p-Aminophenyl β-cellobioside as an affinity ligand for exo-type cellulases

p-Aminophenyl β-cellobioside (PAPC) is shown to be an effective affinity ligand for the chromatographic fractionation of cellobiohydrolases (CBHs). A crude cellulase preparation from the filamentous fungus Trichoderma reesei served as a representative source of enzymes for this study. Prior to chromatography, PAPC was tethered via its amino functional group to N-hydroxysuccinimide-activated agarose. The resulting affinity matrix specifically retained the CBH component of relatively complex cellulase mixtures. The purity of the resulting CBH preparations, based on measured specific activities, was comparable to that of corresponding enzyme preparations obtained using more traditional thioglycoside-based affinity ligands. The application of PAPC as an affinity ligand illustrates that the tethered ligand associates with the T. reesei CBHs in a catalytically nonproductive mode. In contrast, the free ligand is readily hydrolyzed by T. reesei CBH I.