TAILIEUCHUNG - Báo cáo khoa học: Improving thermostability and catalytic activity of pyranose 2-oxidase from Trametes multicolor by rational and semi-rational design

The fungal homotetrameric flavoprotein pyranose 2-oxidase (P2Ox; EC ) catalyses the oxidation of various sugars at position C2, while, concomitantly, electrons are transferred to oxygen as well as to alternative electron acceptors (. oxidized ferrocenes). | Improving thermostability and catalytic activity of pyranose 2-oxidase from Trametes multicolor by rational and semi-rational design Oliver Spadiut1 Christian Leitner1 Clara Salaheddin1 Balazs Varga2 Beata G. Vertessy2 Tien-Chye Tan3 Christina Divne3 and Dietmar Haltrich1 1 Department of Food Sciences and Technology BOKU-University of NaturalResources and Applied Life Sciences Vienna Austria 2 Institute of Enzymology BiologicalResearch Center Hungarian Academy of Sciences Budapest Hungary 3 Schoolof Biotechnology RoyalInstitute of Technology KTH Albanova University Center Stockholm Sweden Keywords enzyme engineering pyranose oxidase stability stabilization subunit interaction Correspondence D. Haltrich Department of Food Sciences and Technology Universitat fur Bodenkultur Wien Muthgasse 18 A-1190 Wien Austria Fax 43 1 36006 6251 Tel 43 1 36006 6275 E-mail Database Structuraldata are available in the Protein Data Bank under the accession numbers 3BG6 3BG7 and 3BLY Received 25 June 2008 revised 19 November 2008 accepted 1 December 2008 doi The fungal homotetrameric flavoprotein pyranose 2-oxidase P2Ox EC catalyses the oxidation of various sugars at position C2 while concomitantly electrons are transferred to oxygen as well as to alternative electron acceptors . oxidized ferrocenes . These properties make P2Ox an interesting enzyme for various biotechnological applications. Random mutagenesis has previously been used to identify variant E542K which shows increased thermostability. In the present study we selected position Leu537 for saturation mutagenesis and identified variants L537G and L537W which are characterized by a higher stability and improved catalytic properties. We report detailed studies on both thermodynamic and kinetic stability as well as the kinetic properties of the mutational variants E542K E542R L537G and L537W and the respective double mutants L537G E542K L537G E542R L537W .

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