Engineering galactose oxidase for efficient cascade synthesis of L-guluronic acid from D-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 by 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 iterative saturation mutagenesis, a variant, GOase 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 C6 position. In a cascade reaction, 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.