Crystalline d-Mannitol:NAD Oxidoreductase from Leuconostoc mesenteroides

The crystalline d-mannitol dehyrogenase (d-mannitol:NAD oxidoreductase, EC 1.1.1.67) catalyzed the reversible reduction of d-fructose to d-mannitol. d-Sorbitol was oxidized only at the rate of 4% of the activity for d-mannitol. The enzyme was inactive for all of four pentitols and their corresponding 2-ketopentoses. The apparent optimal pH for the reduction of d-fructose or the oxidation of d-mannitol was 5.35 or 8.6, respectively. The Michaelis constants were 0.035 m for d-fructose and 0.020 m for d-mannitol. The enzyme was also found to be specific for NAD. The Michaelis constans were 1 × 10^?5 m for NADH₂ and 2.7 × 10^?4 m for NAD.     

Studies on Kojic Acid Metabolism by Microorganisms

An enzyme, comenic aldehyde dehydrogenase, which catalyzes the oxidation of comenic aldehyde to comenic acid was partially purified from cell extract of Arthrobacter ureafaciens K-1.

The enzyme was purified 31-fold at Sephadex G-100 filtration step, 112-fold at DEAE-Sephadex A-50 fractionation step, and recovery of the activity was 73.3% and 38.5% respectively.

NADP and magnesium ion were essential for the oxidation. The enzyme shows optimum activity at pH 7.8. Enzyme activity was extremely sensitive to sulfhydryl reagents such as p-chloromercuribenzoate and monoiodoacetate. l-Cysteine or dithiothreitol protected the enzyme from p-chloromercuribenzoate inhibition. Carbonyl reagents, such as hydroxylamine and semicarbazide, inhibit the enzyme reaction by formation of addition compounds between carbonyl reagents and aldehyde group of the substrate. The enzyme was completely inactivated after heating for 5 min at 40°C The Km for 5-methoxy comenic aldehyde is 2.5×10^?6 m, and for NADP is 0.4×1O^?6 m. The reaction product, 5-methoxy comenic acid was identified by paperchromatography. The characterization of the enzyme has been carried out by using 5-methoxy comenic aldehyde as the substrate in stead of comenic aldehyde.     

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.     

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.     

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.     

A Method for Preparing 16α-Hy-droxypregnenolone-3-succinate by Streptomyces roseochromogenes

A choline dehydrogenase, which was present in the particulate fraction of the cell-free extract of Pseudomonas aeruginosa A-16, oxidized choline to betaine aldehyde without any dissociable coenzymes, while the enzyme, which was treated with Triton X-100, oxidized choline only with a supplement of phenazine methosulfate. The difference spectrum showed the presence of cytochrome-like components in the particulate. Km values for choline and phenazine methosulfate were 1.7 × 10^?3 m and 1.4 × 10^?4 m, respectively. The dehydrogenase was inhibited by SH-reagents such as p-chloromercuribenzoate and iodoacetic acid. Of a variety of substrates tested, only choline caused the enzymatic reduction of phenazine methosulfate. The estimation of choline was tried using the enzyme.     

Kinetic Studies on Crystalline Yeast Phosphoglyceric Acid Mutase

Effects of the substrate and the coenzyme on the crystalline yeast phosphoglyceric acid mutase activity have been investigated. Lineweaver-Burk plots at different concentrations of the substrate (d-3-phosphoglyceric acid: 3×10^?7 to 8×10^?3m) and the coenzyme (d-2, 3-diphosphoglyceric acid: 8×10^?7 to 10^?5m) change in such a way to indicate the involvement of an enzyme-substrate-coenzyme ternary complex as an active intermediate in the enzymic reaction process. It is concluded that the reaction catalyzed by the yeast enzyme follows the sequential pathway and that a phosphorylated enzyme does not participate as an obligatory intermediate in the reaction mechanism, if it occurs. Kinetic studies indicate Km values of 6×10^?4m for d-3-phosphoglyceric acid and 8×10^?7m for d-2, 3-diphosphoglyceric acid. The substrate is a competitive inhibitor of the coenzyme with a Ksi (inhibition constant) of 3.2×10^?3m. The coenzyme inhibition is not observed at concentration tested. A kinetic treatment to determine the mechanism of the enzyme reaction from the experimental data which are obtaind in the range of inhibitory substrate concentrations is presented.     

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.     

Studies on d-Glucose-isomerizing Activity of d-Xylose-grown Cells from Bacillus coagulans,Strain HN-68

d-Glucose-isomerizing enzyme has been extracted in high yield from d-xylose-grown cells of Bacillus coagulans, strain HN-68, by treating with lysozyme, and purified approximately 60-fold by manganese sulfate treatment, fractionation with ammonium sulfate and chromatography on DEAE-Sephadex column. The purified d-glucose-isomerizing enzyme was homogeneous in polyacrylamide gel electrophoresis and ultracentrifugation and was free from d-glucose-6-phosphate isomerase. Optimum pH and temperature for activity were found to be pH 7.0 and 75°C, respectively. The enzyme required specifically Co⁺⁺ with suitable concentration for maximal activity being 10^?3 m. In the presence of Co⁺⁺, enzyme activity was inhibited strongly by Cu⁺⁺, Zn⁺⁺, Ni⁺⁺, Mn⁺⁺ or Ca⁺⁺. At reaction equilibrium, the ratio of d-fructose to d-glucose was approximately 1.0. The enzyme catalyzed the isomerization of d-glucose, d-xylose and d-ribose. Apparent Michaelis constants for d-glucose and d-xylose were 9×10^?2 m and 7.7×10^?2 m, respectively.     

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.     

Kinetic Properties of Fungal Polyamine Oxidases and Their Application to Differential Determination of Spermine and Spermidine

Polyamine oxidase from Penicillium chrysogenum oxidized spermine rapidly and spermidine slightly at pH 7.5. The apparent Km values for spermine and spermidine were calculated to be 2.25 × 10^?5 m and 9.54 × 10^?6 m, respectively. The relative maximum velocities for spermine and spermidine were 3.37 × 10^?3 m (H₂O₂) per min per mg of protein and 2.08 × 10^?4 m (H₂O₂) per min per mg of protein, respectively. Spermine oxidation of the enzyme was competitively inhibited by spermidine and putrescine. The apparent Ki values by spermidine and putrescine were calculated to be 3.00 × 10^?5 m and 1.80 × 10^?8 m, respectively. On the other hand, polyamine oxidase from Aspergillus terreus rapidly oxidized both spermidine and spermine at pH 6.5. The apparent Km values for spermidine and spermine were 1.20 × 10^?8 m and 5.37 × 10^?7 m, respectively. The relative maximum velocities for spermidine and spermine were 1.55 × 10^?2 m (H₂O₂) per min per mg of protein and 6.20 × 10^?3 m (H₂O₂) per min per mg of protein, respectively.

Differential determination of spermine and spermidine was carried out using the two enzymes. The initial rate was assayed with Penicillium enzyme and the end point was measured afte addition of Aspergillus enzyme. Small amounts of polyamines (25 to 200 nmol of spermine and 25 to 250 nmol of spermidine) were assayed by solving two simultaneous equations obtained from the rate assay method and the end point assay method. The calculated values were in close agreement with those obtained by an amino-acid analyzer.     

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.     

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⁺.     

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.     

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.     

Purification and Properties of Formyltetrahydrofolate Synthetase from Spinach

Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was found in fresh spinach leaves and purified about 60-fold by treatments of ammonium sulfate, protamine sulfate, dialysis, and DEAE-cellulose column chromatography. Some properties of the enzyme were investigated. Optimum pH was found to be 7.5, and optimum temperature was observed to be at 37°C. In the enzyme reaction, FAH₄ and formate were required specifically as the substrates, and Mg⁺⁺ and ATP were essential components. The Michaelis constants for dl-FAH₄, formate, ATP and magnesium chloride were 1.7×10^?3 m, 1.7×10^?2 m, 4.1×10^?4 m and 3.3×10^?3 m, respectively. The primary product formed in the reaction catalyzed by the enzyme was suggested as N¹⁰-formyl-FAH₄ spectrophotometrically. It was observed that the enzyme also catalyzed the reverse reaction. The possible role of the enzyme in plants was discussed.     

Radiation Chemistry of Foods

An intermediate radical, ?H₂OH, was produced in aqueous methanol solution containing nitrous oxide by γ-irradiation. Yields of ethylene glycol and formaldehyde, the major and the minor product from ?H₂OH, respectively, changed on the addition of some solutes. Cysteine lowered the both product yields to zero even at a low concentration of 5 × 10^?5m. Oxygen of low concentrations (2.5~7.5 × 10^?5 m) changed effectively the major product from ethylene glycol to formaldehyde. k _(CySH+?H2OH)/k_(O2+?H2OH) was calculated as 0.5.

Ascorbic acid (5 × 10^?5 m) lowered ethylene glycol yield to 48%, cystine (10^?3m) to 15%, methionine (10^?3m) to 31%, histidine (10^?3m) to 42%, tryptophan (10^?3m) 46%, tyrosine (10^?3m) to 77%, phenylalanine (10^?3m) to 73%, hypoxanthine (10^?3m) to 37%, adenine (10^?3m) to 52%, uracil (10^?3m) to 20%, thymine (10^?3m) to 10%, cytosine (10^?3 m) to 49%, rutin (10^?3m) to 23%, pyrogallol (10^?3m) to 41%, and gallic acid (10^?3m) to 78% of the control. These results suggest that the reactions of the secondary radicals such as ?H₂OH perform an important role in material change of foods irradiated with γ rays.     

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 Properties of α-d-Galactosidase Produced by Corticium rolfsii

An α-d-galactosidase was purified from the culture filtrate of Corticium rolfsii IFO 6146 by a combination of QAE-Sephadex A-50 and SE-Sephadex C-50 chromatography. The purified enzyme was demonstrated to be free of other possibly interfering glycosidases and glycanases. The maximum activity of the enzyme towards p-nitrophenyl α-d-galactopyrano-side was found to be at pH 2.5 to 4.5, and the enzyme was fairly active at pH 1.1 to 2.0. The enzyme was stable over a pH range 4.0 to 7.0 at 5°C for 72 hr and relatively unstable at pH 1.1 to 2.0 as compared with endo-polygalacturonase, α-l-arabinofuranosidase and β-d-galactosidase produced by C. rolfsii. The enzymic activity was completely inhibited by Hg²⁺ and Ag⁺ ions, respectively. Km values were determined to be 0.16 × 10^?3 m for p-nitrophenyl α-d-galactopyranoside and 0.26 × 10^?3m for o-nitrophenyl α-d-galactopyranoside. The values of V_max were also determined to be 26.6 μmoles and 28.6 μmoles per min per mg for p- and o-nitrophenyl α-d-galactopyranoside, respectively.     

Fukiic Acid Isolated from the Hydrolysate of a Polyphenol in Petasites japonicus

d-Glucose-isomerizing enzyme was purified in a crystalline form with a good yield from the cells of Bacillus coagulans, strain HN-68, and some phsicochemical properties were investigated.

The purified enzyme was homogeneous on both ultracentrifugal and disc-electrophoretical analyses. The molecular weight of the enzyme was determined to be 175,000 and 160,000 from the sedimentation-viscosity method and the gel filtration method, respectively.

The sedimentation coefficient , partial specific volume, at 280 mμ, and the nitrogen content of the enzyme were determined to be 10.2×10^?13 sec, 0.705 cm³g^?1, 10.6 and 16.2%, respectively. The integral numbers of amino acid residues per molecule calculated on the basis of 160,000 were as follows; Lys₁₂₀, His₄₉, Arg₆₁, Asp₁₈₂, Thr₈₇, Ser₇₀, Glu₁₃₆, Pro₄₄, Gly₁₀₆, Ala₁₄₀, Half-Cys₀, Val₅₃, Met₂₇, Ileu₅₁, Leu₁₃₄, Tyr₅₈, Phe₉₆, Try₁₃, and amide-ammonia₈₀.

Purified enzyme preparation obtained from Bacillus coagulans, strain HN-68 requires Co²⁺ for d-glucose- and d-ribose-isomerizing activities and Mn²⁺ for d-xylose-isomerizing activity. The values of Km for d-glucose, d-xylose and d-ribose were 9×10^?2, 1.1×10^?3, 7.7×1O^?m and of the relative V_max were 0.52, 1.1 and 0.25 mg/min at 40°C, respectively. d-Glucose-isomerizing activity was inhibited by d-xylose and d-ribose. However, there was not a difference among three activities of the enzyme with respect to following properties: Activation energy was 14,600 cal per mol. The enzyme was inhibited in a competitive manner by tris(hydroxymethyl)aminomethane, d-xylitol, d-sorbitol and d-mannitol, and the Ki values for these inhibitor were 3×10^?4, 2.5×10^?3, 2.9×10^?2 and 7×10^?2m, respectively. The ratio of three activities did not change by heat- and pH-treatments. Mn²⁺, Co²⁺ and Ni²⁺ protected strongly the enzyme from heat denaturation. The enzyme can isomerize d-glucose, d-xylose and d-ribose to their corresponding ketose, but the kinetic constants and induction studies indicated that ^d-xylose is the natural substrate for the enzyme.