Domain engineering of dextransucrase and soft nanoconfinement-enhanced biosynthesis of pharmaceutical-grade dextran

Dextran, a polysaccharide with critical pharmaceutical applications, requires precise molecular weight control for optimal functionality. Traditional chemical synthesis methods face challenges in efficiency and environmental sustainability. Here, we present a combinatorial strategy integrating enzyme engineering and soft nanoconfinement to achieve one-step biosynthesis of tailored dextran. Twelve recombinant dextransucrases were designed by swapping domains V/IV between the processive enzyme DSR-OK (producing ultra-high molecular weight dextran) and the distributive enzyme DSR-MΔ2 (yielding low molecular weight dextran). Among these variants, CZ8 demonstrated superior catalytic activity, synthesizing 40 kDa dextran directly from sucrose. To further enhance chain elongation, lipid-based nanomaterials, alcohol ethoxylate (AEO) and polyethylene glycol (PEG, 20 kDa), were incorporated into the reaction system. Under optimized conditions (125 g/L sucrose, 4 U/mL CZ8, 5 wt% AEO, or 1 wt% PEG), the molecular weight of dextran increased to 70 kDa, attributed to nanoconfinement-induced spatial restriction favoring polymerization. Notably, the AEO nanomaterial spontaneously separated via gravitational settling within 24 h, achieving >99 % removal efficiency without energy-intensive purification. Structural analysis confirmed dextran's α-(1,6) backbone, with minor α-(1,3) branching under nanoconfinement. This study provides a green, scalable platform for dextran production, emphasizing enzyme-nanomaterial synergy for precise molecular weight control and sustainable downstream processing.