A NEW TOOL FOR STUDYING THE MOLECULAR ARCHITECTURE OF THE FUNGAL CELL-WALL - ONE-STEP PURIFICATION OF RECOMBINANT TRICHODERMA BETA-(L-6)-GLUCANASE EXPRESSED IN PICHIA-PASTORIS

The fungal cell wall is a supramolecular network of glycoproteins and polysaccharides. Its analysis is seriously hampered by the lack of eas ily available hydrolytic enzymes in a pure form. Here we describe a si mple and efficient purification procedure of a recombinant beta-(1-6)- glucanase from Trichoderma harzianum expressed in Pichia pastoris. Tra nsformed cells efficiently secreted the enzyme into the induction medi um. We purified the enzyme using a one-step method based on hydrophobi c interaction chromatography. The yield was 80%. SDS-PAGE of the purif ied enzyme revealed a single band with an apparent molecular mass of 4 3 kDa. The isoelectric point of the enzyme was 5.8, and it showed maxi mal enzyme activity and stability at pH 5.0. As beta-(1-6)-glucan is a n important component of fungal cell walls, the easy availability of p ure beta-(1-6)-glucanase will highly facilitate studies of the molecul ar organization of the fungal cell wall. (C) 1998 Elsevier Science B.V . All rights reserved.