title: Engineering of pyranose dehydrogenase for increased oxygen reactivity.
abstract: Pyranose dehydrogenase (PDH), a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organo)metals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of Agaricus meleagris PDH were expressed in Saccharomyces cerevisiae. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in Pichia pastoris and characterized and revealed a five-fold increase of the oxygen reactivity.
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Iris
authors.initials: I
authors.lastname: Krondorfer
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Austria; University of Applied Sciences Wiener Neustadt - Campus Tulln, Tulln, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Lipp
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Brugger
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Petra
authors.initials: P
authors.lastname: Staudigl
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Sygmund
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Clemens K
authors.initials: CK
authors.lastname: Peterbauer
mesh_terms: D000364:Agaricus / Q000201:enzymology*; D002237:Carbohydrate Dehydrogenases / Q000378:metabolism*; D020134:Catalytic Domain; D011232:Chemical Precipitation; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D005818:Genetic Engineering / Q000379:methods*; D057166:High-Throughput Screening Assays; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; D010100:Oxygen