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

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4-glucanase (XEG) has been isolated from the filamentous fungus Aspergillusaculeatus by expression cloning in yeast. The colonies expressingfunctional XEG were identified on agar plates containing azurine-dyedcross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced,cloned into an Aspergillus expression vector, and transformed intoAspergillus oryzae for heterologous expression. The recombinant enzyme waspurified to apparent homogeneity by anion- exchange and gel permeationchromatography. The recombinant XEG has a molecular mass of 23,600, anisoelectric point of 3.4, and is optimally stable at a pH of 3.4 andtemperature below 30 degreesC. The enzyme hydrolyzes structurally diversexyloglucans from various sources, but hydrolyzes no other cell wallcomponent and can therefore be considered a xyloglucan-specific endo-beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retentionof the anomeric configuration. The Kmof the recombinant enzyme is 3.6mg/ml, and its specific activity is 260 micromol/min per mg protein. Theenzyme was tested for its ability to solubilize xyloglucan oligosaccharidesfrom plant cell walls. It was shown that treatment of plant cell walls withXEG yields only xyloglucan oligosaccharides, indicating that this enzymecan be a powerful tool in the structural elucidation of xyloglucans.