by means of ammonium sulfate fractionation and DEAE-cellulose column chromatography. AMP acted as the competitive inhibitor against G6P and NADP in the G6P dehydrogenase reaction. This inhibition was remarkable at low concentrations of NADP, increasing the sigmoidicity of the NADP-saturation curve. On the other hand, 6PG dehydrogenase was not affected by AMP. Fructose-1,6-bisphosphate (FDP) and phosphoenolpyruvate (PEP) inhibited slightly G6P dehydrogenase. 6PG dehydrogenase was also weakly inhibited by FDP. Apparent Km values of G6P dehydrogenase were calculated as 1.8 × 10^?4 m for G6P and 3.1 × 10^?5 m for NADP. Those of 6PG dehydrogenase were 9.4 × 10^?5 m for 6PG and 2.8 × 10^?5 m for NADP.     

Purification and Properties of Acid Carboxypeptidase III from Aspergillus oryzae

Acid carboxypeptidase III from Aspergillus oryzae was purified from the rivanol non-precipitated fraction. The optimum activity of the enzyme occurred at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme was inhibited by diisopropylphosphorofluoridate and SH reagents such as p-chloromercuribenzoate and monoiodoacetate, but not by such metal chelating agents as ethylenediaminetetraacetate, αα′-dipyridyl and o-phenanthroline. The molecular weight of the enzyme was estimated to be about 61,000. The enzyme hydrolyzed the peptides that possess masked or bulky N-terminal.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10^?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10^?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-¹⁴C revealed that l-glutamic acid-1-¹⁴C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme.     

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.     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

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.     

Enzymatic Properties of β-Xylosidase from Malbranchea pulchella var. sulfurea No. 48

Some enzymatic properties of Malbranchea β-xylosidase were investigated. The β- xylosidase activity was inhibited by Hg²⁺, Zn²⁺, Cu²⁺, N-bromosuccinimide, p-chloromercuribenzoate and sodium laurylsulfate, while this activity was activated by Ca²⁺. The enzyme released xylose as the end product even from 10% xylobiose solution without forming any xylooligosaccharides. The enzyme well acted on aryl-β-d-xylosides, but showed no activity on alkyl-β-d-xylosides, and it was practically free from glucosidase activity. The Km and V_max values of this enzyme for xylobiose were calculated to be 2.86 × 10^?8 m and 34.5 μmoles/mg/min, respectively, and these values determined for phenyl-β-d-xyloside were 3.01 × 10^?8 m and 16.2 μmoles/mg/min, respectively.