A practical approach to steady-state kinetic analysis of cellulases acting on their natural insoluble substrate

Measurement of steady-state rates (v(ss)) is straightforward in standard enzymology with soluble substrate, and it has been instrumental for comparative biochemical analyses within this area. For insoluble substrate, however, experimental values of v(ss) remain controversial, and this has strongly limited the amount and quality of comparative analyses for cellulases and other enzymes that act on the surface of an insoluble substrate. In the current work, we have measured progress curves over a wide range of conditions for two cellulases, TrCel6A and TrCel7A from Trichoderma reesei, acting on their natural, insoluble substrate, cellulose. Based on this, we consider practical compromises for the determination of experimental v(ss) values, and propose a basic protocol that provides representative reaction rates and is experimentally simple so that larger groups of enzymes and conditions can be readily assayed with standard laboratory equipment. We surmise that the suggested experimental approach can be useful in comparative biochemical studies of cellulases; an area that remains poorly developed.