Papain digestion of crude Trichoderma reesei cellulase: purification and properties of cellobiohydrolase I and II core proteins.

The major cellulase components produced by Trichoderma reesei are composed of distinct catalytic and cellulose-binding domains. A simple two-step procedure is described for the purification of the catalytic domains, also termed core proteins (cp), of the major components, cellobiohydrolase (CBH) I and II. The novel aspect of this procedure is that native CBH I and II do not have to be purified initially. Papain digestion of a commercial T. reesei cellulase preparation followed by gel filtration on a Superdex 75 column resulted in the separation of fractions containing CBH I cp and CBH II cp; chromatofocusing purified the latter to homogeneity. N-terminal protein sequencing of CBH II cp provided good evidence for its identity. A comparison of the catalytic activity and cellulose-binding ability of these cp was made. A major difference between them was that CBH II cp bound to microcrystalline cellulose, unlike CBH I cp. CBH I cp readily hydrolysed the bond between the aglycone and cellobiose in p-nitrophenyl cellobioside unlike the CBH II cp preparation. Neither CBH I cp nor CBH II cp had activity toward carboxymethylcellulose, but both were able to hydrolyse barley beta-glucan. It was also shown that incubation of cellulose fibres with native CBH I, CBH I cp or CBH II cp resulted in a smoothing of the fibre surface.