Nanoconfined enzymatic cascade strategy for highly efficient synthesis of D-allulose

Enzymatic cascade strategy in vitro presents a transformative approach for sustainable biosynthesis of D-allulose, illustrating the superior efficiency compared to traditional single-step epimerization by D-allulose 3-epimerase. However, streamlining multi-enzyme cascades for higher yield, enhanced stability, and industrial-scale low-cost synthesis remains a formidable challenge. Herein, we design an innovative nanoconfined enzymatic cascade strategy by co-encapsulating five key enzymes (α-glucan phosphorylase, phosphoglucomutase, phosphoglucose isomerase, D-allulose 6-phosphate epimerase, and D-allulose 6-phosphate phosphatase enzymes) inside a nanoreactor consisting of a CD- b -PEG- b -PS block copolymer. After optimizing this nanoconfined enzymatic cascade strategy through an integrated approach combining response surface modeling and strategic experimental validation, this rationally designed nanoconfined enzymatic cascade strategy shows remarkable catalytic performance, achieving a high product yield (42.9 mM, 77.3 %) without requiring additional co-enzymes including isoamylase (IA), 4-α-glucanotransferase (4GT), polyphosphate glucokinase (PPGK), and glucose isomerase (GI), demonstrating significant enhancement compared to the conventional free enzyme strategies (24.8 mM, 44.8 %). It also exhibits strong potential for industrial application, as evidenced by a scale-up test at 50 g L −1 substrate loading, which yields 145.7 mM (52.1 %) of D-allulose after 50 h without the addition of auxiliary enzymes such as IA, 4GT, PPGK, and GI. These compelling results establish our platform as a robust and versatile platform for constructing nanoconfined enzymatic cascades and open new avenues for developing next-generation biocatalytic strategies that bridge the gap between natural enzymatic pathways and industrial biomanufacturing requirements. • A nanoconfined enzymatic cascade was developed by co-encapsulating five key enzymes within a micelle nanoreactor. • This nanocascade strategy achieved 77.3 % yield without co-enzymes, a 1.7-fold enhancement over free enzymes (44.8 %). • Demonstrating industrial potential at 50 g L −1 , it achieved a 145.7 mM titer, nearly 2-fold increase over free enzymes. • The strategy showed high reusability over eight cycles, unlike single-use free enzyme systems.