title: Recombinant Aspergillus β-galactosidases as a robust glycomic and biotechnological tool.
abstract: Galactosidases are widespread enzymes that are used for manifold applications, including production of prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in various analytical approaches. The nature of these applications often require galactosidases to be present in a purified form with clearly defined properties, including precisely determined substrate specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In this study, we present the recombinant expression and purification of two previously uncharacterized β-galactosidases from Aspergillus nidulans as well as one β-galactosidase from Aspergillus niger. All enzymes were active toward p-nitrophenyl-β-D-galactopyranoside as substrate and displayed similar temperature and pH optima. The purified recombinant galactosidases digested various complex substrates containing terminal galactose β-1,4 linked to either N-acetylglucosamine or fucose, such as N-glycans derived from bovine fibrin and Caenorhabditis elegans. In our comparative study of the recombinant galactosidases with the commercially available galactosidase from Aspergillus oryzae, all enzymes also displayed various degrees of activity toward complex oligosaccharides containing β-1,3-linked terminal galactose residues. All recombinant enzymes were found to be robust in the presence of various organic solvents, temperature variations, and freeze/thaw cycles and were also tested for their ability to synthesize galactooligosaccharides. Furthermore, the use of fermentors considerably increased the yield of recombinant galactosidases. Taken together, we demonstrate that purified recombinant galactosidases from A. niger and from A. nidulans are suitable for various glycobiological and biotechnological applications.
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, Vienna, 1190, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Dragosits
authors.forename: Stefan
authors.initials: S
authors.lastname: Pflügl
authors.forename: Simone
authors.initials: S
authors.lastname: Kurz
authors.forename: Ebrahim
authors.initials: E
authors.lastname: Razzazi-Fazeli
authors.forename: Iain B H
authors.initials: IB
authors.lastname: Wilson
authors.forename: Dubravko
authors.initials: D
authors.lastname: Rendic
mesh_terms: D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D054794:Glycomics / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D001616:beta-Galactosidase
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