title: Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases.
abstract: Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had kcat values of 33.3 and 61.3s(-1) and Km values for glucose of 33.4 and 27.9mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.
authors.affiliation: Center for Chemistry IHTM, University of Belgrade, Njegoševa 12, 11001 Belgrade, Serbia.
authors.forename: Marija
authors.initials: M
authors.lastname: Blazic
authors.forename: Gordana
authors.initials: G
authors.lastname: Kovacevic
authors.forename: Olivera
authors.initials: O
authors.lastname: Prodanovic
authors.forename: Raluca
authors.initials: R
authors.lastname: Ostafe
authors.forename: Marija
authors.initials: M
authors.lastname: Gavrovic-Jankulovic
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Radivoje
authors.initials: R
authors.lastname: Prodanovic
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics*; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D005949:Glucose Oxidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C543385:AGA2 protein, S cerevisiae; D015815:Cell Adhesion Molecules; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D005949:Glucose Oxidase