Journal of applied glycoscience最新文献

We functionally characterized the GH10 xylanase (SoXyn10A) and the GH11 xylanase (SoXyn11B) derived from the actinomycete Streptomyces olivaceoviridis E-86. Each enzyme exhibited differences in the produced reducing power upon degradation of xylan substrates. SoXyn10A produced higher reducing power than SoXyn11B. Gel filtration of the hydrolysates generated by both enzymes revealed that the original substrate was completely decomposed. Enzyme mixtures of SoXyn10A and SoXyn11B produced the same level of reducing power as SoXyn10A alone. These observations were in good agreement with the composition of the hydrolysis products. The hydrolysis products derived from the incubation of soluble birchwood xylan with a mixture of SoXyn10A and SoXyn11B produced the same products as SoXyn10A alone with similar compositions. Furthermore, the addition of SoXyn10A following SoXyn11B-mediated digestion of xylan produced the same products as SoXyn10A alone with similar compositions. Thus, it was hypothesized that SoXyn10A could degrade xylans to a smaller size than SoXyn11B. In contrast to the soluble xylans as the substrate, the produced reducing power generated by both enzymes was not significantly different when pretreated milled bagasses were used as substrates. Quantification of the pentose content in the milled bagasse residues after the enzyme digestions revealed that SoXyn11B hydrolyzed xylans in pretreated milled bagasses much more efficiently than SoXyn10A. These data suggested that the GH10 xylanases can degrade soluble xylans smaller than the GH11 xylanases. However, the GH11 xylanases may be more efficient at catalyzing xylan degradation in natural environments (e.g. biomass) where xylans interact with celluloses and lignins.

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.

Extra-long chains (ELC) of amylopectin in rice endosperm are synthesized by granule-bound starch synthase I encoded by the Waxy (Wx) gene, which primarily synthesizes amylose. Previous studies showed that single nucleotide polymorphisms (SNP) in intron 1 and exon 6 of the Wx gene influences ELC amount. However, whether these SNPs are conserved among rice cultivars and if any other SNPs are present in the Wx gene remained unknown. Here, we sequenced the Wx gene from 17 rice cultivars with S or L-type amylopectin, including those with known ELC content and those originating in China with unique starch properties, as well as typical japonica and indica cultivars. In addition to the two SNPs described above, an additional SNP correlating with ELC content was found in exon 10. Low ELC cultivars (<3.0 %) had thymine at the splicing donor site of intron 1, Tyr224 in exon 6, and Pro415 in exon 10. Cultivars with moderate ELC content (4.1-6.9 %) had guanine at the splicing donor site of intron 1, Ser224 in exon 6, and Pro415 in exon 10. Cultivars with high ELC content (7.7-13.9 %) had guanine at the splicing donor site of intron 1, Tyr224 in exon 6, and Ser415 in exon 10. The chain length distribution pattern of amylopectin was correlated with the amounts of SSIIa found in starch granules and gelatinization temperature, but not with ELC content. The combinations of SNPs in the Wx gene found in this study may provide useful information for screening specific cultivars with different ELC content.

Novel bioreactor beads for simultaneous saccharification and fermentation (SSF) of lime-pretreated rice straw (RS) into ethanol were prepared. Genetically modified Saccharomyces cerevisiae cells expressing genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase were immobilized in calcium alginate beads containing inorganic lightweight filler particles to reduce specific gravity. For SSF experiments, the beads were floated in slurry composed of lime-pretreated RS and enzymes and incubated under CO₂ atmosphere to reduce the pH for saccharification and fermentation. Following this reaction, beads were readily picked up from the upper part of the slurry and were directly transferred to the next vessel with slurry. After 240 h of incubation, ethanol production by the beads was equivalent to that by free cells, a trend that was repeated in nine additional runs, with slightly improved ethanol yields. Slurry with pre-saccharified lime-pretreated RS was subjected to SSF with floating beads for 168 h. Although higher cell concentrations in beads resulted in more rapid initial ethanol production rates, with negligible diauxic behavior for glucose and xylose utilization, no improvement in the ethanol yield was observed. A fermentor-scale SSF experiment with floating beads was successfully performed twice, with repeated use of the beads, resulting in the production of 40.0 and 39.7 g/L ethanol. There was no decomposition of the beads during agitation at 60 rpm. Thus, this bioreactor enables reuse of yeast cells for efficient ethanol production by SSF of lignocellulosic feedstock, without the need for instruments for centrifugation or filtration of whole slurry.

Generally, Ca(OH)₂ pretreatment of lignocellulosics for fermentable sugar recovery requires a subsequent washing step for calcium removal and pH control for optimized saccharification. However, washing Ca(OH)₂-pretreated feedstock with water is considered problematic because of the low solubility of Ca(OH)₂ and its adsorption to biomass. In this study, we estimated the availability of carbonated water for calcium removal from the slurry of Ca(OH)₂-pretreated rice straw (RS). We tested two kinds of countercurrent washing sequences, four washings exclusively with water (W4) and two washings with water and subsequent two washings with carbonated water (W2C2). The ratios of calcium removal from pretreatment slurry after washing were 64.2 % for the W4 process and 92.1 % for the W2C2 process. In the W2C2 process, 49 % of the initially added calcium was recovered as CaO by calcination. In enzymatic saccharification tests under a CO₂ atmosphere at 1.5 atm, in terms of recovery of both glucose and xylose, pretreated, feedstock washed through the W2C2 process surpassed that washed through the W4 process, which could be attributed to the pH difference during saccharification: 5.6 in the W2C2 process versus 6.3 in the W4 process. Additionally, under an unpressurized CO₂ atmosphere at 1 atm, the feedstock washed through the W2C2 process released 78.5 % of total glucose residues and 90.0 % of total xylose residues. Thus, efficient removal of calcium from pretreatment slurry would lead to not only the recovery of added calcium but also the proposal of a new, simple saccharification system to be used under an unpressurized CO₂ atmosphere condition.

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.