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Purification and characterization of an extracellular exochitinase, -N-acetylhexosaminidase, from the fungal mycoparasite Stachybotrys elegans

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Abstract:

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, -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 -N-acetylhexosaminidase is most active at pH 5.0 and 40°C and hydrolyzes the -nitrophenyl-N-acetyl--D-glucosaminide with apparent Km of 84.6 µM. Polyclonal antibodies raised against the 68-kDa -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.Key words: chitinases, protein purification, mycoparsite, Rhizoctonia solani.

Le mycoparasite Stachybotrys elegans produit deux exo- et une endo-chitinases lorsqu'il est cultivé en présence de chitine. Nous avons purifié jusqu'à homogénéité une des exochitinases, la -N-acétylhexosaminidase, et caractérisé partiellement ses propriétés physiques et biochimiques. Par filtration en gel, l'enzyme d'origine a un poids moléculaire de 120 kDa et de 68 kDa par dodécyl sulfate de sodium electrophorése sur gel de polyacrylamide, ce qui suggère que la protéine originale se retrouve probablement sous forme d'un dimère en solution. La -N-acétylhexosaminidase purifiée a une activité maximale à pH 5,0 et à 40°C, et elle hydrolyse la p-nitrophényl -N-acétyl--D-glucosaminide selon un Km apparent de 84,6 µM. Des anticorps polyclonaux dirigés contre la -N-acétylhexosaminidase de 68 kDa (NAG-68) ont révélé que l'anticorps était très spécifique et qu'il reconnaissait la protéine dans un filtrat brut. Ces observations suggèrent que cette protéine n'est pas un produit de la protéolyse d'une autre protéine. Une analyse en Western blot a confirmé que l'activité de NAG-68 était induite lorsque S. elegans était cultivé sur des fragments purifiés de la paroi cellulaire de son hôte, le Rhizoctonia solani, ainsi que durant l'interaction antagoniste de ce mycoparasite et de son hôte lorsque les deux étaient cultivés sur un milieu synthétique avec ou sans apport supplémentaire d'une source de carbone.Mots clés : chitinase, purification de protéines, mycoparasite, Rhizoctonia solani.[Traduit par la Rédaction]

Keywords: Rhizoctonia solani; chitinase; chitinases; mycoparasite; mycoparsite; protein purification; purification de protéines

Document Type: Research Article

Publication date: April 1, 2002

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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