Serine protease–viscoelastic polysaccharide interaction in inorganic CaCO3: Enhanced thermal stability for efficient enzyme particulation via immobilization

Abstract

Efficient enzyme particulation is essential for advancing enzyme applications across the food, cosmetic, and pharmaceutical industries. This study introduces a novel enzyme immobilization strategy utilizing mesoporous calcium carbonate (CaCO₃) that incorporates viscoelastic polysaccharides to improve Bacillus licheniformis protease stability and reusability. An in situ biomineralization method was employed, where CaCO₃ was synthesized while simultaneously encapsulating both the enzyme and guar gum in a water-based mixed-solvent system. Immobilization via protease–guar gum interactions significantly enhanced functional properties. Notably, guar gum increased enzymatic activity 3.39-fold (p < 0.01) and imparted thermal resistance during vacuum drying at 40–70 °C. The guar gum–protease–CaCO₃ system showed 2.04-fold higher residual activity at 40 °C compared to the guar gum-free system. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and Brunauer–Emmett–Teller (BET) analysis confirmed robust enzyme–matrix integration. The immobilized protease retained 45 % of its initial activity after 45 days and 70 % after 10 reuse cycles. Guar gum further enhanced storage stability by 8 % and reusability by 10 %. This is the first demonstration of a synergistic protease–polysaccharide system embedded within inorganic CaCO₃, providing a durable, thermally stable platform for cosmetic enzyme formulations, functional food biocatalysts, and pharmaceutical intermediates, though validation in complex industrial matrices is needed.