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- Title
Purification and characterization of an N-acetylglucosaminidase produced by a Trichoderma harzianum strain which controls Crinipellis perniciosa.
- Authors
De Marco, J. Lisboa; Valadares-Inglis, M. C.; Felix, C. R.
- Abstract
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 ρ-nitrophenyl-N-acetylglucosaminide (ρNGlcNAc) with Michaelis–Menten kinetics. Maximal activities were determined at pH 4.0 and a temperature range of 50–60°C. Km and Vmax values for ρNGlcNAc hydrolysis were 8.06 μmoles ml-1 and 3.36 μmoles ml-1 min-1, respectively, at pH 6.0 and 37°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.
- Subjects
TRICHODERMA; FUNGI; PHYTOPATHOGENIC microorganisms; CHITIN; ENZYMES
- Publication
Applied Microbiology & Biotechnology, 2004, Vol 64, Issue 1, p70
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-003-1490-5