This review describes examples of the structural characterization of sugar chains in solutions or gels at the nano level using small-angle X-ray scattering (SAXS). The review initially focuses on carrageenan, a sulfate-containing electrolytic polysaccharide, aqueous solutions of which undergo gelation when cooled. The gelation process is based on the formation of double helix structures as crosslinking regions. The association state of the various sugar chains can be determined by SAXS. On the basis of SAXS results, it was demonstrated that a 3% solution of κ-carrageenan, with potassium ion as the counter cation, yields gels by initial double helix formation and the subsequent association of 2 double helices. In contrast, the gelation of ι-carrageenan, which contains a greater number of sulfate groups, was due solely to the formation of double helices. In the second example, the SAXS observation on the structural association between regioselectively desulfated heparin and fibroblast growth factor (FGF) is described. The SAXS results indicated that the sulfate group at the 2-O position in the iduronic acid moiety of heparin was essential for aggregation with FGF. It is suggested that the nature of this structural association influences physiological activity. Finally, the SAXS data from a cyclic sugar chain, which has a characteristic profile that differs from the linear chain structure, is presented, and its inclusion structure is described.
In nature, crystalline cellulose is mainly degraded by cellobiohydrolases (CBHs) produced by various microorganisms. Since both substrate and enzymatic features relate to the reaction and the reaction proceeds at a solid/liquid interface, it is quite difficult to perform the quantitative analysis of CBH. In the present study, we estimate the surface area of crystalline celluloses by the adsorption maxima (Amax) of CBH, the specific activity of CBH (k=v/A) was plotted versus surface density (ρ=A/Amax) to adjust for the difference of surface area, and quantify the reaction rate of CBH. The results obtained from the approach indicate that the feature of crystalline cellulose greatly affects the reaction of CBH.
Reducing-end xylose-releasing exo-oligoxylanase (Rex, EC. 126.96.36.199) is an inverting xylanolytic enzyme, belonging to the glycoside hydrolase (GH) family 8, which hydrolyzes xylooligosaccharides to release xylose (X1) from its reducing end. Rex hydrolyzes α-xylobiosyl fluoride (α-X2F) to yield xylobiose (X2) only in the presence of X1, confirming the Hehre resynthesis-hydrolysis mechanism. A library of mutant Rex at the catalytic base (D263) was constructed by saturation mutagenesis, in which D263C accumulated the highest level of xylotriose (X3) from α-X2F and X1. However, F− releasing activities of the mutants were much less than that of the wild type. Next, Y198 residue of Rex that forms a hydrogen bond with nucleophilic water was substituted with phenylalanine, causing a marked decrease in hydrolytic activity and a small increase in the F− releasing activity from α-X2F in the presence of X1. Y198F of Rex accumulated more product during the glycosynthase reaction than D263C. Recently, an inverting α-1,2-fucosidase belonging to GH95 was converted into glycosynthase by mutating a catalytic base residue. In both cases, the catalytic base should be intact.