Influence of methionine residue position on oxidative stability of glucose oxidase from Aspergillus niger
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Glucose oxidase (GOx) is a promising candidate for construction of implantable miniature biofuel cells and biosensors for continuous glucose monitoring. The main drawback that limits current application of GOx in these devices is its low stability, especially sensitivity to reactive oxygen species. In order to address this problem, we performed saturation mutagenesis at all 11 methionine residues as their interaction with reactive oxygen species inactivates enzymes. For successful screening of these libraries a method based on yeast surface display (YSD) systems was developed. Mutations at methionine positions close to the GOx active site contributed the most to the oxidative stability, and combinations of the four best single mutations were tested. Combined mutants did not show higher stability or activity compared to the parental single mutants. To confirm oxidative stability of YSD expressed GOx mutants they were re-cloned in Pichia pastoris, purified and immobilized on macroporous ...copolymer. The additional kinetic analysis of immobilized GOx mutants confirmed that the best mutant with only one mutation close to the active site (M561S) has 2.5 times increased half-life in the presence of hydrogen peroxide compared to the wild-type variant.
Keywords:Glucose oxidase / Methionine oxidation / Oxidative stability / Site directed mutagenesis / Yeast surface display
Source:Biochemical Engineering Journal, 2019, 146, 143-149