Determining the solution conformational entropy of O-linked oligosaccharides at quasi-physiological conditions: size-exclusion chromatography and molecular dynamics

The physiological functions of oligosaccharides are influenced by a number of structural parameters such as anomeric configuration, glycosidic linkage, and degree of polymerization. These parameters affect the conformation of the oligosaccharides which, in turn, is responsible for characteristics such as aptameric and enzymatic binding, chiral recognition, and the structural targeting of bacterial and parasitic recognition events. Here, we measure the solution conformational entropy (DeltaS) of two series of oligosaccharides, linear malto- and cellooligosaccharides, using size-exclusion chromatography (SEC). For each series, we have determined DeltaS as a function of degree of polymerization (DP). The choice of oligosaccharides studied also allowed us to compare the influence of anomeric configuration on DeltaS, and to do so as a function of DP. Studies were conducted in water at physiological temperature and pH in order to resemble conditions within the human body. Experimental results were augmented with results from molecular dynamics computer modeling simulations in aqueous solvent. A comparison between experimental and computational data showed how the techniques can complement each other. An example of the latter is the considerable enthalpic contribution to the chromatographic separation of alpha- and gamma-cyclodextrin, which may have gone unnoticed if not for the large discrepancy between the results obtained by the separate techniques.     

Alkaline degradation kinetics and CE-separation of cello- and xylooligomers. Part I

The degradation kinetics of cello- and xylooligomers under alkaline conditions has been studied, and kinetic order, kinetic rate constants as well as activation parameters (E(A), DeltaE(#), DeltaS(#)) for the different oligomers have been determined. The results were corroborated by a mathematical model of the degradation kinetics. A reliable and convenient method for the separation and simultaneous quantification of cello- and xylooligomers, based on capillary electrophoresis (CE) with pre-column derivatization, has been established. p-Aminobenzonitrile was used as the UV tag, and 550 mM borate buffer containing hexadimethrine bromide was employed as the running electrolyte.     

Preparation of cellulase concoction using differential adsorption phenomenon

Controlled depolymerization of cellulose is essential for the production of valuable cellooligosaccharides and cellobiose from lignocellulosic biomass. However, enzymatic cellulose hydrolysis involves multiple synergistically acting enzymes, making difficult to control the depolymerization process and generate desired product. This work exploits the varying adsorption properties of the cellulase components to the cellulosic substrate and aims to control the enzyme activity. Cellulase adsorption was favored on pretreated cellulosic biomass as compared to synthetic cellulose. Preferential adsorption of exocellulases was observed over endocellulase, while β-glucosidases remained unadsorbed. Adsorbed enzyme fraction with bound exocellulases when used for hydrolysis generated cellobiose predominantly, while the unadsorbed enzymes in the liquid fraction produced cellooligosaccharides majorly, owing to its high endocellulases activity. Thus, the differential adsorption phenomenon of the cellulase components can be used for the controlling cellulose hydrolysis for the production of an array of sugars.