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United States Department of Agriculture

Agricultural Research Service

Title: Preliminary purification and characterization of a xylnase activity from Aspergillus flavus

Authors
item Mellon, Jay
item Cotty, Peter

Submitted to: National Cotton Council Beltwide Cotton Conference
Publication Type: Proceedings
Publication Acceptance Date: January 10, 2007
Publication Date: August 10, 2007
Citation: Mellon, J.E., Cotty, P.J. 2007. Preliminary purification and characterization of a xylnase activity from Aspergillus flavus. Proceedings of the 2007 Beltwide Cotton Conference. p. 147-152.

Interpretive Summary: Aflatoxin is a very potent carcinogen and toxin that is produced by the fungus, Aspergillus flavus. When this fungus infects cotton plants, the developing seed can become contaminated with this toxin, rendering the product unusable for additional agricultural uses. Xyloglucans are important structural components of plant cell walls. Cottonseed contains high concentrations of xylans and is particularly susceptible to aflatoxin contamination. The fungus is capable of producing an enzyme, xylanase that breaks down xylans. This hydrolase activity presumably helps the fungus breach host cell walls and gain access to potential nutrient sources in cottonseed that include free sugars (raffinose), lipids, and storage proteins. In order to better understand the role of this enzyme in fungal virulence, a xylanase activity from a field isolate of A. flavus was partially purified and characterized. This A. flavus hydrolase is a low molecular weight, heat stable protein that is consistent with a highly mobile (diffusible), plant cell wall degrading factor. This research will benefit cotton breeders, producers, and pathologists, and will aid in the formulation of methods to prevent aflatoxin contamination of cottonseed.

Technical Abstract: Aspergillus flavus AF36 was found to secrete a xylanase activity when grown on a medium containing xylan as a sole carbon source. Enzyme activity was assayed using an agar medium containing Remazol Brilliant Blue R conjugated xylan as substrate. Positive hydrolytic activity of a test sample produced a diffusion clear zone emanating from the sample application site. A survey of several closely related fungi revealed secretion of a similar xylanase activity from A. nomius, A. oryzae, A. parasiticus, A. tamarii, but not from A. sojae. This xylanase was relatively thermostabile up to 60 ºC, but all activity was lost at equal to or greater than 66 ºC. The activity was tolerant of a wide pH range (4.0-8.0) with essentially no optimum. A wide range of protease inhibitors and EDTA exhibited no effect on the xylanase activity. A concentrated sample of the AF36 xylanase activity was subjected to gel filtration chromatography on a P-60 column. A small protein peak that coincided with a peak of xylanase activity eluted close to the included volume of the column. Data suggests this hydrolytic activity is associated with a low molecular weight (< 20 kD) thermostabile protein, and is consistent with a highly mobile (diffusible), plant cell wall degrading factor.

Last Modified: 4/23/2014
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