Thermostability improvement of the glucose oxidase from Penicillium amagasakiense for efficient antimicrobial performance through computer-aided molecular design.

Glucose oxidases (GODs) induce the catalyzation from β- d -glucose to gluconic acid in an oxygen-consuming process, providing a potential antibiotic substitution strategy. However, the inadequate properties of existing GODs in parallel hinder the antimicrobial capacity for industrial applications. In this study, PaGOD (WT) from Penicillium amagasakiense was enzymatically improved through computer-aided design based on energy optimization. Two thermostable variants A263P and K424F were selected and combined to generate variant A263P/K424F, superior in both thermostability (t 1/2 at 60 °C increased 2.6-fold) and catalytic efficiency (2.1-fold increase in catalytic efficiency), in comparison with the WT. The molecular dynamics simulations revealed the improved rigidity of A263P/K424F is attributed to the formation of hydrogen bonds within the flexible region and the newly-formed salt bridge Lys473: Asp477, following the increased ΔΔG. For improvement of antibacterial capacity, A263P/K424F impressively lower the half maximal inhibitory concentrations (IC 50) to 12 and 11 mg/L respectively for Escherichia coli and Staphylococcus aureus (86.4 % and 78.8 % lower than the WT, 65.7 % and 50 % lower than erythromycin). The results indicated that the antibacterial effects of GOD can be improved through in vitro molecular modification, which could be an effective strategy to address antibacterial requirements with antibiotic-free agents. • This study provides a potential antibacterial GOD for antibiotic substitution. • Antibacterial capacity was optimized by stabilizing the GOD for better properties. • Computer-aided design increased loop rigidity and remote interaction of target GOD. • The mutant exhibited better inhibition than erythromycin for some common bacteria. [ABSTRACT FROM AUTHOR]