Journal of applied glycoscience最新文献

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.

The branched structure of amylose was probed using concanavalin A (ConA) lectin, which forms precipitable aggregates with highly branched glucans, such as glycogen and amylopectin. Rice (japonica cultivar) amylose was fractionated from de-fatted, gelatinized starch by precipitation with 1-butanol (BuOH) and purified by ultracentrifugation and repeated crystallization. The purified amylose still has short side chains, whose chain-length (CL) distribution resembles that of amylopectin. More than 96 wt% of the amylose was not precipitated with ConA and remained in the resultant supernatant. The amylose recovered from the supernatant exhibited essentially the same size distributions of molecules and the CL distributions of main and side chains as those of amylose without ConA precipitation. The molar % of branched molecules was slightly decreased by ConA precipitation (-ConA, 11.6; +ConA, 8.1). These results suggest that the side chains detected in BuOH-precipitable amylose preparation are essentially attributable to amylose itself. Also, the non-precipitable nature of the branched molecules of amylose by ConA supports our previous proposal that the organization of the short side chains on amylose molecules is quite different from that found in amylopectin, in which the short side chains are arranged in a cluster fashion, and the branched glucan interacts with ConA to form precipitable aggregates. A tiny amount of ConA-precipitable glucan was detected, but its CL distribution was inconsistent with the size distribution of the branched molecules. Even if the precipitable glucans were fragments of amylopectin, their contribution to the branches detected in amylose should be minor.

The application of flour is determined by the composition of its starch and storage proteins. Previously isolated diploid waxy wheat is known to be amylose-free and possesses the same amylopectin structure as the wild-type. To reveal its characteristics, starch, protein, lipid, fiber, gluten, and allergen contents and rheological properties were analyzed and compared to its parental wild-type diploid wheat and commercially available hexaploid wheats. The results showed that the starch content of diploid waxy wheat was similar, but its protein, lipid, and fiber contents were higher than that of the wild-type. In addition, diploid waxy wheat produced high levels of gluten unlike its wild-type while its allergen level was similar to its wild-type. The storage modulus of diploid waxy wheat was significantly lower than that of other wheat lines at high temperatures. These results suggest that diploid waxy wheat holds different characteristics from hexaploid wheats for food processing.

D-Allulose (D-psicose) is a rare sugar and a C-3 epimer of D-fructose. D-Allulose has been reported to have several health benefits via its alteration of both glucose and lipid metabolism. It was previously reported that D-allulose alters the hepatic metabolomic profile. Although the kidneys are crucial organs in metabolic regulation, the effects of D-allulose on renal metabolism have not yet been established. Therefore, this study was designed to capture the overall metabolic response in the kidneys to D-allulose. This was done by providing an AIN-93G diet to Wistar rats, with or without 3 % D-allulose, for four weeks. Renal tissue and blood samples were collected after a 3-hour fasting for evaluation of the renal metabolic profile and their related plasma parameters. D-Allulose increased renal weight without changes in the plasma indices associated with reduced renal function. Metabolic profiling identified a total of 264 peaks. As the contribution rate was too low in the principal component analysis results of the metabolic profiling results, we evaluated the metabolites that were significantly different between two groups and identified 23 up-regulated and 26 down-regulated metabolites in the D-allulose group. D-Allulose also had significant influence on several metabolites involved in glucose metabolism, amino acid metabolism, and purine metabolism. Moreover, the levels of trimethylamine N-oxide and symmetric dimethylarginine, which are associated with several diseases such as chronic kidney disease and cardiovascular disease decreased following D-allulose diets. This study showed that D-allulose affects the renal metabolic profile, and our findings will help elucidate the function of D-allulose.

We recently found two α-L-glucosidases, which can hydrolyze p-nitrophenyl α-L-glucopyranoside (PNP L-Glc) rather than p-nitrophenyl α-L-fucopyranoside, in glycoside hydrolase family 29. This study evaluated their substrate specificity for p-nitrophenyl α-L-rhamnopyranoside (PNP L-Rha), α-L-quinovopyranoside (PNP L-Qui), and α-L-xylopyranoside (PNP L-Xyl), of which structure is similar to PNP L-Glc. The two α-L-glucosidases had little activity toward PNP L-Rha. They exhibited higher k _cat/K _m values for PNP L-Qui but smaller for PNP L-Xyl than for PNP L-Glc. The molecular docking studies indicated that these specificities were correlated well with the active-site structure of the α-L-glucosidases. The finding that α-L-quinovoside, which has been suggested to occur in nature, is also a substrate for α-L-glucosidases indicates that this enzyme are not solely dedicated to α-L-glucoside hydrolysis.

To overcome incompatibility issues and increase the possibility of blood transfusion, technologies that enable efficient conversion of A- and B-type red blood cells to the universal donor O-type is desirable. Although several blood type-converting enzymes have been identified, detailed understanding about their molecular functions is limited. α-Galactosidase from Bifidobacterium bifidum JCM 1254 (AgaBb), belonging to glycoside hydrolase (GH) 110 subfamily A, specifically acts on blood group B antigen. Here we present the crystal structure of AgaBb, including the catalytic GH110 domain and part of the C-terminal uncharacterized regions. Based on this structure, we deduced a possible binding mechanism of blood group B antigen to the active site. Site-directed mutagenesis confirmed that R270 and E380 recognize the fucose moiety in the B antigen. Thermal shift assay revealed that the C-terminal uncharacterized region significantly contributes to protein stability. This region is shared only among GH110 enzymes from B. bifidum and some Ruminococcus species. The elucidation of the molecular basis for the specific recognition of blood group B antigen is expected to lead to the practical application of blood group conversion enzymes in the future.

Transient absorption at 340 nm under alkaline conditions has long been used to detect the presence of 3-keto-O-glycosides without understanding the molecular basis of the absorbance. The time course of A_{340 nm} for the alkaline treatment of 3-ketolevoglucosan, an intramolecular 3-keto-O-glycoside, was investigated to identify the three products generated through alkaline treatment. By comparing the spectra of these compounds under neutral and alkaline conditions, we identified 1,5-anhydro-D-erythro-hex-1-en-3-ulose (2-hydroxy-3-keto-D-glucal) as being the compound responsible for the absorption.

The objective of this study was to characterize the endosperm starch in rice that ectopically overexpressed the α-amylase. Transgenic rice plants, transformed with cauliflower mosaic virus 35S promoter driven AmyI-1 (35S::AmyI-1) and AmyII-4 (35S::AmyII-4), and 10 kDa prolamin promoter driven AmyI-1 (P10::AmyI-1), were cultivated under standard conditions (23 °C, 12 h in the dark/ 26 °C, 12 h in the light), and brown grains were subsequently harvested. Each grain displayed characteristic chalkiness, while electron microanalyzer (EPMA)-SEM images disclosed numerous small pits on the surface of the starch granules, attributable to α-amylase activity. Fluorescence labeling and capillary electrophoresis analysis of starch chain length distribution revealed no significant alterations in the starches of 35S::AmyI-1 and 35S::AmyII-4 transgenic rice compared to the wild-type. Conversely, the extremely short α-glucan chains (DP 2-8) exhibited a dramatic increase in the P10::AmyI-1 starch. Rapid visco-analyzer analysis also identified variations in the chain length distribution of P10::AmyI-1 starch, manifesting as changes in viscosity. Moreover, ¹H-NMR analysis uncovered dynamic modifications in the molecular structure of starch in rice grain transformed with P10::AmyI-1, which was found to possess unprecedented structural characteristics.