PURIFICATION, CHARACTERIZATION, AND SYNERGISTIC ACTIVITY OF A GLUCAN 1,3-BETA-GLUCOSIDASE AND AN N-ACETYL-BETA-GLUCOSAMINIDASE FROM TRICHODERMA-HARZIANUM

A glucan 1,3-beta-glucosidase (EC 3.2.1.58) and an N-acetyl-beta-gluco saminidase (EC 3.2.1.30) were purified to homogeneity from the culture filtrate of Trichoderma harzianum strain P1. The molecular masses and the pis were 78 kDa and 6.2, respectively, for the glucan 1,3-beta-gl ucosidase and 72 kDa and 4.6, respectively, for the N-acetyl-beta-gluc osaminidase. The glucan 1,3-beta-glucosidase and the N-acetyl-beta-glu cosaminidase were tested against Botrytis cinerea, and their antifunga l activity was compared with that obtained for an endochitinase and a chitin 1,4-beta-chitobiosidase also purified from T. harzianum strain P1. The four cell wall-degrading enzymes were also tested as mixtures containing two, three, or all four proteins in all possible combinatio ns. A synergistic, inhibitory effect was detected on both spore germin ation and germ tube elongation of B. cinerea when two, three, or four enzymes were applied together. The highest level of antifungal activit y was obtained when a solution containing four different cell wall-deg rading enzymes was used. ED(50) (50% effective dose) values were as lo w as 1.6 mu g ml(-1) for inhibition of conidial germination and 1.7 mu g ml(-1) for inhibition of germ tube elongation of the surviving spor es.