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 T50 of M4, the temperature corresponding to a 50% loss of maximal enzyme activity, increased by 7.5 °C and thermal inactivation half-lives (t1/2) 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 kcat, 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.
Submitter: Shu-Ching Ou
Submission Date: Jan. 25, 2019, 4:13 p.m.
Data in Table 1 is not included as it mostly includes values from other studies.
|Number of data points||35|
|Assays/Quantities/Protocols||Experimental Assay: Half-life: incubation at 60 °C ; Experimental Assay: Half-life: incubation at 70 °C ; Experimental Assay: kcat ; Experimental Assay: Km ; Derived Quantity: SD of kcat ; Derived Quantity: SD of Km ; Derived Quantity: kcat/Km|
|Libraries||Variants for GoxA ; Variants for GoxA_beta-D-Glucose|