Purification and characterization of an N-acetylglucosaminidase produced by a Trichoderma harzianum strain which controls Crinipellis perniciosa

Isolate 1051 of Trichoderma harzianum, a mycoparasitic fungus, was found to impair development of the phytopathogen, Crinipellis perniciosa, in the field. This Trichoderma strain growing in liquid medium containing chitin produced substantial amounts of chitinases. The N-acetylglucosaminidase present in the culture-supernatant was purified to homogeneity by gel filtration and hydrophobic interaction chromatography, as demonstrated by SDS-PAGE analysis. The enzyme had a molecular mass of 36 kDa and hydrolyzed the synthetic substrate rho-nitrophenyl-N-acetylglucosaminide (rhoNG1cNAc) with Michaelis-Menten kinetics. Maximal activities were determined at pH 4.0 and a temperature range of 50-60degreesC. K-m and V-max values for rhoNG1cNAc hydrolysis were 8.06 mumoles ml(-1) and 3.36 mumoles ml(-1) min(-1), respectively, at pH 6.0 and 37degreesC. The enzyme was very sensitive to Fe3+, Mn2+ and Co2+ ions, but less sensitive to Zn2+, Al3+, Cu2+ and Ca2+. Glucose at a final concentration of 1 mM inhibited 65% of the original activity of the purified enzyme. Determination of the product (reducing sugar) of hydrolysis of C. perniciosa mycelium and scanning electron microscopic analysis revealed that the N-acetylglucosaminidase hydrolyses the C. perniciosa cell wall.