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

Authors

  • Philippe DEBEIRE,

    Corresponding author
    1. Station de Technologie Alimentaire, Institut National de la Recherche Agronomique, Villeneuve d'Ascq, France
    Search for more papers by this author
  • Bernard PRIEM,

    1. Station de Technologie Alimentaire, Institut National de la Recherche Agronomique, Villeneuve d'Ascq, France
    Search for more papers by this author
  • Gerard STRECKER,

    1. Université des Sciences et Techniques de Lille Flandres-Artois, Laboratoire de Chimie Biologique (Unité Mixte de Recherches du Centre National de la Recherche Scientifique no. 111), Villeneuve d'Ascq, France
    Search for more papers by this author
  • Michel VIGNON

    1. Centre de Recherches sur les Macromolécules Végétales, Centre National de la Recherche Scientifique, Grenoble, France
    Search for more papers by this author

Correspondence to P. Debeire, Station de Technologie Alimentaire, Institut Natinal de la Recherche Agronomique, BP 39, F-59651 Villenuve d'Ascqu Cédex, France

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.

Abbreviations
Xyl

xylose

GlcA

glucuronic acid

Enzymes
 

Endo-1,4-β-xylanase (EC 3.2.1.8)

 

β-xylosidase (EC 3.2.1.37)

 

sorbitol dehydrogenase (EC 1.1.1.14)

Ancillary