Endochitinase, a mannan-associated enzyme from Saccharomyces cerevisiae

A chitinase was extracted with digitonin from intact yeast cells and purified by adsorption-digestion on chitin. The purified enzyme liberates oligosaccharides of various sizes from chitin, thus behaving as an endochitinase. As found with other chitinases, the yeast enzyme is much more active on nascent chitin, i.e. the chitin formed in the same reaction mixture by the corresponding synthetase, than on preformed polysaccharide. The enzyme has a very low pH optimum, about pH 2.5, and is quite stable at pH 3. Polyacrylamide gel electrophoresis of different preparations of purified chitinase revealed a variable number of protein bands, whose pattern often changed after storage of the enzyme. The distribution of activity in the gel matched that of the stainable material. Association of the chitinase with mannan is indicated by the following results: (a) coincidence in Coomassie blue-staining and periodate-Schiff-staining bands after disc gel electrophoresis; (b) adsorption of the activity on concanavalin A-Sepharose and elution with alpha-methylmanoside; (c) precipitation of the chitinase with appropriate antimannan sera. A carbohydrate content of approximately 18% was found in a trichloroacetic acid-precipitated sample of purified enzyme. Protein and mannan were not dissociated by boiling with sodium dodecyl sulfate, urea, and beta-mercaptoethanol. It could not, however, be conclusively established whether protein and carbohydrate are covalently linked, because the chitinase is resistant to endo-beta-N-acetyl-glucosaminidase.