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Glucanases in Schizosaccharomyces

ISOLATION AND PROPERTIES OF THE CELL WALL-ASSOCIATED -(13)-GLUCANASES

From the ¹ From the Department of Food Science and Technology, University of California, Davis, California 95616

Cell walls of all species of the fission yeast Schizosaccharomyces underwent autohydrolysis in buffer at pH 5.0, releasing ß-(13)-linked oligosaccharides, variable amounts of glucose, and ß-(13)-glucanase activity. Schizosaccharomyces versatilis walls were the most active in this respect and were studied in detail. The most complete release of ß-glucanases (about 80% of the total activity) took place when the autolysis was done in dialysis sacs; in a closed system, enzyme release was considerably reduced. The released ß-glucanase activity was very stable and could be separated by diethylaminoethyl-chromatography into an endo-ß-(13)-glucanase (not adsorbed, 13% of the total activity) and an adsorbed fraction (87 %), which was recovered by elution and proved to be exo-ß-glucanase. The latter enzyme was extensively purified and had properties identical to those of the previously studied exo-ß-glucanase from cell extracts or the extracellular enzyme in the culture medium. The endo-ß-(13)-glucanase was purified by column chromatography until it was electrophoretically homogeneous with a molecular weight of 97,000. ß-(13)-linked glucans were hydrolyzed randomly to oligosaccharides, finally yielding laminaribiose and glucose. Laminaritriose was hydrolyzed much more rapidly than by endoglucanases from other organisms. The enzyme has an optimum pH at 5 to 6, a V_max of 5 µmoles of glucose equivalents released per min per mg of protein, and a K_m value of 0.33 mg of laminarin per ml. The enzyme demonstrated sigmoidal kinetics with two cooperative active sites per enzyme molecule. Glucono--lactone showed noncompetitive inhibition with a K_i of 95 mm. The enzyme did not contain any carbohydrate or phosphorus.

The exoglucanase had no effect on cell walls but the endoglucanase caused extensive lysis. Complete lysis was obtained if a bacterial -(13)-glucanase was added to the endo-ß-(13)-glucanase. No evidence was obtained for the presence of endo-ß-(16)- or endo--(13)-glucanases in Schizosaccharomyces. Endo-ß-(13)-glucanases were demonstrated also in the cell walls of a number of other species of yeast.

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