Purification and properties of an endo-1,4-xylanase excreted by a hydrolytic thermophilic anaerobe, Clostridium thermolacticum. A proposal for its action mechanism on larchwood 4-O-methylglucuronoxylan.
An extracellular xylanase from a thermophilic anaerobe, Clostridium thermolacticum, was purified 400-fold by ion-exchange chromatography and gel filtration. The purified enzyme had a specific activity of 31,670 nkat/mg of protein at 60 degrees C, a molecular mass of 39 kDa and a pI of 4.9. The enzyme exhibited maximal activity at 80 degrees C (1 h assay) and at pH 6.0-6.5. There was little loss of activity after 4 days at 60 degrees C and the enzyme was stable in the wide pH range 3-11. Examination of the hydrolysis products of larchwood xylan indicated that it was an endoxylanase; at the early stage of the reaction, xylose (Xyl)-containing oligosaccharides of 3-12 residues were released and after a prolonged incubation time, the neutral end-products were Xyl2 and Xyl3. Kinetic studies of the hydrolysis of xylose-containing oligosaccharides of 4-7 residues showed that the tetrasaccharide was hydrolysed more slowly than the pentasaccharide, while the calculated Km and V values for pentasaccharide and hexasaccharide were similar. The primary structures of the XylnGlcA produced by long-term hydrolysis of larchwood glucuronoxylan were determined on the basis of their carbohydrate composition, by methylation analysis and by 1H-NMR and 13C-NMR spectroscopies. These data allowed us to propose a model for the mode of action of this endoxylanase on larchwood 4-O-methylglucuronoxylan.
Debeire
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P
Station de Technologie Alimentaire, Institut National de la Recherche Agronomique, Villeneuve d'Ascq, France.
Priem
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B
Strecker
G
G
Vignon
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M
D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases; D006026:Glycoside Hydrolases / Q000302:isolation & purification*; D006868:Hydrolysis; D007525:Isoelectric Focusing; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D014990:Xylans / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism; D014995:Xylosidases / Q000302:isolation & purification* / Q000494:pharmacology
D009844:Oligosaccharides; D011134:Polysaccharides; D014990:Xylans; C044967:4-O-methyl glucuronoxylan; D014994:Xylose; D006026:Glycoside Hydrolases; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases