title: Identification of a major xylanase from Aspergillus flavus as a 14-kD protein.
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 ED(50) 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.
authors.affiliation: Southern Regional Research Center, Agricultural Research Service, USDA, P. O. Box 19687, New Orleans, LA 70179, USA. Jay.Mellon@ars.usda.gov
authors.forename: Jay E
authors.initials: JE
authors.lastname: Mellon
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Cotty
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Callicott
authors.forename: Hamed
authors.initials: H
authors.lastname: Abbas
mesh_terms: D001231:Aspergillus flavus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001236:Aspergillus oryzae / Q000235:genetics; D002850:Chromatography, Gel / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D013058:Mass Spectrometry; D008628:Mercury / Q000378:metabolism; D008970:Molecular Weight; D014357:Trypsin / Q000378:metabolism; D014990:Xylans / Q000378:metabolism
chemical_list: D003470:Culture Media; D004791:Enzyme Inhibitors; D014990:Xylans; D043364:Endo-1,4-beta Xylanases; D014357:Trypsin; D008628:Mercury