Alkoxycarbonyl elimination of 3-O-substituted glucose and fructose by heat treatment under neutral pH.

3-O-Substituted reducing aldoses are commonly unstable under heat treatment at neutral and alkaline pH. In this study, to evaluate the decomposition products, nigerose (3-O-α-d-glucopyranosyl-d-glucose) and 3-O-methyl glucose were heated at 90 °C in 100 mM sodium phosphate buffer (pH 7.5). Decomposition via ß-elimination was observed that formed a mixture of 3-deoxy-arabino-hexonic acid and 3-deoxy-ribo-hexonic acid; upon further acid treatment, it was converted to their γ-lactones. Similarly, turanose (3-O-α-d-glucopyranosyl-d-fructose), a ketose isomer of nigerose, decomposed more rapidly than nigerose under the same conditions, forming the same products. These findings indicate that 3-O-substituted reducing glucose and fructose decompose via the same 1,2-enediol intermediate. The alkoxycarbonyl elimination of 3-O-substituted reducing glucose and fructose occurs readily if an O-glycosidic bond is located on the carbon adjacent to the 1,2-enediol intermediate. Following these experiments, we proposed a kinetic model for the3- decomposition of nigerose and turanose by heat treatment under neutral pH conditions. The proposed model showed a good fit with the experimental data collected in this study. The rate constant of the decomposition for nigerose was (1.2 ± 0.1) × 10-4 s-1, whereas that for turanose [(2.6 ± 0.2) × 10-4 s-1] was about 2.2 times higher.

Large-scale preparation of ß-1,2-glucan using quite a small amount of sophorose.

A large amount of ß-1,2-glucan was produced enzymatically from quite a small amount of sophorose as an acceptor material through three synthesis steps using a sucrose phosphorylase and a 1,2-ß-oligoglucan phosphorylase. The first synthesis step was performed in a 200 µL of a reaction solution containing 5 mM sophorose and 1.0 M sucrose. ß-1,2-Glucan in a part of the resultant solution was hydrolyzed to ß-1,2-glucooligosaccharides by a ß-1,2-glucanase. The second synthesis was performed in 25 times the volume for the first synthesis. The hydrolysate solution (1% volume of the reaction solution) was used as an acceptor. After treatment with the ß-1,2-glucanase again, the third synthesis was performed 200 times the volume for the second synthesis (1 L). The reaction yield of ß-1,2-glucan at each synthesis was 93%, 76% and 91%. Finally, more than 140 g of ß-1,2-glucan was synthesized using approximately 20 µg of sophorose as the starting acceptor material. Abbreviations: DPs: degrees of polymerization; SOGP: 1,2-ß-oligoglucan phosphorylase; Sopns: ß-1,2-glucooligosaccharides with DP of n; Glc1P: α-glucose 1-phosphate; SucP: sucrose phosphorylase from Bifidobacterium longum subsp. longum; SGL: ß-1,2-glucanase; CaSGL: Chy400_4174 protein; TLC: thin layer chromatography; GOPOD: glucose oxidase/peroxidase; PGM: phosphoglucomutase; G6PDH: glucose 6-phosphate dehydrogenase.

Identification, characterization, and structural analyses of a fungal endo-ß-1,2-glucanase reveal a new glycoside hydrolase family.

endo-ß-1,2-Glucanase (SGL) is an enzyme that hydrolyzes ß-1,2-glucans, which play important physiological roles in some bacteria as a cyclic form. To date, no eukaryotic SGL has been identified. We purified an SGL from Talaromyces funiculosus (TfSGL), a soil fungus, to homogeneity and then cloned the complementary DNA encoding the enzyme. TfSGL shows no significant sequence similarity to any known glycoside hydrolase (GH) families, but shows significant similarity to certain eukaryotic proteins with unknown functions. The recombinant TfSGL (TfSGLr) specifically hydrolyzed linear and cyclic ß-1,2-glucans to sophorose (Glc-ß-1,2-Glc) as a main product. TfSGLr hydrolyzed reducing-end-modified ß-1,2-gluco-oligosaccharides to release a sophoroside with the modified moiety. These results indicate that TfSGL is an endo-type enzyme that preferably releases sophorose from the reducing end of substrates. Stereochemical analysis demonstrated that TfSGL is an inverting enzyme. The overall structure of TfSGLr includes an (α/α)6 toroid fold. The substrate-binding mode was revealed by the structure of a Michaelis complex of an inactive TfSGLr mutant with a ß-1,2-glucoheptasaccharide. Mutational analysis and action pattern analysis of ß-1,2-gluco-oligosaccharide derivatives revealed an unprecedented catalytic mechanism for substrate hydrolysis. Glu-262 (general acid) indirectly protonates the anomeric oxygen at subsite -1 via the 3-hydroxy group of the Glc moiety at subsite +2, and Asp-446 (general base) activates the nucleophilic water via another water. TfSGLr is apparently different from a GH144 SGL in the reaction and substrate recognition mechanism based on structural comparison. Overall, we propose that TfSGL and closely-related enzymes can be classified into a new family, GH162.

Colorimetric determination of ß-1,2-glucooligosaccharides for an enzymatic assay using 3-methyl-2-benzothiazolinonehydrazone.

A colorimetric determination method measuring the reducing ends of sugars is usually used for quantitative evaluation of polysaccharide-degrading activity of endo-type enzymes. However, no appropriate colorimetric method has been established for enzymatic assay of ß-1,2-glucanases, which produce ß-1,2-glucooligosaccharides from ß-1,2-glucans. The Anthon-MBTH method has been potentially the most adaptable for color development of ß-1,2-glucooligosaccharides among various known colorimetric methods for detecting the reducing power of oligosaccharides, since the difference between sophorose and other ß-1,2-glucooligosaccharides in absorbance is relatively small. Almost the same color development was obtained for ß-1,2-glucooligosaccharides when the heating time with the Anthon-MBTH method was changed. The kind of base and concentration of dithiothreitol did not markedly affect the color development. Most buffer components, salts and a chelating reagent used for usual enzymatic experiments did not inhibit color development. Furthermore, assay was performed successfully for a ß-1,2-glucanase using the modified MBTH method.

Synthesis of three deoxy-sophorose derivatives for evaluating the requirement of hydroxy groups at position 3 and/or 3' of sophorose by 1,2-ß-oligoglucan phosphorylases.

Sophorose (Sop2) is known as a powerful inducer of cellulases in Trichoderma reesei, and in recent years 1,2-ß-D-oligoglucan phosphorylase (SOGP) has been found to use Sop2 in synthetic reactions. From the structure of the complex of SOGP with Sop2, it was predicted that both the 3-hydroxy group at the reducing end glucose moiety of Sop2 and the 3'-hydroxy group at the non-reducing end glucose moiety of Sop2 were important for substrate recognition. In this study, three kinds of 3- and/or 3'-deoxy-Sop2 derivatives were synthesized to evaluate this mechanism. The deoxygenation of the 3-hydroxy group of D-glucopyranose derivative was performed by radical reduction using a toluoyl group as a leaving group. The utilization of a toluoyl group that plays two roles (a leaving group for the deoxygenation and a protecting group for a hydroxy group) resulted in efficient syntheses of the three target compounds. The NMR spectra of the two final compounds (3-deoxy- and 3,3'-dideoxy-Sop2) suggested that the glucose moiety of the reducing end of Sop2 can easily take on a furanose structure (five-membered ring structure) by deoxygenation of the 3-hydroxy group of Sop2. In addition, the ratio of the five- and six-membered ring structures changed depending on the temperature. The SOGPs exhibited remarkably lower specific activity for 3'-deoxy- and 3,3'-dideoxy-Sop2, indicating that the 3'-hydroxy group of Sop2 is important for substrate recognition by SOGPs.