Tridec-1-ene-3,5,7,9,11-pentayne,an Ovicidal Substance from Xanthium canadense

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC. 1.4.3.5) has been purified from dry baker’s yeast to an apparent homogeneity on a polyacrylamide disc gel electrophoresis in the presence of 10 µm of phenylmethylsulfonyl fluoride throughout purification.

1) The purified enzyme, obtained as holo-flavoprotein, has a specific activity of 27µmol/mg/hr for pyridoxamine 5′-phosphate at 37°C, and a ratio of pyridoxine 5′-phosphate oxidase to pyridoxamine 5′-phosphate oxidase is approximately 0.25 at a substrate concentration of 285 µm. Km values for both substrates are 18 µm for pyridoxamine 5′-phosphate and 2.7 µm for pyridoxine 5′-phosphate, respectively.

2) The enzyme can easily oxidize pyridoxamine 5′-phosphate, but when pyridoxamine and pyridoxine 5′-phosphate are coexisted in a reaction mixture the enzyme activity is markedly suppressed much beyond the values expected from its high affinity (low Km) and low V_max for the latter substrate.

3) Optimum temperature for both substrates is approximately 45°C, and optimum pH is near 9 for pyridoxamine 5′-phosphate and 8 for pyridoxine 5′-phosphate.

4) From the data obtained, the mechanism of regulation of this enzyme in production of pyridoxal 5′-phosphate and a reasonable substrate for the enzyme in vivo are discussed.     

Preparation of Phgsphatioyll2-3Hjinositgl from Yeast Grown in Medium Containing N;Yol2-3Hjinositol

Phosphatidy[2-³]jinositol was prepared from Saccharoniycts cerevisiae (YSC-2), grown in synthetic meaiurn containing myo[2-³H]inositol. Over 44 μCi (or 81 %) of the racio-labeleo inositol was taken up by the organism, with 34 yCi incorporated into phospnatiaylinositol. Upon purification d) silicic acia-meaium pressure liquia chrcnatography (MPLC), a final yield of 24 to 2b μCi of phosphatiayl[2-³h]inositot with a specific radioactivity of 40 ± 10³ apm/nmoie wäs obtained. The purified phosphatiuyl[2-³H] inositol was founo to be a suitable substrate for phospholipase C from human platelets     

The Fermentative Formation of CDP-Choline by Haploid Cells of Yeasts and the Change of the Temperature-sensitivity of a Mutant of a Yeast,Saccharomyces rouxii

Phosphorylation of CMP and formation of CDP-choline were tested with various haploid cells of yeasts. Most of them had more or less the ability, but a mutant (Lys–M7, alpha type) of Saccharomyces rouxii was found to lack the ability. Further study revealed the change of the temperature-sensitivity of the mutant, which could not produce CDP-choline when treated at 37°C, whereas it could at 16°C. The growth of the mutant was more sensitive to temperatures than that of the wild strain. The former did not grow at 36.3°C, while the latter grew.     

Structure of DNA in Spores of Bacillus cereus

The structure of DNA extracted from dormant and germinating spores of B. cereus T was investigated using circular dichroism and other methods. No significant differences between DNAs extracted from vegetative cells and from spores of various stages could be found by analyses of CD spectra, CsCl density gradient centrifugation, melting profiles and template activity. All the DNA preparations were in B conformation and had the same density (1.695), Tm (83°C) and template activity in the reaction of DNA-dependent RNA polymerase. An abnormal DNA fraction of high density which was formerly found in B. cereus spores or a stable DNA complex with protein and/or RNA was not detected in the present extracts of spores. Preliminary X-ray analyses of intact spores indicate that the structure of DNA in spores is not so different from B form.     

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.     

Substrate Specificity of Thermostable D-Alanine-D-alanine ligase from Thermotoga maritima ATCC 43589

D-Alanine-D-alanine ligase (Ddl) and its mutants maintain the biosynthesis of peptidoglycan, and the substrate specificity of Ddls partially affects the resistance mechanism of vancomycin-resistant enterococci. Through investigation of Ddls, Ddl from Thermotoga maritima ATCC 43589 showed novel characteristics, vis. thermostability up to 90 °C and broad substrate specificity toward 15 D-amino acids, particularly D-alanine, D-cysteine, and D-serine, in that order.     

Conversion of Nicotine into Nornicotine and N-Methylmyosmine by Fungi

Several species of fungi were tested for their abilities to degrade (S)-nicotine, of which Pelliculariafilamentosa JTS-208, the pathogen of tobacco damping off disease, and Cunninghamella echinulata IFO-4444, a saprophyte, were found to be able to degrade nicotine. P. filamentosa JTS-208 accumulated nornicotine only in the nicotine medium. C. echinulata IFO-4444 accumulated nornicotine and N-methylmyosmine, the first fungal metabolite, and three unidentified compounds.     

Practical Synthesis of the Disaccharide Epitope,D-Galactopyranosyl-α-1,3-D- galactopyranose,by using 1,2;5,6-Di-O-cyclohexylidene-α-D-galactofuranose as the Glycosyl Acceptor

D-Galactosyl-α-1,3-D-galactopyranose (1) was chemically prepared in a good yield by coupling phenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside (5) or 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide (8) with 1,2:5,6-di-O-cyclohexylidene-α-D-galactofuranose (3) with subsequent de-O-benzylation and de-O-cyclohexylidenation of the resulting protected α-1,3-disaccharide.     

cis- and trans-Linalool 3,7-Oxides and Methyl Salicylate Glycosides and (Z)-3-Hexenyl β-D-Glucopyranoside as Aroma Precursors from Tea Leaves for Oolong Tea

Two new alcoholic aroma precursors, cis- and trans-linalool 3,7-oxides 6-O-β-D-apiofuranosyl-β-D-glucopyranosides (1 and 2), as well as two already known compounds, (Z)-3-hexenyl β-D-glucopyranoside (3) and methyl salicylate 6-O-β-D-xylopyranosyl-β-D-glucopyranoside (β-primeveroside: 4), and another new monoterpendiol glycoside, 8-hydroxygeranyl β-primeveroside (5) have recently been isolated as aroma precursors in tea leaves (Camellia sinensis var. sinensis cv. Maoxie) ready for oolong tea processing.     

Isolation of Possible Intermediate of Chlorogenic Acid Biosynthesis in Sweet Potato Root Tissue

Thienodolin, a new plant growth-regulating substance, was isolated from the fermentation broth of a streptomycete strain identified as Streptomyces albogriseolus.

The active principle was extracted with ethyl acetate and purified by silica gel column chromatography and preparative HPLC. The substance showed growth promoting activity with 1.2 × 10^?6–1.2 × 10^?5 M treatment to rice seedlings, and inhibitory activity with 4.0 × 10^?5 M treatment.     

Practical Preparation of D-Galactosyl-β1→4-L-rhamnose Employing the Combined Action of Phosphorylases

D-Galactosyl-β1→4-L-rhamnose (GalRha) was produced enzymatically from 1.1 M sucrose and 1.0 M L-rhamnose by the concomitant actions of four enzymes (sucrose phosphorylase, UDP-glucose-hexose 1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, and D-galactosyl-β1→4-L-rhamnose phosphorylase) in the presence of 1.0 mM UDP-glucose and 30 mM inorganic phosphate. The accumulation of GalRha in 1 liter of the reaction mixture reached 230 g (the reaction yield was 71% from L-rhamnose). Sucrose and fructose in the reaction mixture were removed by yeast treatment, but isolation of GalRha by crystallization after yeast treatment was unsuccessful. Finally, 49 g of GalRha was isolated from part of the reaction mixture with yeast treatment by gel-filtration chromatography.     

Transglucosylation Catalyzed by Sucrose Phosphorylase from Leuconostoc mesenteroides and Production of Glucosyl-xylitol

The synthesis of glucooligosaccharides from α-D-glucose-1-phosphate by transglucosylation with sucrose phosphorylase from Leuconostoc mesenteroides was studied using the purified enzyme and high performance liquid chromatography. The enzyme had a rather broad acceptor specificity and transferred glucosyl residues to various acceptors such as sugars and sugar alcohols. Especially, 5-carbon sugar alcohols (pentitols), D- and L-arabitol were acceptors equal to D-fructose, which was known as a good acceptor. The transfer product of xylitol formed by the enzyme was investigated. The structure of the product was found to be 4-O-α-D-glucopyranosyl-xylitol (G-X) by acid hydrolysis and ¹³C-nuclear magnetic resonance analysis. G-X is a probable candidate for a preventive for dental caries because it reduced the synthesis of water-insoluble glucan by Streptococcus mutans and kept a neutral pH in the cell suspension.     

ESR Studies on Lipid-protein Interaction in Gluten

The interaction of protein with lipid in wheat gluten has been studied by electron spin resonance (ESR). The gluten in the flour suspension was spin-labeled with a fatty acid spin label (N-oxyl-4,4'-dimethyloxazolidine derivative of 5-ketostearic acid) and washed out from the flour. The ESR spectra of the spin label incorporated in gluten exhibited clearly separated parallel and perpendicular hyperfine splittings. The orientation of the gluten lipid and its fluidity showed temperature dependence. Phase transition was observed at 25°C. Compared with gluten, vesicles of the lipids extracted from flour were found to be in a less oriented, highly fluid state, and with much lower activation energy for rotational viscosity, while the reconstituted gluten, which was prepared by mixing purified gluten protein and the extracted lipids, had a lipid environment similar to that of gluten. The results indicate that the lipid was immobilized in the gluten matrix by strong interaction with protein.     

Inducibility of Rat Liver Drug-Metabolizing Enzymes as an Index of Food-Screeing for Safety Assessment

D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat?K _m) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s^?1, and 100 (mmol/L)^?1 s^?1 respectively.     

Synthesis of β-D-Fructopyranosyl-(2→6)-D-glucopyranose from D-Glucose and D-Fructose by a Thermal Treatment

The synthesis is reported of β-D-fructopyranosyl-(2→6)-D-glucopyranose that had previously been isolated from a fermented plant extract as a new saccharide. A disaccharide was predominately formed from an equal amount of D-glucose and D-fructose under melting conditions at 140 °C for 60 to 90 min. This saccharide was isolated from the reaction mixture by carbon-Celite column chromatography and preparative HPLC, and was confirmed to be β-D-fructopyranosyl-(2→6)-D-glucopyranose by TOF-MS and NMR analyses.     

Plasmid-determined Transformation of cis-Abienol and Sclareol in Nocardia restricta JTS-162

The cis-abienol-transformable bacterium JTS-162 was identified as Nocardia restricta. This bacterium has three plasmids (pCA1, pCA2 and pCA3). Two types of cured strains were obtained by mitomicin C treatment or growth at the maximum temperature; one type had two plasmids (pCA2 and pCA3) and the other type had no plasmid. These two types of cured strains lost the ability to oxidize C-18 methyl and to split the A-ring of cis-abienol. These two types of cured strains were also devoid of the. ability to oxidize the C-18 methyl of sclareol. From these results, it was considered that C-18 methyl oxidation and A-ring splitting of these labdanes were determined by plasmid pCA1 in N. restricta JTS-162.     

Purification and Characterization of Chitosanase and Exo-β-D-Glucosaminidase from a Koji Mold,Aspergillus oryzae IAM2660

Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0-30%). The 135-kDa enzyme, named exo-β-D-glucosaminidase, released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.     

3-Oxoishwarane,a New Sesquiterpenic Ketone from Aristolochia debilis Sieb. et Zucc

3-Oxoishwarane (V), a new sesquiterpenic ketone, and ?^1,10-aristolenone-2 (II) were Isolated from roots of Aristolochia debilis Sieb. et Zucc     

Characterization of a PA14 domain-containing galactofuranose-specific β-d-galactofuranosidase from Streptomyces sp.

As a constituent of polysaccharides and glycoconjugates, β-d-galactofuranose (Galf) exists in several pathogenic microorganisms. Although we recently identified a β-d-galactofuranosidase (Galf-ase) gene, ORF1110, in the Streptomyces strain JHA19, very little is known about the Galf-ase gene. Here, we characterized a strain, named JHA26, in the culture supernatant of which exhibited Galf-ase activity for 4-nitrophenyl β-d-galactofuranoside (pNP-β-d-Galf) as a substrate. Draft genome sequencing of the JHA26 strain revealed a putative gene, termed ORF0643, that encodes Galf-ase containing a PA14 domain, which is thought to function in substrate recognition. The recombinant protein expressed in Escherichia coli showed the Galf-specific Galf-ase activity and also released galactose residue of the polysaccharide galactomannan prepared from Aspergillus fumigatus, suggesting that this enzyme is an exo-type Galf-ase. BLAST searches using the amino acid sequences of ORF0643 and ORF1110 Galf-ases revealed two types of Galf-ases in Actinobacteria, suggesting that Galf-specific Galf-ases may exhibit discrete substrate specificities.     

Purification and Properties of Two Chitinases from Streptomyces sp. J-13-3

Two chitinases (Chi-A and Chi-B) purified from Streptomyces sp. J-13-3 had the same molecular weights (31,000) and enzymatic properties (optimum pH and temperature of pH 6.0 and 45°C) but had significantly different isoelectric points (3.9 for Chi-A, 3.5 for Chi-B). Chi-A and -B had identical N-terminal amino acid sequences (ADXAAAWNASSVYTGGGSASYNGHN), similar amino acid compositions, and immunological cross-reactivities. A concomitant decrease of Chi-A and increase of Chi-B was observed in their productions during cultivation.