Reversion reactions catalyzed by glucoamylases I and II from Aspergillus niger: product analysis, kinetics equilibria, and modeling

Abstract

<p>High performance liquid chromatography (HPLC) of carbohydrates and glucoamylase-catalyzed reversion reactions were studied in this work. The HPLC of twenty different disaccharides on prepacked amine-bonded silica columns using acetonitrile-water eluents was described in Section I. Glucopyranosyl-glucoses had increasing retention times in the order of linkage (1(--->)3), (1(--->)4), (1(--->)2) and (1(--->)1), and (1(--->)6). Replacement of one of the glucosyl residues by galactose led to longer retention times, while substitution by fructosyl residue yielded shorter ones. Replacement with two xylosyl residues or forced assumption of the furanose ring form by the fructosyl residue, as in sucrose and palatinose, gave greatly reduced retentions;Section II deals with the separation of thirteen different trisaccharides on prepacked amino-bonded silica columns using acetonitrile-water eluents. As with mono- and disaccharides, replacement of glucose by xylose or fructose caused shorter retention times, while its replacement by galactose led to delayed elution. Retention times of trisaccharides containing only glucose were shortest when only (1(--->)3) bonds were present, and increased with increasing number of (1(--->)4) and especially (1(--->)6) bonds;In Section III a contribution to the understanding of the retention mechanism of carbohydrates was made by (1) measuring water concentrations in mobile and stationary liquid phases associated with amine-bonded and free silica surfaces, and by (2) relating retention of carbohydrates to two parameters, the number of hydroxyl groups per molecule that could hydrogen bond to amine groups on the surface and the calculated hydration number of the molecule;Section IV is a study of glucose reversion reactions catalyzed by the two major A. niger glucoamylase forms. Kinetics and equilibrium of the formation of five disaccharides and two trisaccharides were studied at three different levels of initial glucose concentration, pH, and temperature. A kinetic model based on adsorption of glucose and the seven oligosaccharides by the first three subsites of glucoamylase was formulated. Agreement of simulated and actual oligosaccharide formation data through the course of the reaction was excellent at all three temperatures and at the two lower initial glucose concentrations.</p>