Tailored biocatalysis for dicarboxylic acid production: A versatile enzymatic approach to galactaric and glucaric acids.

The conversion of renewable biomass into value-added dicarboxylic acids, such as galactaric and glucaric acids, remains a significant challenge for the sustainable chemical industry, largely due to the lack of efficient and selective enzymatic processes. In this study, we employed protein engineering strategies to modify glucose oxidase (GOx) from Aspergillus niger, aiming to enhance its substrate specificity and broaden its oxidative capacity for the production of dicarboxylic acids. Site-saturation mutagenesis libraries targeting position F414 of the GOx-Y68W variant were constructed and screened. This effort led to the identification of four active variants with markedly enhanced oxidative performance. Among them, the Y68W + F414P variant demonstrated the highest efficiency and selectivity for glucose oxidation at C1, achieving glucuronic acid yields of 40 % under mild conditions (40 °C, pH 6.0). Notably, the Y68W + F414C variant exhibited dual oxidation activity at both the C1 and C6 positions of glucose, enabling a one-pot biocatalytic route to glucaric acid with a 15 % yield. This study represents the first report of glucose oxidase variants capable of driving sequential oxidation toward glucaric acid in a single enzymatic system. These findings advance the application of protein engineering in tailoring classical oxidases for complex, multi-step biotransformations, offering new opportunities for the sustainable production of dicarboxylic acids from renewable feedstocks.