D-xylan-degrading enzyme system from the fungus Phanerochaete chrysosporium: isolation and partial characterisation of an alpha-(4-O-methyl)-D-glucuronidase.
A number of fungi were screened for their capacities to produce extracellular alpha-(4-O-methyl)-D-glucuronidase. Of those tested, Phanerochaete chrysosporium ATCC 24725 produced the enzyme in greatest yield. The single alpha-(4-O-methyl)-D-glucuronidase produced by this fungus was purified by a series of chromatographic methods involving anion exchange, hydrophobic interaction and chromatofocusing. Isolated in this way, the enzyme had an apparent molecular mass of 112 kDa in sodium dodecyl sulphate polyacrylamide gels, and a pI of 4.6 when determined by isoelectric focusing in polyacrylamide gels. The enzyme was optimally active at pH 3.5, but showed significant activity over the pH range 3-5. In the absence of substrate the enzyme was inactivated at pH 3.5 in 2 h at 50 degree C: at pH 5.0 it retained 42% of its activity for 24 h at this temperature. The enzyme showed little activity on glucuronoxylan polysaccharides, but some short-chain xylo-oligosaccharides which were substituted with alpha-linked 4-O-methyl-D-glucopyranosyl uronic acid attached to the 2-position of the non-reducing D-xylopyranosyl residue were readily hydrolysed. There were marked synergistic effects apparent in the release of 4-O-methyl-D-glucopyranosyl uronic acid from various glucuronoxylans when the alpha-(4-O-methyl)-D-glucuronidase was acting in concert with endo-(1-->4)-beta-D-xylanase, and with beta-D-xylosidase and/or an alpha-L-arabinofuranosidase.
Castanares
A
A
Rowett Research Institute, Bucksburn, Aberdeen, UK.
Hay
A J
AJ
Gordon
A H
AH
McCrae
S I
SI
Wood
T M
TM
D001487:Basidiomycota / Q000201:enzymology*; D001673:Biodegradation, Environmental; D001709:Biotechnology; D002240:Carbohydrate Sequence; D005658:Fungi / Q000201:enzymology; D005966:Glucuronidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D013379:Substrate Specificity; D014990:Xylans / Q000737:chemistry / Q000378:metabolism*
D009844:Oligosaccharides; D014990:Xylans; C098344:alpha-(4-O-methyl)-D-glucuronidase; D005966:Glucuronidase