Abstract
Aspergillus flavus K49 secreted at least two xylanase activities when grown on a medium containing larch (wood) xylan as a sole carbon source. Enzyme activity was assayed using an agar medium containing Remazol Brilliant Blue R conjugated oat spelt xylan as substrate. Crude enzyme preparations were inhibited by Hg+2, with an ED50 of 17.5 mM and maximum inhibition of 83% at 50 mM. A concentrated sample of A. flavus K49 xylanase preparation was subjected to gel filtration chromatography on a P-30 column. A small protein peak coinciding with the major peak of xylanase activity was separated from the other secreted fungal proteins. An additional peak of xylanase activity was observed in fractions containing multiple fungal proteins. Analysis by denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of fractions containing the smaller molecular weight xylanase revealed a major and minor protein band in the vicinity of 14 kD. Analysis of these same fractions by acidic native PAGE revealed a single band. Confirmation of identity for the isolated xylanase was provided by isolation of a protein band from a SDS–PAGE gel, followed by trypsin digestion/analysis by tandem mass spectrometry. Comparison of the peptide library derived from this protein band with sequence data from the A. oryzae genomic data base provided a solid match with an endo-1,4-β-xylanase, XlnA. This identification is consistent with a low molecular weight protein associated with the major xylanolytic activity. XlnA may be a highly mobile (diffusible), plant wall hemicellulose degrading factor with significant activity during plant infection.
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Acknowledgments
The authors thank Bobbie J. Johnson for excellent technical assistance, and Dr. George Tsaprailis and the Arizona Proteomics Consortium of the University of Arizona for their assistance in conducting the MS analysis of the 14-kD protein. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.
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Mellon, J.E., Cotty, P.J., Callicott, K.A. et al. Identification of a Major Xylanase from Aspergillus flavus as a 14-kD Protein. Mycopathologia 172, 299–305 (2011). https://doi.org/10.1007/s11046-011-9425-7
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DOI: https://doi.org/10.1007/s11046-011-9425-7