Molecular stabilizing a glucose oxidase from Aspergillus eucalypticola with improved catalytic activity and optimized...

Glucose oxidase (GOX EC.1.1.3.4) specifically catalyzes the reaction of β-d-glucose to gluconic acid in an oxygen-consuming process, enabling its antimicrobial potential. However, the unstable nature and insufficient activity of the FAD-dependent dimer hinder the antibacterial capacity. In this study, A GOX from Aspergillus eucalypticola (AeGOXL), outstanding for its catalytic efficiency, was selected for laboratory evolution through random mutagenesis. Three thermostable variants Q148K, H283Y, and P514K were selected and form the combined variant Q148K/H283Y (M1), superior in both thermostability (T₅₀ increased 13 °C, T_m increased 6.7 °C, and 2.1-fold of t_1/2 prolonged at 80 °C) and catalytic activity (97% and 57% increase in specific activity and k_cat/K_m), comparing with the WT. Structural investigations elucidated the molecular stabilization due to additional hydrogen bond network formation, while long-range interaction elevated the flexibility of key residues, improving the substrate affinity. For antibacterial capacity, M1 significantly brought down the half maximal inhibitory concentrations (IC₅₀) for Staphylococcus aureus and Escherichia coli separately to 21.3 and 17.4 mg/L (52.5% and 70.5% lower than the WT, comparable to sensitive conventional antibiotics), in accordance with the electron microscope manifestation. This research delved into the GOX functional based enzymatic antibacterial approaches, providing milder and more eco-friendly substrate to address antibacterial requirements.