毕赤酵母
葡萄糖氧化酶
黑曲霉
突变体
生物化学
过氧化氢
饱和突变
化学
氧化磷酸化
蛋氨酸
突变
酵母
乙醇氧化酶
生物传感器
基因
重组DNA
氨基酸
作者
Gordana Kovačević,Raluca Ostafe,Rainer Fischer,Radivoje Prodanović
标识
DOI:10.1016/j.bej.2019.03.016
摘要
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.
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