KINETIC MEASUREMENTS OF CELLULASE ACTIVITY ON INSOLUBLE SUBSTRATES USING DISODIUM-2,2'-BICINCHONINATE

Determinations of cellulase activity using insoluble substrates have t ypically required long incubation times and significant amounts of enz yme. Utilizing disodium 2,2'-bicinchoninate, an assay has been develop ed to measure the initial production, of reducing groups released duri ng cellulase hydrolysis of insoluble, regenerated cellulose. The proce dure pel mits the determination of total, soluble and insoluble reduci ng groups from a single reaction mixture under initial velocity condit ions (< 3% hydrolysis). We report the applicability of this method to determinations of enzyme activity and for average molecular weight cal culations of substrates. The K-m values for Trichoderma reesei EG I, E G II, CBH I and CBH II are reported using phosphoric acid swollen cell ulose (average DP 166) as a substrate. Procedures for preparation of u niform cellulose suspensions are described.