Improving the thermostability and catalytic efficiency of glucose oxidase from Aspergillus niger by molecular evolution

Abstract Glucose oxidase (Gox) has many applications in numerous industries. However, thermal instability is a major drawback that prevents its broader use. Here, Gox from Aspergillus niger (GoxA) was selected for laboratory evolution for purposes of enhancing thermostability and catalytic efficiency through random and rational mutagenesis. The most active mutant, M4, accumulated six amino acid substitutions. The T 50 of M4, the temperature corresponding to a 50% loss of maximal enzyme activity, increased by 7.5 °C and thermal inactivation half-lives ( t 1/2 ) at 60 °C and 70 °C increased 8.4-fold and 5.6-fold, respectively, compared to wild-type GoxA. Concomitantly, M4 demonstrated a 1.86-fold increase in k cat , resulting in a 1.78-fold increase in catalytic efficiency. Molecular dynamics simulation revealed diverse mechanisms underlying the effects of each mutation on thermostability and catalytic efficiency. These results suggest that key properties of glucose oxidase can be modified in vitro by laboratory evolution, which may have remarkable economic importance.