Engineering a Robust Glucose Oxidase for Juice Preservation: Enhanced Catalytic Performance Drives Browning Inhibition and Shelf Life Extension.

Glucose oxidase (GOX) is widely recognized for its potential in food preservation, yet its practical application is often limited by insufficient stability and catalytic efficiency under processing conditions. In this study, a GOX from Aspergillus luchuensis (AlGoxA) was engineered through directed evolution, yielding the combinatorial mutant M1 (A179 V/T302L). The variant exhibited a 10.8-fold increase in catalytic efficiency and an 81.1% reduction in Km compared to the wild type. M1 retained 81.9% of its activity after 60 min at 60 °C, a remarkable improvement over the wild type's 6.2%. It also demonstrated broad pH tolerance, maintaining over 66% activity under alkaline conditions. When applied to apple juice preservation, M1 effectively suppressed browning, with the polyphenol oxidase inhibition rate reaching 87.1%, increased vitamin C retention by 37.9%, and exhibited direct antifungal activity against common spoilage fungi. This study provides a highly efficient and robust biocatalyst for extending juice shelf life while preserving nutritional quality.