Purification and characterisation of a beta-D-xylosidase from the anaerobic rumen fungus Neocallimastix frontalis.
A beta-D-xylosidase from the anaerobic rumen fungus Neocallimastix frontalis was purified by anion-exchange and gel filtration chromatography. The enzyme was isoelectrically homogeneous and had an isoelectric point of pH 4.6. The apparent molecular mass calculated by gel filtration was 150,000 Da. Under denaturing conditions, the enzyme appeared as a dimer composed of two polypeptides with molecular masses of 83,000 and 53,000 Da. The pH and temperature optimum were 6.4 and 37 degrees C, respectively: the activity was very sensitive to temperature. The enzyme was inhibited by copper, silver and zinc ions, EDTA and SDS, and was stimulated by calcium and magnesium ions. It was competitively inhibited by D-xylose with an apparent Ki of 3.98 mM. The beta-D-xylosidase exhibited hydrolytic activity on xylobiose and xylo-oligosaccharides of dp up to 7: the specific activities and maximum velocities decreased as the chain length increased. Analysis of the products of hydrolysis by HPLC indicated a typical exo-action. A mixture of beta-D-xylosidase and a xylanase acted synergistically in producing high reducing sugar values, using a xylan from oat spelts.
Rowett Research Institute, Bucksburn, Aberdeen, United Kingdom.
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Garcia-Campayo
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Wood
D000818:Animals; D002480:Cellulase / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D008411:Chytridiomycota / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D012417:Rumen / Q000382:microbiology*; D013379:Substrate Specificity; D014995:Xylosidases / Q000302:isolation & purification / Q000378:metabolism*; D001617:beta-Glucosidase / Q000302:isolation & purification / Q000378:metabolism
D009844:Oligosaccharides; D014995:Xylosidases; D001617:beta-Glucosidase; C026579:exo-1,4-beta-D-xylosidase; D002480:Cellulase