A xyloglucan-specific endo- -1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme

• DOI: 10.1093/glycob/9.1.93
• Publication Date: 2002-07-26T22:47:12Z
• Authors: M. Pauly; L. N. Andersen; S. Kauppinen; L. V. Kofod; W. S. York; P. Albersheim; A. Darvill
• Keywords: Xyloglucan; Aspergillus oryzae; Heterologous expression; Glucanase; Agarose

A full-length c-DNA encoding a xyloglucan-specific endo -β-1,4-glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed crosslinked xyloglucan. cDNA was isolated, sequenced, cloned into an vector, and transformed oryzae for heterologous expression. recombinant enzyme purified to apparent homogeneity anion-exchange gel permeation chromatography. molecular mass of 23,600, isoelectric point 3.4, is optimally stable at pH 3.4 temperature below 30°C. hydrolyzes structurally diverse xyloglucans various sources, but no other cell wall component can therefore be considered -b-1,4-glucanohydrolase. its substrates with retention anomeric configuration. K m 3.6 mg/ml, specific activity 260 µmol/min per mg protein. tested ability solubilize xyloglucan oligosaccharides plant walls. It shown that treatment walls yields only oligosaccharides, indicating this powerful tool structural elucidation xyloglucans.