Inorganic Hybrid Nanoflower Patterns of Transglycosylating α-glucosidase for Production of Isomaltooligosaccharides

In this study, divalent metal ions (Zn²⁺, Cu²⁺, and Mn²⁺) were employed for the in situ synthesis of highly stable hybrid nanoflowers (tAG-NFs) to immobilize transglycosylating α-glucosidase (tAG) for isomaltooligosaccharide (IMO) production. Characterization via SEM, FTIR, and XRD confirmed the successful formation of these distinct nanostructures and increased crystallinity. Optimization revealed that the Mn-tAG-NF system achieved the highest immobilization efficiency (~ 46%). This architecture demonstrated superior enzyme stability, retaining 41.61% activity at 64 °C and exhibiting high storage stability and reusability (~ 50% activity after five cycles), significantly outperforming the free enzyme. Kinetic analysis showed that immobilization generally enhanced substrate affinity (reduced K_m). Crucially, HPLC studies confirmed that the Zn-tAG-NF and Mn-tAG-NF architectures successfully modulated the enzyme's catalytic specificity. These systems suppressed competing hydrolysis and favored the desired transglycosylation reaction, resulting in significantly higher IMO yields (Zn-tAG-NF: 0.52 ± 0.06 g/g maltose; Mn-tAG-NF: 0.47 ± 0.03 g/g maltose) and lower glucose byproduct compared to free tAG (0.37 ± 0.06 g/g maltose). This work demonstrates that the choice of divalent metal ion is critical for tailoring tAG nanoflower properties, providing a novel method to enhance enzyme stability and product selectivity for industrial biocatalysis.