Application of high-performance anion-exchange chromatography with pulsed amperometric detection to compare the kinetic properties of β-glucosidase on oligosaccharides from lichenase digested barley β-glucan

A chromatographic method using HPAEC-PAD was developed to accurately quantify the major oligosaccharides derived from lichenase degradation of barley beta-glucan. This method was further used to follow beta-glucosidase degradation and product formation as progress curves. This approach allowed us to compare the kinetic characteristics of beta-glucosidase on each exclusive oligosaccharide substrate and their mixtures. Our results show that when determining the kinetic parameters of exohydrolases on oligosaccharides following the progress curve for the substrates is necessary since calculations based only on released monosaccharide products may lead to an error. The catalytic activity of almond beta-glucosidase on laminaribiose (G3G) was approximately half that measured against cellobiose (3.15 S-1). The enzyme had 12 times and 560 times less catalytic activity on 3-O-beta-cellobiosyl-D-glucose (G4G3G) and on 3-O-beta-cellotriosyl-D-glucose (G4G4G3G) respectively than on G3G. Our approach offers a useful tool for the determination of the kinetics of enzymatic or chemical modification of various carbohydrate substrates. (C) 2012 Elsevier Ltd. All rights reserved.