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.     

Isolation and Identification of ( – ) - Quinic Acid as an Unidentified Major Tea-component

A new enzyme, N-acetyl- d-hexosamine dehydrogenase (N-acety 1-α-d-hexosamine: NAD⁺ 1-oxidoreductase), was purified to homogeneity on polyacrylamide gel electrophoresis from a strain of Pseudomonas sp. about 900-fold with a yield of 12 %. The molecular weight of the enzyme was about 124,000 on gel filtration and 30,000 on SD S-polyacrylamide gel electrophoresis, respectively. Its isoelectric point was 4.7. The optimum pH was about 10.0. The enzyme was most stable between pH 8.0 and pH 10.5. The highest enzyme activity was observed with N-acetyl-d-glucosamine (Km = 5.3mm) and N-acetyl-d-galactosamine (Km = 0.8mm) as the sugar substrate. But it was not so active on N-acetyl-d-mannosamine. NAD⁺ was used specifically as the hydrogen acceptor. The anomeric requirement of the enzyme for N-acetyl-d-glucosamine was the α-pyranose form, and the reaction product was N-acetyl-d-glucosaminic acid. The enzyme activity was inhibited by Hg and SDS, but many divalent cations, metal-chelating reagents, and sulfhydryl reagents had no effect.     

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.     

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.     

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.     

Studies on Glucose Isomerase of Bacteria

A bacterial strain, HN-56, having an activity of d-glucose isomerization was isolated from soil, and was identified to be similar to Aerobacter aerogenes (Kruse) Beijerink. d-Glucose-isomerizing activity was induced when HN-56 was precultured in the media containing d-xylose, d-mannose, lactate, especially d-mannitol. Paper chromatography showed that the ketose formed in reaction system containing d-glucose was d-fructose alone. The optimum pH for the reaction was 6.5~7.0. Sulfhydryl reagents inhibit the reaction, but metal inhibitors affect little if any. With the washed living cells as enzyme source, only arsenate could accumulate d-fructose. In addition, the cells grown with d-mannitol and d-mannose showed no activity of d-xylose isomerase.     

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.     

Isopullulanase from Arthrobacter globiformis T6

The physico-chemical properties of the purified glucose isomerases [d-xylose ketol isomerase, EC 5.3.1.5] of Streptomyces olivochromogenes and Bacillus stearothennophilus were examined. The molecular size and shape of both enzymes were similar. The molecular weights, sedimentation coefficients, partial specific volumes, diffusion constants and Stokes’ radii of the Streptomyces and Bacillus enzymes were determined to be 120,000 and 130,000, 7.55 S and 9.35 S, 0.725 and 0.736 ml/g, 5.87 × 10^-7 and 6.82 × 10^-7 cm²/sec, and 51 and 53 Å, respectively. The Streptomyces glucose isomerase was found to consist of two subunits, each having a molecular weight of 56,000. Large differences were found in the amino acid compositions of these two enzymes, especially in their serine, proline, tyrosine, lysine and arginine contents. The enzymatic properties of both these purified glucose isomerases were also examined, and it was seen that they both displayed activity on d-xylose, d-xylulose, d-glucose, d-fructose, d-arabinose and d-ribose. The smaller Km values and the larger molecular activities for d-xylose and d-xyluIose indicated that both enzymes are essentially d-xylose isomerases. The optimum temperature was 80°C for both enzymes. The optimum pH was 8 to 10 for the Streptomyces enzymes and 7.5 to 8.0 for the Bacillus enzyme. The Bacillus enzyme was more thermostable than the Streptomyces enzyme, but required cobalt ions in addition to magnesium ions for the full expression of its activity.     

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 Characterization of β-D-Glucosidase (β-D-Fucosidase) from Bifidobacterium breve clb Acclimated to Cellobiose

The β-d-glucosidase (EC. 3.2.1.21) activity of Bifidobacterium breve 203 was increased by acclimation with cellobiose, and the enzyme was purified to homogeneity from cell-free extracts of an acclimatized strain of B. breve clb, by ammonium sulfate fractionation and column chromatographies of anion-exchange, gel filtration, Gigapaite, and hydrophobic interaction. This enzyme had not only β- d-glucosidase activity but also β- d-fucosidase activity, which is specific to Bifidobacteria in intestinal flora. The molecular weight of the purified enzyme was estimated to be 47,000–48,000 and the enzyme was assumed to be a monomeric protein. The optimum pH and temperature of the enzyme were around 5.5 and 45°C, respectively. The enzyme was stable up to 40°C and between pH 5 and 8. The isoelectric point of the enzyme was 4.3 and the K_m values for p-nitrophenyl-β-d-glucoside and p-nitrophenyl-β-d-fucoside were 1.3mm and 0.7 mm, respectively. This enzyme had also transferase activity for the β-d-fucosyl group but not for the β-d-glucosyl group. The N-terminal amino acid sequence of this enzyme was similar to those of β-d-glucosidase from other bacteria, actinomycetes, and plants.     

Biochemical characteristics of maltose phosphorylase MalE from Bacillus sp. AHU2001 and chemoenzymatic synthesis of oligosaccharides by the enzyme

ABSTRACT

Maltose phosphorylase (MP), a glycoside hydrolase family 65 enzyme, reversibly phosphorolyzes maltose. In this study, we characterized Bacillus sp. AHU2001 MP (MalE) that was produced in Escherichia coli. The enzyme exhibited phosphorolytic activity to maltose, but not to other α-linked glucobioses and maltotriose. The optimum pH and temperature of MalE for maltose-phosphorolysis were 8.1 and 45°C, respectively. MalE was stable at a pH range of 4.5–10.4 and at ≤40°C. The phosphorolysis of maltose by MalE obeyed the sequential Bi–Bi mechanism. In reverse phosphorolysis, MalE utilized d-glucose, 1,5-anhydro-d-glucitol, methyl α-d-glucoside, 2-deoxy-d-glucose, d-mannose, d-glucosamine, N-acetyl-d-glucosamine, kojibiose, 3-deoxy-d-glucose, d-allose, 6-deoxy-d-glucose, d-xylose, d-lyxose, l-fucose, and l-sorbose as acceptors. The k_cat(app)/K_m(app) value for d-glucosamine and 6-deoxy-d-glucose was comparable to that for d-glucose, and that for other acceptors was 0.23–12% of that for d-glucose. MalE synthesized α-(1→3)-glucosides through reverse phosphorolysis with 2-deoxy-d-glucose and l-sorbose, and synthesized α-(1→4)-glucosides in the reaction with other tested acceptors.     

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 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.     

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.     

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⁺.     

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.     

Studies on Glucose Isomerase of Bacteria

A bacterial strain, HN-500, having an activity of d-glucose isomerization was newly isolated from soil, and was identified to be similar to Escherichia intermedia (Werkman and Gillen) Vaughn and Levine. The strain, grown on wide varieties of carbon sources, shows definitely d-glucose isomerizing activity in the presence of arsenate. d-Fructose formed in reaction mixture was identified by paper chromatography and was isolated in crystalline form from calcium-fructose complex. In order to increase the production of d-glucose isomerase, d-glucose and ammonium nitrogen were effective carbon and nitrogen sources, respectively, but none of the metallic ions tested were effective, furthermore manganese, ferrous and ferric ions present mOre than 10^-5m in growth medium fully repressed the enzyme formation. The cells grown on carbon sources other than d-xylose showed no activity of d-xylose isomerase.     

Studies on an Enzyme Capable of Splitting β-d-1,6-Glucosidic Linkage

In the previous paper, it was reported that the higher molecular weight luteose which consisted of β-1,6-linked d-glucose units was made by Pen. aculeatutn var. apiculatum. The isolation of the microorganism which is able to produce β-d-1,6-glucanase and some properties of this glucanase were described in this paper. The optimum reaction temperature for this enzyme was at about 40°C, and the optimum pH was at about 6.7. At the maximum degree of the hydrolysis, only d-glucose and gentiobiose were detected, and at this point, the hydrolysis degree of higher molecular weight luteose was about 55% as d-glucose. The formation of β-d-1,6-glucanase was induced by the addition of higher molecular weight luteose. From the property of this enzyme, the authors proposed the name “lutease” for this enzyme.     

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.     

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.