A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme
| Autor: | Lene Venke Kofod, William S. York, Alan G. Darvill, Sakari Kauppinen, Lene Nonboe Andersen, Markus Pauly, Peter Albersheim |
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| Rok vydání: | 1999 |
| Předmět: |
DNA
Complementary Aspergillus oryzae Molecular Sequence Data Gene Expression Biochemistry law.invention Substrate Specificity chemistry.chemical_compound Cellulase law Cell Wall Polysaccharides Enzyme Stability Amino Acid Sequence Isoelectric Point Glucans Chromatography Expression vector biology Chemistry Aspergillus aculeatus Plants biology.organism_classification Yeast Recombinant Proteins Xyloglucan Molecular Weight Aspergillus Expression cloning Recombinant DNA Xylans Heterologous expression Sequence Alignment |
| Zdroj: | Glycobiology. 9(1) |
| ISSN: | 0959-6658 |
| Popis: | A full-length c-DNA encoding a xyloglucan-specific endo -beta-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 cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion-exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/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 with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans. |
| Databáze: | OpenAIRE |
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