title: Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2stability.
abstract: Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H2O2. By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H2O2stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H2O2, the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H2O2incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada.
authors.forename: Thu V
authors.initials: TV
authors.lastname: Vuong
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada.
authors.forename: Maryam
authors.initials: M
authors.lastname: Foumani
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada.
authors.forename: Benjamin
authors.initials: B
authors.lastname: MacCormick
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada.
authors.forename: Rachel
authors.initials: R
authors.lastname: Kwan
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada.
authors.forename: Emma R
authors.initials: ER
authors.lastname: Master
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019943:Amino Acid Substitution; D001203:Ascomycota / Q000201:enzymology; D020134:Catalytic Domain; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005949:Glucose Oxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D009844:Oligosaccharides / Q000737:chemistry; D010084:Oxidation-Reduction; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C072250:glucooligosaccharide oxidase; D005949:Glucose Oxidase