Ion-exchange chromatographic purification and quantitative analysis of Trichoderma reesei cellulases cellobiohydrolase I, II and endoglucanase II by fast protein liquid chromatography

Abstract

Trichoderma cellulases appear in several isoforms which makes their purification and analysis difficult. We used fast protein liquid chromatography (FPLC) to purify three major cellulases and to quantitate these enzymes in reconstituted mixtures during cellulose hydrolysis studies (in lack of specific substrates and because of the synergism between the enzymes such analysis is very difficult, if at all possible, with conventional activity measurements). For the analysis methods linear calibration was achieved from 10–15 pmol to 0.5–1 nmol (from 0.5–0.8 to 27–64 μg) for the different enzymes. Due to the high resolution chromatographic media used, our purification methods are simpler and quicker than the usual protocols for cellulase purification. Several isoforms of cellobiohydrolase (CBH) I were purified. The isoforms had different isoelectric points (p I) but their catalytic and adsorption properties were similar. A remarkable feature of CBH I and endoglucanase (EG) II was that their electrophoretically pure preparations gave double peaks during ion-exchange chromatography in certain pH intervals where the two peaks (probably representing two conformations) were transformed into each other by changing pH. This behaviour of cellulases has never been reported before and further explains the difficulties in cellulase purification.