Journal of applied glycoscience最新文献

Super Ohtaka^®, a fermented beverage of plant extracts, is prepared from approximately 50 kinds of vegetables and fruits is a naturally fermented mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp.). In this study, we separated water-soluble polysaccharides from Super Ohtaka^® using dialysis and chromatography, yielding four polysaccharide fractions. The polysaccharide fraction designated as OEP3 exhibited hyaluronidase inhibitory activity. The half-maximal inhibitory concentration was 860 µg/mL. This polysaccharide not only stimulated macrophages but also inhibited hyaluronidase activity and showed weak 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity.

Glycoside hydrolase family 131 (GH131) proteins are found in oomycetes, ascomycetes, and basidiomycetes, and have been reported to hydrolyze various β-glucan polysaccharides. Coprinopsis cinerea, a model basidiomycete, contains two GH131 proteins, CcGH131A and CcGH131B. This study focuses on the structural and functional properties of CcGH131B, a protein that lacks the carbohydrate bonding module 1 (CBM1) domain present in CcGH131A. The crystal structure of CcGH131B was determined. The structure displayed a β-jelly roll fold with extra loops and α-helices, resulting in a deeper substrate-binding groove compared to CcGH131A and also PaGluc131A, a GH131 protein from Podospora anserina. A cellobiose-bound structure of the E161A mutant, in which the potential catalytic residue Glu161 was substituted with Ala, showed that the region of the minus subsites bind cellulose. In contrast, the region of the plus subsites mainly consists of hydrophobic amino acid residues and appeared to interact with hydrophobic molecules rather than with carbohydrates. Analysis using native affinity polyacrylamide gel electrophoresis showed that CcGH131B interacted with cellulosic polysaccharides such as methylcellulose and carboxymethylcellulose, while the protein exhibited no detectable enzymatic activity under the tested conditions. These results suggest that the substrate specificity of CcGH131B is likely to be different from those of CcGH131A and PaGluc131A.

Isoprimeverose [α-D-xylopyranosyl-(1→6)-D-glucose] is produced from xyloglucan using the cooperative action of glycoside hydrolases including isoprimeverose-producing oligoxyloglucan hydrolase and β-galactosidase in Aspergillus oryzae. This study investigated A. oryzae strains and culture conditions suitable for isoprimeverose production from xyloglucan. Each strain of A. oryzae had a different ability to degrade xyloglucans. When an A. oryzae strain with high xyloglucan-degradation activity was cultured in a medium containing partially degraded xyloglucan as the carbon source, the production of glycoside hydrolases that degrade xyloglucan into isoprimeverose was highly induced. Our procedure efficiently produced isoprimeverose from xyloglucan without any genetically modified microorganisms or purification of enzymes.

In this study, we developed a method to synthesize sophorose using three enzymes-sucrose phosphorylase from Leuconostoc mesenteroides, 1,2-β-oligoglucan phosphorylase from Enterococcus italicus, and exo β-1,2-glucooligosaccharide sophorohydrolase from Parabacteroides distasonis-in a one-pot reaction, employing inexpensive starting materials. After optimization, a reaction was carried out using 5 mM glucose, 250 mM sucrose, 10 mM inorganic phosphate, and enzyme concentrations of 5 µg/mL sucrose phosphorylase, 20 µg/mL 1,2-β-oligoglucan phosphorylase, and 50 µg/mL exo β-1,2-glucooligosaccharide sophorohydrolase at 30 °C for 48 h, yielding 108 mM sophorose. Following yeast treatment, sophorose was purified by size-exclusion chromatography with a final yield of 45 % based on the amount of sucrose used as the donor substrate.

Water sorption reduces the glass transition temperature (T _g) of amorphous carbohydrate powders due to water plasticization. Caking of amorphous powder occurs when T _g decreases below the storage temperature (T), that is, when the glass-to-rubber transition occurs. Although glass-to-rubber transition also occurs when T is greater than T _g, knowledge regarding the caking of amorphous powders induced by T elevation is limited. Thus, caking properties were investigated using amorphous carbohydrate powders with varying water activity (a _w) values prepared at 25 °C, stored at a higher temperature, and then returned to 25 °C (T-cycled samples) for storage. Maltodextrin and glucose mixtures at weight ratios of 0, 0.1, and 0.2 glucose were employed. The caking behavior of T-cycled powders with high a _w values was similar to that of a _w-cycled samples (dried powders were stored under various a _w conditions and then returned to the dry condition via vacuum-drying) reported previously. T-cycled powders with a low a _w value, by contrast, were resistant to caking even in the rubbery state. This suggests that water molecules support the progression of caking as the binder under high-a _w conditions. To analyze the hydration level at which water molecules begin to act as a binder for caking, determination of the multilayer adsorbed water content and multilayer adsorbed a _w values is proposed. The fracture stress increased with increases in T - T _g, depending on the sample. The binding effect of water also contributed to the formation of a harder cake.

Because of the complicated hierarchical structure of starch, starch retrogradation is usually evaluated by combining several structural analysis methods covering various spatial scales. However, structural analyses are typically performed individually, making correlating the structural changes at different spatial scales challenging. Therefore, this study used a simultaneous measurement system comprising small-angle neutron scattering (SANS)/Fourier-transform infrared (FTIR)-attenuated total reflection (ATR) to record multiple structural changes in potato starch during retrogradation. In the SANS patterns, the shoulder-like peak became more pronounced with time. The peak intensity, I _max, representing the amount of ordered semicrystalline structures, increased over time, revealing the orderly reassembly of starch on the nanoscale upon retrogradation. In the FTIR-ATR spectra, the ratio of absorptions (R _1042/1016) at 1,042 and 1,016 cm^-1, indicating the short-range ordered structure in starch, increased during retrogradation. Therefore, the double-helix structures were reformed during retrogradation. The rate constant of the kinetic change for R _1042/1016 was larger than for I _max; thus, changes in the short-range ordered structure of starch converged before the changes in the semicrystalline structure. These results suggest that the formation of double-helix structures of the amylopectin side chain and the structural change of its ordered arrangement could occur in stages during retrogradation.

Short linear maltodextrin (SLMD) was synthesized from starch via the combined action of branching and debranching enzymes. The number-average degree of polymerization and number-average chain length of SLMD were 8.49 ± 0.21 and 8.52 ± 0.60, respectively, indicating that it consists of linear chains. In gel permeation chromatography analyses, SLMD showed a single peak at a molecular weight of 1,200. SLMD consisted mainly of linear saccharides with a degree of polymerization of 6-12, without high molecular weight α-glucans or small malto-oligosaccharides. SLMD had a much higher blue value and a longer λmax compared with those of commercial dextrose equivalent (DE) 13 maltodextrin. While the DE 13 maltodextrin solution remained clear, an SLMD solution became turbid upon cooling, with the turbidity reversing upon heating. This interconversion was reproducible. SLMD absorbed moisture only to a limited extent, even under high relative humidity, and remained solid without noticeable viscousness. These results demonstrate the novelty and distinct properties of SLMD compared with those of other maltodextrins available on the market, implying its potential for various applications in the food industry.

D-Allulose 3-epimerase catalyzes C-3 epimerization between D-fructose and D-allulose was found in Arthrobacter globiformis strain M30. The enzyme gene was cloned, and its recombinant enzyme and the mutant variants were expressed in E. coli. Using the information of the sequence and model structure, we succeed in the improvement of melting temperature for the enzyme without significant loss of the enzyme activity by protein engineering method. The melting temperatures were increased by 2.7, 2.1, 3.7, 5.1, and 8.0 c[C for the mutants Glu75Pro, Arg137Lys, Ala200Lys, Ala270Lys, and Val237Ile, respectively. Each effect of the mutation was independent and additive. By integrating the above mutations, we constructed a thermostable mutant that exhibits a melting temperature 12 c[C higher than wild type, and remains stable at 65 c[C for 2 h. These highly stable properties suggest that the thermostable enzymes represent an ideal enzyme candidate for the industrial production of D-allulose.

β-Glucose 1-phosphate (βGlc1P) is a donor substrate in the synthesis of various α-glucosides by glycoside phosphorylases belonging to the glycoside hydrolase family 65. This study presents an efficient synthesis of βGlc1P combining enzymatic phosphorolysis of inexpensive maltose and baker's yeast fermentation to bias the equilibrium toward maltose phosphorolysis by removing released glucose. Mass production of βGlc1P was obtained in a 2 L reaction mixture initially containing 500 mM maltose and inorganic phosphate, with a yield of 76 %. βGlc1P was isolated from the reaction mixture by crystallization after electrodialysis to obtain 181 g of βGlc1P as a bis(cyclohexylammonium) salt.

Enzymatic hydrolysis of cellulosic biomass is a complex process involving many factors, including multiple enzymes, heterogeneous substrates, and multi-step enzyme reactions. Cellulase researchers have conventionally used a double-exponential equation to fit the experimental time course of product formation, but no theoretical basis for this has been established. Here we present a mechanism-based equation that fits well the progress curves of cellulase reaction, incorporating the concepts of non-productive and productive binding on the cellulose surface and processivity. The derived equation is double exponential. Our findings indicate that the reaction mechanism of cellulase itself can account for the double-exponential nature of the progress curve independently of other factors that may contribute, such as substrate heterogeneity and involvement of other enzymes.