The isomalto- and nigero-oligosaccharides are usually produced from starch by the combination of α-glucosidase and starch degrading enzymes, such as α- and β-amylases. In this study, a new reaction system for the production from starch of two α-1,4 glucans having α-1,6- and α-1,3-linked glucosyl residues at or near the non-reducing end was established. These glucans were efficiently produced by the coupled reaction of α-glucosidase and cyclodextrin glucanotransferase (CGTase) with negligible cyclodextrin production. The produced glucans underwent very little hydrolyzation by β-amylase but α-amylase clearly enhanced the digestion of glucan. This indicated that glucosidic linkage other than α-1,4-linkage was introduced at or near the non-reducing end of the glucan and the reducing end part of the glucan was mainly composed of α-1,4-linkages. The glucosidic linkage introduced was dependent on the specificity of α-glucosidase for glucosidic linkage. α-Glucosidases from Aspergillus niger (ANG) and Acremonium strictum (ASG) produced α-1,6- and α-1,3-glucosidic linkages, respectively. The chain length distribution also varied according to the specificity of α-glucosidases for substrate chain length. The major DP of the glucans produced by ANG and ASG were 4-6 and 6-10, respectively. The glucan produced by the coupled reaction was highly resistant to retrogradation. The syrup including this glucan maintained transparency following storage at room temperature for 1 month. In contrast, control syrup including the starch hydrolysate lost transparency despite the lower content of long-chain glucan. This indicates that the glucosyl residue linked by α-1,3-linkage present at or near the non-reducing end of glucan strongly inhibits aggregation of glucan and provides retrogradation tolerance.
β-1,4-Endoglucanase of glycosyl hydrolase family (GHF) 7 from a fungus Aspergillus oryzae catalyzed the condensation between lactose (Lac) and alkanols. With Lac as a glycosyl donor, methanol, ethanol, 1-butanol and 1-hexanol and 1,6-hexanediol (HD) could be aglycons. The maximal condensation was observed between 250 mM Lac and 1.5 M HD at 30°C, pH 5.0. Transglycosylation to HD occurred to a similar extent with Lac-β-pNP or with cellobiose-β-pNP as the sugar donors, while the condensation between N-acetyllactosamine and HD was negative. Lac-condensation activity was also observed with the cellulase from Fusarium oxysporum, which belongs to GHF7. These suggested that fungal cellulases belonging to GHF7 should possess an ability to perform the condensation using Lac as a glycosyl donor.
We performed two dimensional electrophoresis (2-DE) coupled with MS analysis on Theobroma cacao pod husk (fruit pericarp) to explore the proteome of this recalcitrant tissue. Using a phenol extraction/methanol-ammonium acetate precipitation method, we have obtained 2-DE images with approximately 700 protein spots detected after colloidal CBB staining. Two hundred and forty-four protein spots were analyzed by de novo sequencing of SPITC-derivatized tryptic peptides by MALDI-TOF/TOF MS. Applying this technique, 144 protein spots from cocoa pod husk were identified. The majority of the identified proteins were involved in metabolism and energy. Several of these proteins could be linked to pod growth and development processes.
Further structural study of the xyloglucanase-derived eggplant xyloglucan oligosaccharides was carried out to investigate the oligosaccharide units of xyloglucan from the cell-walls of eggplant in detail. The result shows that a hexasaccharide, XSG, a heptasaccharide, XX2GG, an octasaccharide, LX2GG and a nonasaccahride, LSGGG, in addition to the major oligosaccharides XXGG, LXGG, XLGG, XSGG, LLGG and LSGG, the structures of which were previously reported, are included in oligosaccharide units of eggplant xyloglucan.
Three kinds of ω-epoxyalkyl α-glucopyranosides (3′,4′-epoxybutyl α-D-glucopyranoside (E4G), 4′,5′-epoxypentyl α-D-glucopyranoside (E5G) and 5′,6′-epoxyhexyl α-D-glucopyranoside (E6G)), having alkyl chains of different lengths at their aglycone moieties, inactivated the endodextranase from Streptococcus mutans ATCC 25175 (SmDex) irreversibly with the pseudo-first order kinetics. Alkyl chain length-dependent inactivation was observed and the degree of activity loss was E5G, E6G and E4G, in that order, implying that the distance between epoxide group and glucosyl residue of ω-epoxyalkyl α-glucopyranoside was important in the modification of endodextranase. Inactivation by E5G followed the model of reversible intermediate-complex formation mechanism (suicide inhibitor-based mechanism). The rate constant of irreversible inactivation (k) and the dissociation constant of intermediate-complex (KR) of SmDex and E5G were 0.44 min-1 and 1.45 mM, respectively. Hydrolytic reaction product (isomaltose) protected SmDex from E5G-inactivation, suggesting that E5G bound to the catalytic site of SmDex. This is the first report that ω-epoxyalkyl α-glucopyranoside becomes a suicide substrate for endodextranase.
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