Phosphorylated sugars

recognized as central intermediates in carbohydrate metabolism and critical precursors for enzymatic synthesis of rare sugars

face significant technical barriers in their industrial-scale production. The multi-enzymatic preparation systems for these compounds inherently accumulate complex impurities

including protein-based catalysts

residual substrates

and oligosaccharide by-products

posing persistent challenges in product separation and biocatalyst recycling. To address this limitation

we conducted a systematic investigation of ultrafiltration-based separation strategies during the multi-enzyme-catalyzed synthesis of fructose-1

6-bisphosphate (FDP)

with particular emphasis on membrane fouling mechanisms. By screening the ultrafiltration membranes

UE020 showed the best performance in the model system

achieving significant separation targets: 99.97% retention of bovine serum albumin

FDP/maltodextrin separation coefficient of 7.41

and FDP recovery of 93.63%. An analysis of the components of resistance revealed that concentration polarization induced by maltodextrin was the main factor constituting the resistance

irreversible resistance due to bovine serum albumin was a secondary effect

and the resistance constituted by FDP was negligible. A mitigation strategy employing powdered activated carbon for dynamic membrane formation significantly improved system performance

reducing irreversible resistance by 59.14% and enhancing flux recovery by 20.85%. In this study

ultrafiltration was strategically employed to achieve efficient separation of FDP and enzyme recovery. Significantly

we deciphered the synergistic fouling mechanisms arising from interactions within the multicomponent system containing phosphorylated sugars

oligosaccharides

and proteins. These findings provide a mechanistic framework for scaling up multi-enzymatic systems dedicated to phosphorylated sugar biosynthesis

effectively bridging the gap between laboratory-scale synthesis and industrial implementation.