title: Heterologous expression and biochemical characterization of novel pyranose 2-oxidases from the ascomycetes Aspergillus nidulans and Aspergillus oryzae.
abstract: A gene encoding a pyranose 2-oxidase (POx; pyranose/oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) was identified in the genome of the ascomycete Aspergillus nidulans. Attempts to isolate POx directly from A. nidulans cultures or to homologously overexpress the native POx (under control of the constitutive gpdA promoter) in A. nidulans were unsuccessful. cDNA encoding POx was synthesized from mRNA and expressed in Escherichia coli, and the enzyme was subsequently purified and characterized. A putative pyranose 2-oxidase-encoding gene was also identified in the genome of Aspergillus oryzae. The coding sequence was synthetically produced and was also expressed in E. coli. Both purified enzymes were shown to be flavoproteins consisting of subunits of 65 kDa. The A. nidulans enzyme was biochemically similar to POx reported in literature. From all substrates, the highest catalytic efficiency was found with D-glucose. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones and 2,6-dichloroindophenol. As judged by the catalytic efficiencies (k (cat)/k(m)), some of these quinone electron acceptors are better substrates for pyranose oxidase than oxygen. The enzyme from A. oryzae was physically similar but showed lower kinetic constants compared to the enzyme from A. nidulans. Distinct differences in the stability of the two enzymes may be attributed to a deletion and an insertion in the sequence, respectively.
authors.affiliation: Food Biotechnology Lab, Department of Food Sciences and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Ines
authors.initials: I
authors.lastname: Pisanelli
authors.forename: Petra
authors.initials: P
authors.lastname: Wührer
authors.forename: Yazmid
authors.initials: Y
authors.lastname: Reyes-Dominguez
authors.forename: Oliver
authors.initials: O
authors.lastname: Spadiut
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.forename: Clemens
authors.initials: C
authors.lastname: Peterbauer
mesh_terms: D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D016227:Benzoquinones / Q000378:metabolism; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D016227:Benzoquinones; D018076:DNA, Complementary; D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase