Immobilization, characterization, and enhanced properties of pullulanase-iron hybrid nanoflowers

Pullulanase is a vital debranching enzyme widely utilized in starch saccharification and related biotechnological industries to efficiently hydrolyze α-1,6 glycosidic bonds. As a free enzyme, its broader application is restricted by high costs and operational fragility. Addressing these limitations, this study introduces a green immobilization strategy. A novel pullulanase-iron hybrid nanoflower was synthesized via a single-step, facile biomimetic mineralization process. Structural characterizations (FTIR, SEM-EDS, XRD) confirmed the successful immobilization within an amorphous, hierarchical cauliflower-like network. The immobilized pullulanase demonstrated a broadened operational window (optimum: 25 °C, pH 5.5). Furthermore, it demonstrated extraordinary stability, retaining nearly full activity up to 55 °C, alongside remarkable tolerance across acidic environments, retaining over 94% activity between pH 3.5 and 6.5. Kinetic evaluations revealed that while apparent K m increased to 10.73 mg/mL due to mass transfer limitations, the V max (~ 2.22 U/mL) was completely preserved. Crucially, immobilization acted as a powerful steric shield; whereas free pullulanase was severely deactivated (~ 50% residual activity) by Cu 2+ and NH 3 , the immobilized enzyme was fully protected. Additionally, Co 2+ and Mn 2+ actively enhanced performance by 10–19%. Retaining > 96% activity through four cycles and ~ 91% after 10 consecutive cycles, this strategy yields a highly durable enzyme suitable for rigorous continuous bioprocessing.