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.     

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.     

Specific Surface Area and Specific Pore Volume Distribution of Tobacco

The specific surface area and the specific pore volume distribution of Japanese tobaccos were measured by means of the low temperature gas adsorption technique utilizing the B.E.T. and the Inkley methods. The specific surface area and the specific pore volume of the micropores less than 300 Å in diameter varied from 6,000 to 17,000 cm²/g and from 0.0012 to 0.0036 cm³/g, respectively, with types of curing in the ascending order of the sun cured, the flue cured and the air cured tobaccos. The both specific values were increased by extracting the tobaccos with water greatly in the case of the flue cured, while slightly in the case of the air cured tobaccos, suggesting that the effect of the curings on the specific values were due to differences in the content of low molecular components. Effectiveness of puffing was also shown. The specific surface area was linearly correlated with the specific volume of the micropores less than 300 Å in diameter, the constant term showing that contribution of the larger pores more than 300 Å in diameter to the specific surface area of tobacco was insignificant.     

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.     

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.     

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.     

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.     

Involvement of Branched Units at Position 6 in the Reactivity of a Unique Variety of β-D-Glucan from Aureobasidium pullulans to Antibodies in Human Sera

We recently determined the structure of a unique type of 1,3-β-D-glucan obtained from Aureobasidium pullulans (AP-FBG) and found that it reacted with the antibodies in human sera. The reactivity of AP-FBG to the antibodies was stronger than that of 1,3-β-D-glucan obtained Grifola frondosa (GRN) but weaker than that of 1,3-β-D-glucan from Candida albicans (CSBG). Here, we demonstrated that AP-FBG reacted to IgG antibodies, especially those of the subclasses IgG2, IgG1, and IgG3, in human sera. Moreover, the results of competitive enzyme-linked immunosorbent assays (ELISAs) using various glucan competitors showed that these IgGs recognized branched chains at position 6. This is the first study to report that the branched chains at position 6 of β-D-glucan strongly contribute to its recognition by antibodies in human sera. This high reactivity of AP-FBG to human IgG could be advantageous for the use of this glucan in medicine, e.g., as an immunostimulatory agent.     

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.     

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.     

Functions,structures, and applications of cellobiose 2-epimerase and glycoside hydrolase family 130 mannoside phosphorylases

Carbohydrate isomerases/epimerases are essential in carbohydrate metabolism, and have great potential in industrial carbohydrate conversion. Cellobiose 2-epimerase (CE) reversibly epimerizes the reducing end d-glucose residue of β-(1→4)-linked disaccharides to d-mannose residue. CE shares catalytic machinery with monosaccharide isomerases and epimerases having an (α/α)₆-barrel catalytic domain. Two histidine residues act as general acid and base catalysts in the proton abstraction and addition mechanism. β-Mannoside hydrolase and 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in β-mannan metabolism, where it epimerizes β-d-mannopyranosyl-(1→4)-d-mannose to β-d-mannopyranosyl-(1→4)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other β-mannoside phosphorylases and hydrolases. Structural analysis of GH130 enzymes revealed an unusual catalytic mechanism involving a proton relay and the molecular basis for substrate and reaction specificities. Epilactose, efficiently produced from lactose using CE, has superior physiological functions as a prebiotic oligosaccharide.     

Interaction of Amino Acids with Transition Metal Ions in Solution (I) Solution Structure of l-Lysine with Co(II) and Cu(II) Ions as Studied by Nuclear Magnetic Resonance Spectroscopy

Interaction of l-lysine with Co(II) and Cu(II) ions has been studied using ¹H- and ¹³C-NMR and solution absorption spectrometry. In l-lysine-Co²⁺ solution in D₂O (100: 1 in concentration), coordination interaction of the α-amino and carboxyl groups with Co²⁺ occurs from the neutral to alkaline pD region, whereas no interaction of the ?-amino group was observed throughout the whole pD region. On the other hand, in l-lysine-Cu²⁺ solution, the ?-amino group also takes part in complexation in the higher pD region (pD≧10). Structural changes in complexation of l-lysine with the divalent cations along with pD variations in aqueous solution are discussed. Dissociation constants of the three functional groups were obtained by ¹H-NMR chemical shifts; pKa₁ = 2.2, pKa₂ = 9.5 and pKa₃ = 11.2.     

Identity of Brusatol and Yatansin,An Antidysentric Agent

In order to develop a fermentation process for lactase (β-d-galactosidase) production, we selected an excellent lactase producer, Kluyveromyces lactis. KY5466, from our yeast culture collection. Some of its mutant derivatives which formed a blue pigment from 5-bromo-4-chloro-3-indolyl-β-d-galactoside in the presence of glucose and those which assimilated phenyl-β-d-galactoside as a carbon source produced 2 to 2.7 times as much lactase as the parent strain. In the late stage of cultivation, the lactase activity decreased to zero for all strains tested soon after the complete consumption of sugar. This phenomenon was found to be correlated with a decrease in the efficiency of protein extraction from the cells. The maximal amount of lactase produced reached 155 units per ml at 48 hr in a 5-1 jar fermentor culture with sugar feeding.     

Preparation of Enzymes Required for Enzymatic Quantification of 5-Keto-D-gluconate and 2-Keto-D-gluconate

For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA.     

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.     

Regioselectivity in β-Galactosidase-catalyzed Transglycosylation for the Enzymatic Assembly of D-Galactosyl-D-mannose

The regioselectivity of β-galactosidase derived from Bacillus circulans ATCC 31382 (β-1,3-galactosidase) in transgalactosylation reactions using D-mannose as an acceptor was investigated. This D-mannose associated regioselectivity was found to be different from reactions using either GlcNAc or GalNAc as acceptors, not only for β-1,3-galactosidase but also for β-galactosidases of different origins. The relative hydrolysis rate of Galβ-pNP and D-galactosyl-D-mannoses, of various linkages, was also measured in the presence of β-1,3-galactosidase and was found to correlate well with the ratio of disaccharides formed by transglycosylation. The unexpected regioselectivity using D-mannose can therefore be explained by an anomalous specificity in the hydrolysis reaction. By utilizing the identified characteristics of both regioselectivity and hydrolysis specificity using D-mannose, an efficient method for enzymatic synthesis of β-1,3-, β-1,4- and β-1,6-linked D-galactosyl-D-mannose was subsequently established.     

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.     

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.     

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.     

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.