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 Aspergillus aculeatus b y expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed crosslinked xyloglucan, T he cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus ex pression vector, and transformed into Aspergillus oryzae for heterologous e xpression. The recombinant enzyme was purified to apparent homogeneity by a nion-exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally sta ble at a pH of 3.4 and temperature below 30 degrees C, The enzyme hydrolyze s structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-spec ific endo-beta-1,4-glucanohydrolase. XEG hydrolyzes its substrates with ret ention of the anomeric configuration. The K-m of the recombinant enzyme is 3.6 mg/mi, and its specific activity is 260 mu mol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls wit h XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.