Purification and characterization of an extracellular exochitinase, beta-N-acetylhexosaminidase, from the fungal mycoparasite Stachybotrys elegans.
The mycoparasite Stachybotrys elegans produces two exo- and one endo-acting chitinases when grown on chitin. We purified to homogeneity one of the exo-acting chitinases, beta-N-acetylhexosaminidase and partially characterized its physical and biochemical properties. The native enzyme has a molecular mass of 120 kDa when determined by gel filtration and 68 kDa by sodium dodecyl sulfate - polyacrylamide gel electrophoresis indicating that the native protein probably occurs as a dimer in solution. The purified beta-N-acetylhexosaminidase is most active at pH 5.0 and 40 degrees C and hydrolyzes the p-nitrophenyl-N-acetyl-beta-D-glucosaminide with apparent Km of 84.6 microM. Polyclonal antibodies raised against the 68-kDa beta-N-acetylhexosaminidase (NAG-68) indicated that the antibody is highly specific and recognizes the protein in crude filtrate preparation. This suggests that the protein is a not a proteolytic product of another protein. Western blot analysis showed that the activity of NAG-68 was induced when S. elegans was grown on purified cell wall fragments of its host, Rhizoctonia solani, as well as during antagonistic interaction of the mycoparasite and host when both were grown on synthetic medium with or without supplemental carbon source.
Taylor
Greg
G
Department of Plant Science, McGill University, Montréal, QC, Canada.
Jabaji-Hare
Suha
S
Charest
Pierre M
PM
Khan
Wajahatullah
W
D000818:Animals; D015153:Blotting, Western; D002688:Chitinases / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011817:Rabbits; D013191:Stachybotrys / Q000201:enzymology* / Q000502:physiology; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases / Q000302:isolation & purification* / Q000378:metabolism
D002688:Chitinases; D001619:beta-N-Acetylhexosaminidases