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Molecular Plant Pathology

Chitinolytic Enzymes of Trichoderma harzianum: Purification of Chitobiosidase and Endochitinase. G. E. Harman,Departments of Horticultural Sciences and Plant Pathology, Cornell University, Geneva, NY 14456; C. K. Hayes(2), M. Lorito(3), R. M. Broadway(4), A. Di Pietro(5), C. Peterbauer(6), and A. Tronsmo(7). (2)(3)(5)(6)Departments of Horticultural Sciences and Plant Pathology, Cornell University, Geneva, NY 14456; (4)Department of Entomology, Cornell University, Geneva; (7)Department of Biotechnological Sciences, Agricultural University of Norway, Ås, Norway; (3)Permanent address: Istituto di Patologia Vegetale, Universitá degli Studi di Napoli, and Istituendo Centro CNR di Studio delle Tecniche di Lotta Biologica, 80055 Portici (NA) Italy; (5)Current address: Cátedra de Patologia Vegetal/ETSIAM, Universidad de Córdoba, Spain; (6)Permanent address: Institut für Biochemische Technologie and Mikrobiologie, Technische Universität Wien, Austria. Phytopathology 83:313-318. Accepted for publication 12 October 1992. Copyright 1993 The American Phytopathological Society. DOI: 10.1094/Phyto-83-313.

Trichoderma harzianum strain P1 produces a variety of chitinolytic enzymes including N-acetyl-?-d-glucosaminidases, chitin 1,4-?-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 ~ 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 ~ pH 4.0 and gradually declined as pH increased.