Chemical methods for measuring cellobiase activity are based on increased reducing capacity, following conversion of cellobiose into glucose (l-3). The chemical methods are time consuming, of low sensitivity, and nonspecific. Enzymic methods use glucose oxidase to estimate the amount of glucose liberated (4,5). Although more specific and sensitive, the enzymic methods are also time consuming. The two-step method requires two successive incubations, which take several hours (4). Although very sensitive, the one-step method requires 75 min for a preincubation, a highly purified glucose oxidase with negligible disaccharidase activity, and an absolutely glucose-free substrate (5). In the method for assaying cellobiase described in this paper, glucose is measured enzymically by formation of NADPH with a coupling system using yeast hexokinase, glucose-6-P dehydrogenase, and NADP, similar to previous methods involving TPN (6). The highly reproducible assay evaluated here requires only 15 min and all reagents and auxiliary enzymes of adequate quality are available commercially.
MATERIALS AND METHODS
Biochemical Reagents2
Adenosine 5triphosphate, disodium (ATP) -A grade, 0.1 M solution, pH 6.8.
Nicotine-adenine dinucleotide phosphate, monosodium (NADP) -A grade, 0.03 M solution.
Glucose-6-phosphate dehydrogenase, (yeast) -A grade, 200 units/ml.
Hexokinase (yeast) -A grade, 500 units/ml. Cellobiose-A grade, 0.1 M solution.