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 β-glucan. This method was further used to follow β-glucosidase degradation and product formation as progress curves. This approach allowed us to compare the kinetic characteristics of β-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 β-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-β-cellobiosyl-D-glucose (G4G3G) and on 3-O-β-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.
Department of Organic Chemistry, Corvinus University of Budapest, Budapest, Hungary.
Dániel
D
Radva
Svein Halvor
SH
Knutsen
Judit
J
Kosáry
Simon
S
Ballance
D002852:Chromatography, Ion Exchange / Q000379:methods*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D001467:Hordeum / Q000737:chemistry*; D007700:Kinetics; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D047071:beta-Glucans / Q000737:chemistry / Q000378:metabolism*; D001617:beta-Glucosidase / Q000378:metabolism*
D009844:Oligosaccharides; D047071:beta-Glucans; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase; C028020:licheninase