Experimental and computational investigation of α-amylase–copper phosphate nanohybrids for enhanced enzyme stabilization and liquefaction performance

In this study, α-amylase–copper phosphate nanohybrids were synthesized through a biomimetic self-assembly approach and the synthesis conditions were systematically optimized. Among the investigated divalent metal ions, Cu²⁺ produced well-defined hierarchical nanoflowers with the highest encapsulation efficiency (95.51 ± 0.35%) and was selected for further investigation. FESEM and FTIR analyses confirmed the successful immobilization of α-amylase within the copper phosphate matrix. Compared with the free enzyme, the immobilized α-amylase exhibited improved kinetic performance during soluble starch hydrolysis, with enhanced substrate affinity (Km decreased from 0.528 to 0.269 mg mL⁻¹) and increased catalytic capacity (Vmax increased from 9.98 to 11.48 U mL⁻¹), together with enhanced thermal stability, broader pH tolerance, and improved liquefaction of both soluble starch and bread waste. To elucidate the molecular basis of nanohybrid formation, computational studies involving binding-site prediction, density functional theory (DFT) optimization, and molecular docking were performed. The results revealed that copper phosphate preferentially interacts with multiple solvent-exposed regions of α-amylase through electrostatic and hydrogen-bonding interactions while avoiding the catalytic cleft, thereby promoting cooperative nanohybrid assembly without obstructing substrate access. These findings provide a molecular explanation for the experimentally observed high encapsulation efficiency, hierarchical flower-like morphology, enhanced stability, and improved catalytic performance. Overall, this study provides molecular-level insight into biomimetic enzyme–mineral self-assembly and demonstrates the potential of α-amylase–copper phosphate nanohybrids as efficient biocatalysts for sustainable starch processing and bread waste valorization.