Surface charge modifications modulate glucose oxidase pH-activity profiles for efficient gluconic acid production

Gluconic acid (GA) is used globally in many industries. This study aimed to improve the activity and acid resistance of GoxM8, a thermostable glucose oxidase (Gox) that is obtained from Aspergillus niger and used for GA production. Here, surface charge modification via the Rosetta Supercharge software and mutagenesis was used to rationally design the pH-activity profile of GoxM8. The optimum pH of the mutants Q241E, R499E, and Q241E/R499E shifted from 6.0 to 5.0. At pH 6.0, their specific activities were 23%, 5%, and 17% higher than that of GoxM8, respectively, while at pH 5.0, their specific activities were further increased by 44%, 26%, and 37%, respectively. The double mutant had an additive effect on improving acid tolerance in 100 mM McIlvaine buffer at pH 2.5 for 1 h, and the residual activity of mutant Q241E/R499E was 178% higher than that of GoxM8. The yield of GA production catalyzed by the mutant Q241E/R499E reached 136.9 g/L within 24 h, which was ∼35.5% higher than that of GoxM8. This study provides an effective strategy for modifying the optimum pH and acid tolerance of Gox to produce GA more efficiently.