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°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. Copyright © 1990, Wiley Blackwell. All rights reserved