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

• Published in: Applied microbiology and biotechnology 2004-03, Vol.64 (1), p.70-75
• Main Authors: LISBOA DE MARCO, J, VALADARES-INGLIS, M. C, FELIX, C. R
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - analogs & derivatives; Acetylglucosamine - metabolism; Acetylglucosaminidase - isolation & purification; Acetylglucosaminidase - metabolism; Agaricales - cytology; Agaricales - growth & development; Agaricales - metabolism; Agaricales - ultrastructure; Bacteria; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Cell Wall - metabolism; Chitin; Chitin - metabolism; Chromatography, Gel; Coenzymes - analysis; Crinipellis perniciosa; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors - pharmacology; Enzyme Stability; Enzymes; Fundamental and applied biological sciences. Psychology; Glucose - pharmacology; Hydrogen-Ion Concentration; Hydrolysis; Hydrophobic and Hydrophilic Interactions; Kinetics; Metals - metabolism; Microbiology; Microscopic analysis; Miscellaneous; Mission oriented research; Molecular Weight; Mycelium - metabolism; Pest Control, Biological - methods; Temperature; Trichoderma - enzymology; Trichoderma - growth & development; Trichoderma harzianum
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-003-1490-5

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 (rhoNGlcNAc) with Michaelis-Menten kinetics. Maximal activities were determined at pH 4.0 and a temperature range of 50-60 degrees C. Km and Vmax values for rhoNGlcNAc hydrolysis were 8.06 micromoles ml(-1) and 3.36 micromoles ml(-1) min(-1), respectively, at pH 6.0 and 37 degrees C. 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.