DETERMINATION OF (1-]3),(1-]4)-BETA-D-GLUCANASE ACTIVITY BY A CALCOFLUOR-FLOW INJECTION-ANALYSIS METHOD

A new methodology for the determination of (1 --> 3),(1 --> 4)-beta-D- glucanase activity has been developed. The concentration decay curves corresponding to the depolymerisation of high molecular weight barley (1 --> 3),(1 --> 4)-beta-D-glucan by pure (1 --> 3),(1 --> 4)-beta-D-g lucanase (E.C. 3.2.1.73) from Bacillus subtilis and by crude (1 --> 3) , (1 --> 4)-beta-D-glucanase from different malts were monitored by th e Calcofluor-FIA method. In all cases, the high molecular weight (1 -- > 3),(1 --> 4)-beta-D-glucan decay curves fitted very well to an empir ical formula describing the change in substrate concentration with tim e. The curves possess an inflexion point at which the depolymerisation rate of the substrate reaches a maximum. This maximum depolymerisatio n rate correlates with the initial concentrations of enzyme and substr ate, E(0) and S-0, and the enzyme kinetic constants V-m and K-m throug h a hyperbola similar to that of Michaelis-Menten. The K-m determined for B. subtilis beta-glucanase was rather low, about 0.99 g beta-gluca n/l, when compared with those corresponding to (1 --> 3), (1 --> 4)-be ta-D-glucanase from different malts, which were, in turn, practically identical at about 2.92 g beta-glucan/l. Experiments with barley (1 -- > 3),(1 --> 4)-beta-D-glucans of different high initial molecular weig hts showed that initial molecular weight had no influence on the kinet ics. Thus, this new methodology permits the determination of(1 --> 3), (1 --> 4)-beta-D-glucanase activity in a direct way, i.e. the amount o f(1 --> 3),(1 --> 4)-beta-D-glucan degraded per amount of enzyme (or m alt) per unit of time. Moreover, since it is insensitive to the initia l molecular weight of the substrate, it seems to be well-suited for in ter-laboratory comparisons of(1 --> 3),(1 --> 4)-beta-D-glucanase acti vities. (C) 1995 Academic Press Limited