title: Characterization of a recombinant α-glucuronidase from Aspergillus fumigatus.
abstract: The degradation of xylan requires the action of glycanases and esterases which hydrolyse, in a synergistic fashion, the main chain and the different substituents which decorate its structure. Among the xylanolytic enzymes acting on side-chains are the α-glucuronidases (AguA) (E.C. 3.2.1.139) which release methyl glucuronic acid residues. These are the least studies among the xylanolytic enzymes. In this work, the gene and cDNA of an α-glucuronidase from a newly isolated strain of Aspergillus fumigatus have been sequenced, and the gene has been expressed in Pichia pastoris. The gene is 2523 bp long, has no introns and codes for a protein of 840 amino acid residues including a putative signal peptide of 19 residues. The mature protein has a calculated molecular weight of 91,725 and shows 99 % identity with a putative α-glucuronidase from A. fumigatus A1163. The recombinant enzyme was expressed with a histidine tag and was purified to near homogeneity with a nickel nitriloacetic acid (Ni-NTA) column. The purified enzyme has a molecular weight near 100,000. It is inactive using birchwood glucuronoxylan as substrate. Activity is observed in the presence of xylooligosaccharides generated from this substrate by a family 10 endoxylanase and when a mixture of aldouronic acids are used as substrates. If, instead, family 11 endoxylanase is used to generate oligosaccharides, no activity is detected, indicating a different specificity in the cleavage of xylan by family 10 and 11 endoxylanases. Enzyme activity is optimal at 37 °C and pH 4.5-5. The enzyme binds cellulose, thus it likely possesses a carbohydrate binding module. Based on its properties and sequence similarities the catalytic module of the newly described α-glucuronidase can be classified in family 67 of the glycosyl hydrolases. The recombinant enzyme may be useful for biotechnological applications of α-glucuronidases.
authors.affiliation: Departamento de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago, Chile.
authors.forename: Lorena
authors.initials: L
authors.lastname: Rosa
authors.forename: María Cristina
authors.initials: MC
authors.lastname: Ravanal
authors.forename: Wladimir
authors.initials: W
authors.lastname: Mardones
authors.forename: Jaime
authors.initials: J
authors.lastname: Eyzaguirre
mesh_terms: D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D006026:Glycoside Hydrolases