Engineering galactose oxidase for efficient cascade synthesis of <sc>l</sc>-guluronic acid from <sc>d</sc>-glucose

l-Guluronic acid, a patented drug with immunomodulatory and anti-inflammatory properties, is traditionally sourced from natural seaweed species or synthesized chemically. Both methods suffer from low selectivity and environmental concerns and there is a high demand to develop a green and efficient approach for l-guluronic acid production. Herein, we successfully developed a two-step enzymatic route to synthesize l-guluronic acid from d-glucose using two oxidases, glucose oxidase (GOx) and galactose oxidase (GOase). Due to the sluggish kinetics of GOase in catalyzing the oxidation of d-gluconic acid, rational design was implemented. Through molecular docking and site saturation mutagenesis, a variant, GOase V3 (T406R/K330R/Y329F), was obtained. The variant exhibited a remarkable 13-fold higher oxidation activity towards d-gluconic acid, reaching 7.8 U mg-1. The substitutions synergistically switch the d-gluconic acid molecule to a favorable conformation and facilitate the specific activation of the primary alcohol group at the C-6 position. In a cascade reaction, the complete conversion of 40 mM d-glucose to l-guluronic acid was achieved in 2 h with 100% selectivity, presenting a promising and green bio-manufacturing strategy for the scalable production of l-guluronic acid.