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 (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.
Lisboa De Marco
J
J
Departamento de Biologia Celular, Universidade de Brasília, 70.910-900 Brasília DF, Brazil.
Valadares-Inglis
M C
MC
Felix
C R
CR
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000302:isolation & purification* / Q000378:metabolism*; D000363:Agaricales / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism / Q000648:ultrastructure; D002473:Cell Wall / Q000378:metabolism; D002686:Chitin / Q000378:metabolism; D002850:Chromatography, Gel; D003067:Coenzymes / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D025282:Mycelium / Q000378:metabolism; D010572:Pest Control, Biological / Q000379:methods; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology* / Q000254:growth & development
D003067:Coenzymes; D004791:Enzyme Inhibitors; D008670:Metals; D002686:Chitin; D000118:Acetylglucosaminidase; D005947:Glucose; D000117:Acetylglucosamine