Antisolvent Crystallization of Papain

Protein crystallization plays a crucial role in the food and pharmaceutical industries, enhancing product quality and efficiency by improving purity and controlled particle characteristics. This study focused on the crystallization of the versatile protein papain, extracted from papaya. Antisolvent crystallization was performed. This method is cost-effective and is a simple and energy-efficient approach. Beyond protein crystal production, the antisolvent crystallization process serves as a method for encapsulating active pharmaceutical ingredients (APIs). The study investigated organic solvents like ethanol, acetone, and acetonitrile as potential antisolvents. Additionally, the impact of variables such as the solvent-to-antisolvent (S:AS) volume ratio and papain concentration on particle size, particle size distribution, zeta potential, crystallization yield, and residual activity of papain crystals were examined. Ethanol emerged as the optimal antisolvent, reducing the solubility of papain and preserving papain’s crystalline structure with minimal activity loss. Optimal conditions were identified at a 1:4 S:AS volume ratio and a papain concentration of 30 mg/mL, resulting in nanosized spherical crystals with a high yield and preserved activity. This research underscored the crucial role of thoughtful parameter selection in antisolvent crystallization to achieve specific particle characteristics while maintaining the functionality of the crystallized substance.