Determination of (1→3),(1→4)-β-D-Glucanase Activity by a Calcofluor-Flow Injection Analysis Method

Abstract

A new methodology for the determination of (1→3),(1→4)-β-d-glucanase activity has been developed. The concentration decay curves corresponding to the depolymerisation of high molecular weight barley (1→3),(1→4)-β-d-glucan by pure (1→3),(1→4)-β-d-glucanase (E.C. 3.2.1.73) fromBacillus subtilisand by crude (1→3),(1→4)-β-d-glucanase from different malts were monitored by the Calcofluor-FIA method. In all cases, the high molecular weight (17Instituto de Agroquı́mica y Tecnologı́a de Alimentos (CSIC)rarr;3),(1→4)-β-d-glucan decay curves fitted very well to an empirical formula describing the change in substrate concentration with time. The curves possess an inflexion point at which the depolymerisation rate of the substrate reaches a maximum. This maximum depolymerisation rate correlates with the initial concentrations of enzyme and substrate,EoandSo, and the enzyme kinetic constantsVmandKmthrough a hyperbola similar to that of Michaelis-Menten. TheKmdetermined forB. subtilisβ-glucanase was rather low, about 0·99 g β-glucan/l, when compared with those corresponding to (1→3),(1→4)-β-d-glucanase from different malts, which were, in turn, practically identical at about 2·92 g β-glucan/l. Experiments with barley (1→3),(1→4)-β-d-glucans of different high initial molecular weights showed that initial molecular weight had no influence on the kinetics. Thus, this new methodology permits the determination of (1→3),(1→4)-β-d-glucanase activity in a direct way, i.e. the amount of (1→3),(1→4)-β-d-glucan degraded per amount of enzyme (or malt) per unit of time. Moreover, since it is insensitive to the initial molecular weight of the substrate, it seems to be well-suited for inter-laboratory comparisons of (1→3),(1→4)-β-d-glucanase activities.