A kinetic method for the direct determination of cellobiose hydrolysis by β-glucosidases

Abstract

TheSomogyi—Nelson colorimetric method is applied in a new manner which is more suitable for following the kinetics of cellobiose hydrolysis catalyzed by β-glucosidase (EC 3.2.1.21). TheSomogyi—Nelson colour reagent, which is a mixture of the solutions of the reagent ofSomogyi and that ofNelson in a volume ratio of 1:1, is added to the enzyme-substrate solution at the very start of the reaction. The colour reagent reacts with the product (D-glucose). Under the reaction conditions (0.1M acetate buffer,pH = 5.0 and temperature 37°C) the colour reagent does not affect the enzyme activity. The method excludes any inhibition of the product, owing to the continuous removal of the latter by the colour reagent. The method suggested has been applied to monitor cellobiose hydrolysis with β-glucosidase, contained in four cellulase enzyme preparations from various fungal sources. The values of theMichaelis parameters (Km, V) were determined.