Journal of applied glycoscience最新文献

We evaluated the stabilities of kojibiose and sophorose when heated under neutral pH conditions. Kojibiose and sophorose epimerized at the C-2 position of glucose on the reducing end, resulting in the production of 2-O-α-D-glucopyranosyl-D-mannose and 2-O-β-D-glucopyranosyl-D-mannose, respectively. Under weak alkaline conditions, kojibiose was decomposed due to heating into its mono-dehydrated derivatives, including 3-deoxy-2,3-unsaturated compounds and bicyclic 3,6-anhydro compounds. Following these experiments, we propose a kinetic model for the epimerization and decomposition of kojibiose and sophorose by heat treatment under neutral pH and alkaline conditions. The proposed model shows a good fit with the experimental data collected in this study. The rate constants of a reversible epimerization of kojibiose at pH 7.5 and 90 °C were (1.6 ± 0.1) × 10^-5 s^-1 and (3.2 ± 0.2) × 10^-5 s^-1 for the forward and reverse reactions, respectively, and were almost identical to those [(1.5 ± 0.1) × 10^-5 s^-1 and (3.5 ± 0.4) × 10^-5 s^-1] of sophorose. The rate constant of the decomposition reaction for kojibiose was (4.7 ± 1.1) × 10^-7 s^-1 whereas that for sophorose [(3.7 ± 0.2) × 10^-6 s^-1] was about ten times higher. The epimerization reaction was not significantly affected by the variation in the buffer except for a borate buffer, and depended instead upon the pH value (concentration of hydroxide ions), indicating that epimerization occurred as a function of the hydroxide ion. These instabilities are an extension of the neutral pH conditions for keto-enol tautomerization that are often observed under strong alkaline conditions.

To expand the range of soluble carbon sources for our enzyme production system, we investigated the properties of sucrose utilization and its effect on cellulase production by Trichoderma reesei M2-1. We performed batch cultivation of T. reesei M2-1 on sucrose and related sugars along with cellobiose, which was used as a cellulase inducer. The results clearly revealed that the hydrolysis products of sucrose, i.e. glucose and fructose, but not sucrose, can be used as a carbon source for enzyme production. In a 10-day continuous feeding experiment using invertase-treated sucrose/cellobiose, the fungal strain produced cellulases with a filter paper-degrading activity of 20.3 U/mL and production efficiency of 254 U/g-carbon sources. These values were comparable with those of glucose/cellobiose feeding (21.2 U/mL and 265 U/g-carbon sources, respectively). Furthermore, the comparison of the specific activities clearly indicated that the compositions of both produced enzymes were similar. Therefore, enzymatically hydrolyzed sucrose can be utilized as an alternative carbon source to glucose in our enzyme production system with T. reesei M2-1.

To study the structure of β-glucans, we developed a separation method and molecular library of β-glucan oligosaccharides. The oligosaccharides were prepared by partial acid hydrolysis from laminarin, which is a β-glucan of Laminaria digitata. They were labeled with the 2-aminopyridine fluorophore and separated to homogeneity by size-fractionation and reversed phase high-performance liquid chromatography (HPLC). Branching structures of all isomeric oligosaccharides from trimers to pentamers were determined, and a two-dimensional (2D)-HPLC map of the β-glucan oligosaccharides was made based on the data. Next, structural analysis of the longer β-glucan oligosaccharide was performed using the 2D-HPLC map. A branched decamer oligosaccharide was isolated from the β-glucan and cleaved to smaller oligosaccharides by partial acid hydrolysis. The structure of the longer oligosaccharide was successfully elucidated from the fragment structures determined by the 2D-HPLC map. The molecular library and the 2D-HPLC map described in this study will be useful for the structural analysis of β-glucans.

A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-pNP [Gal(GlcNAc)₂-β-pNP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via β-1,4-galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc)₂-β-pNP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)₂-β-pNP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc)₂ and p-nitrophenol (pNP). A combination of kinetic studies and docking simulation was further conducted to elucidate the mode of substrate binding. The results demonstrate that Gal(GlcNAc)₂-β-pNP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc)₂ and pNP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of pNP liberated from the substrate.

Glucosamine (GlcN) is commonly used as a dietary supplement to promote cartilage health in humans. We previously reported that GlcN could induce autophagy in cultured mammalian cells. Autophagy is known to be involved in the prevention of various diseases and aging. Here, we showed that GlcN extended the lifespan of the nematode Caenorhabditis elegans by inducing autophagy. Autophagy induction by GlcN was demonstrated by western blotting for LGG-1 (an ortholog of mammalian LC3) and by detecting autophagosomal dots in seam cells by fluorescence microscopy. Lifespan assays revealed that GlcN-induced lifespan extension was achieved with at least 5 mM GlcN. A maximum lifespan extension of approximately 30 % was achieved with 20 mM GlcN (p<0.0001). GlcN-induced lifespan extension was not dependent on the longevity genes daf-16 and sir-2.1 but dependent on the autophagy-essential gene atg-18. Therefore, we suggest that oral administration of GlcN could help delay the aging process via autophagy induction.

β-L-Arabinopyranosidases are classified into the glycoside hydrolase family 27 (GH27) and GH97, but not into GH36. In this study, we first characterized the GH36 β-L-arabinopyranosidase BAD_1528 from Bifidobacterium adolescentis JCM1275. The recombinant BAD_1528 expressed in Escherichia coli had a hydrolytic activity toward p-nitrophenyl (pNP)-β-L-arabinopyranoside (Arap) and a weak activity toward pNP-α-D-galactopyranoside (Gal). The enzyme liberated L-arabinose efficiently not from any oligosaccharides or polysaccharides containing Arap-β1,3-linkages, but from the disaccharide Arap-β1,3-L-arabinose. However, we were unable to confirm the in vitro fermentability of Arap-β1,3-Ara in B. adolescentis strains. The enzyme also had a transglycosylation activity toward 1-alkanols and saccharides as acceptors.

Highly thermostable β-mannanase, belonging to glycoside hydrolase family 5 subfamily 7, was purified from the culture supernatant of Talaromyces trachyspermus B168 and the cDNA of its transcript was cloned. The recombinant enzyme showed maximal activity at pH 4.5 and 85 °C. It retained more than 90 % of its activity below 60 °C. Obtaining the crystal structure of the enzyme helped us to understand the mechanism of its thermostability. An antiparallel β-sheet, salt-bridges, hydrophobic packing, proline residues in the loops, and loop shortening are considered to be related to the thermostability of the enzyme. The enzyme hydrolyzed mannans such as locust bean gum, carob galactomannan, guar gum, konjac glucomannan, and ivory nut mannan. It hydrolyzed 50.7 % of the total mannans from coffee waste, producing mannooligosaccharides. The enzyme has the highest optimum temperature among the known fungal β-mannanases and has potential for use in industrial applications.

In cereals, granule-bound starch synthase I (GBSSI)-deficient mutants accumulate glutinous (amylose-free) starch in their storage tissues. The amylose-free starch produced by waxy (wx) mutants of hexaploid bread wheat (Triticum aestivum L.) is used in cakes and breads. However, wx mutants of diploid wheat (T. monococcum L.) have so far no commercial applications. In this study, we identified a mutation in exon 6 of GBSSI in a diploid wheat wx mutant that resulted in the replacement of Trp355 with a stop codon. Molecular markers were developed for the rapid screening of the mutation, which should allow the selection of heterozygous and homozygous plants during backcrossing. This will facilitate the improvement of the agricultural traits of the wx mutant and the generation of new amylose-free wx lines.

Glycoside hydrolases require carboxyl groups as catalysts for their activity. A retaining xylanase from Streptomyces olivaceoviridis E-86 belonging to glycoside hydrolase family 10 possesses Glu128 and Glu236 that respectively function as acid/base and nucleophile. We previously developed a unique mutant of the retaining xylanase, N127S/E128H, whose deglycosylation is triggered by azide. A crystallographic study reported that the transient formation of a Ser-His catalytic dyad in the reaction cycle possibly reduced the azidolysis reaction. In the present study, we engineered a catalytic dyad with enhanced stability by site-directed mutagenesis and crystallographic study of N127S/E128H. Comparison of the Michaelis complexes of N127S/E128H with pNP-X₂ and with xylopentaose showed that Ser127 could form an alternative hydrogen bond with Thr82, which disrupts the formation of the Ser-His catalytic dyad. The introduction of T82A mutation in N127S/E128H produces an enhanced first-order rate constant (6 times that of N127S/E128H). We confirmed the presence of a stable Ser-His hydrogen bond in the Michaelis complex of the triple mutant, which forms the productive tautomer of His128 that acts as an acid catalyst. Because the glycosyl azide is applicable in the bioconjugation of glycans by using click chemistry, the enzyme-assisted production of the glycosyl azide may contribute to the field of glycobiology.

Cellobiose phosphorylase from Cellvibrio gilvus was used to prepare 1,5-anhydro-4-O-β-D-glucopyranosyl-D-fructose [βGlc(1→4)AF] from 1,5-anhydro-D-fructose and α-D-glucose 1-phosphate. βGlc(1→4)AF decomposed into D-glucose and ascopyrone T via β-elimination. Higher pH and temperature caused faster decomposition. However, decomposition proceeded significantly even under mild conditions. For instance, the half-life of βGlc(1→4)AF was 17 h at 30 °C and pH 7.0. Because βGlc(1→4)AF is a mimic of cellulose, in which the C2 hydroxyl group is oxidized, such decomposition may occur in oxidized cellulose in nature. Here we propose a possible oxidizing pathway by which this occurs.