Upcycling agro-industrial biomass molasses into D-mannitol via an enzymatic cascade with in situ cofactor regeneration

Molasses, a sugar industry by-product rich in sucrose, D-glucose, and D-fructose, remains underutilized in high-value biomanufacturing. In this study, we present an efficient enzymatic cascade for the direct conversion of molasses into D-mannitol, a valuable sugar alcohol widely used in the food and pharmaceutical sectors. The cascade integrates three enzymes: invertase for sucrose hydrolysis, mannitol dehydrogenase (MDH) for D-fructose reduction, and glucose dehydrogenase (GDH) for in situ NADH regeneration via D-glucose oxidation, which simultaneously generates D-gluconolactone as a value-added coproduct. Two reaction formats were evaluated: a two-step system that sequentially optimizes enzyme-specific conditions, and a one-pot system enabling operational simplicity. The two-step method yielded 137 ± 13 mM D-mannitol, corresponding to ∼92 % conversion, while the one-pot system produced 123.1 ± 1.3 mM, corresponding to ∼95 % conversion under molasses conditions within 24 h. The molasses pretreatment was not essential for D-mannitol production. Enzymes maintained high catalytic activity in the complex molasses matrix, and glucose supplementation improved cofactor regeneration, eliminating residual D-fructose. This integrated strategy offers a sustainable and scalable platform for agro-industrial waste valorization by co-producing D-mannitol and D-gluconolactone, aligning with circular bioeconomy principles and advancing green bioprocessing technologies.