Construction of dual-enzyme hybrid nanoflowers by protein self-assembly and biomineralization for efficient biosynthesis of d-tagatose

Enzyme–inorganic hybrid nanoflowers (HNFs) are promising immobilization platforms because of their simple fabrication and often enhanced catalytic performance. Here, a robust and highly efficient hybrid nanoflower (β-Gal-SC@ST-LFAI) for d -tagatose biosynthesis was developed using co-immobilizing β-galactosidase (β-Gal) and l -arabinose isomerase (LFAI) by ST/SC–mediated self-assembly and biomineralization. First, the effects of key immobilization conditions on the fabrication of β-Gal-SC@ST-LFAI were investigated. Under optimal conditions, β-Gal-SC@ST-LFAI exhibited higher catalytic activity and activity recovery than a conventional β-Gal@LFAI. Compared with the free enzyme system, β-Gal-SC@ST-LFAI showed enhanced thermal stability, pH tolerance and organic solvent resistance. The structure of β-Gal-SC@ST-LFAI was further characterized by SEM, CLSM, DLS, FTIR, CD and BET technology. The resulting HNFs displayed a typical flower-like architecture with an average diameter of 1.83 μm and specific surface area of 18.57 m 2 ·g −1 . CD spectroscopy showed a decrease in α-helix content and an increase in β-sheet content in β-Gal-SC@ST-LFAI compared to the free cascade enzymes. In addition, β-Gal-SC@ST-LFAI exhibited a lower K m than the free cascade enzymes, indicating enhanced substrate affinity. The HNFs also demonstrated better storage stability than the free enzymes and excellent operational stability, retaining more than 77.99% of their cascade catalytic activity after seven reuse cycles. Finally, β-Gal-SC@ST-LFAI can catalyze the conversion of d -lactose to d -tagatose with a yield of 37.22%. This study provides a practical strategy for constructing multienzyme HNFs with high catalytic efficiency and process robustness.