Purification and properties of an endo‐1,4‐xylanase excreted by a hydrolytic thermophilic anaerobe, Clostridium thermolacticum

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Abstract
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°C, a molecular mass of 39 kDa and a pI of 4.9. The enzyme exhibited maximal activity at 80°C (1 h assay) and at pH 6.0–6.5. There was little loss of activity after 4 days at 60°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.