CHITINOLYTIC ENZYMES OF TRICHODERMA-HARZIANUM - PURIFICATION OF CHITOBIOSIDASE AND ENDOCHITINASE

Trichoderma harzianum strain P1 produces a variety of chitinolytic enzymes including N-acetyl-beta-D-glucosaminidases, chitin 1,4-beta-chitobiosidases, and an endochitinase. Chitobiosidases and an endochitinase were purified from dialyzed, concentrated culture filtrates using gel filtration, chromatofocusing, and isoelectric focusing. Three protein bands were evident in the purified chitobiosidase preparation, representing different levels of N-glycosylation of the same protein. The pI of all purified proteins was approximately 3.9. The molecular mass of the principal glycosylated chitobiosidase was 40 kDa, the deglycosylated form was 35 kDa, and the endochitinase was 41 kDa. The chitobiosidases did not react with a polyclonal antibody prepared against the endochitinase, nor did the endochitinase react with an antibody prepared against the chitobiosidase. The endochitinase and the chitobiosidase had broad pH optima. The larger glycosylated chitobiosidase had a 4.0-7.0 pH optimum. The endochitinase optimum activity was at approximately pH 4.0 and gradually declined as pH increased.