Development of a cold-adapted enzymatic cascade system for in situ value-added conversion of milk.

The global prevalence of lactose intolerance presents a major nutritional challenge, severely limiting milk consumption. In this study, a cold-adapted enzymatic cascade was developed for in-situ lactose-to-lactulose conversion. Molecular dynamics-based screening identified a novel cellobiose 2-epimerase (CE), Papo-CE, exhibiting high cold activity and rapid inactivation above 70 °C. Subsequently, semi-rational engineering of a cold-adapted Marinomonas mediterranea D-mannose isomerase (MmMI) was conducted, which resulted in a mutant M1 (MmMI/F242N/G375W/P373T) with 4.2-fold increase in isomerization activity for converting epilactose into lactulose, attributed to Trp375-mediated π-π stacking and new hydrogen bonds from P373T. Ultimately, an efficient Papo-CE-M1 cascade system was constructed for low-temperature lactose-to-lactulose conversion. The optimized Papo-CE-M1 cascade (1:2 ratio, 12 h) achieved 54.5% lactulose yield and 70.3% lactose conversion in milk. Electronic nose/tongue analysis confirmed flavor improvement without off-notes. This low-temperature, self-inactivating system offers a sustainable route for producing premium low-lactose milk enriched with prebiotics.