Kinetic Measurements of Cellulase Activity on Insoluble Substrates Using Disodium 2,2' Bicinchoninate

Determinations of cellulase activity using insoluble substrates have typically required long incubation times and significant amounts of enzyme. Utilizing disodium 2,2′-bicinchoninate, an assay has been developed to measure the initial production of reducing groups released during cellulase hydrolysis of insoluble, regenerated cellulose. The procedure permits the determination of total, soluble and insoluble reducing groups from a single reaction mixture under initial velocity conditions (< 3% hydrolysis). We report the applicability of this method to determinations of enzyme activity and for average molecular weight calculations of substrates. The K m values for Trichoderma reesei EG I, EG II, CBH I and CBH II are reported using phosphoric acid swollen cellulose (average DP 166) as a substrate. Procedures for preparation of uniform cellulose suspensions are described.