Covalent immobilization of papain onto poly(hydroxyethyl methacrylate)-chitosan cryogels for apple juice clarification

Abstract

Enzyme immobilization appears as a remarkable technique to safely attach enzymes for several applications and cryogels stand as promising support materials to be used in such investigations. In this work, papain enzyme was immobilized onto an interpenetrating network obtained by cryogelation of N,N′-methylenebisacrylamide cross-linked 2-hydroxyethyl methacrylate and glutaraldehyde cross-linked chitosan. Cryogels were modified with –NH₂ functionality and glutaraldehyde in order to attach papain covalently. Immobilization was carried out at 25 ℃ in 0.1 M pH 7.0 phosphate buffer at 1.0 mg/ml enzyme concentration for 5 h. The amount of papain immobilized onto cryogel was calculated to be 15.2 ± 2.54 mg/g cryogel. Macroporous structure and surface area were determined by scanning electron microscopy and Brunauer–Emmett–Teller techniques, respectively. Energy dispersive X-ray analysis showed that papain was bound to the cryogel and cryogel structure was composed of 2-hydroxyethyl methacrylate, chitosan, and glutaraldehyde. Proteolytic activities of free and immobilized papain were measured using casein as substrate. Optimum pH values and temperatures were 8.0 and 65 ℃ for free and immobilized enzymes and kinetic parameters were calculated at these conditions. Reusability and storage stability results indicated that immobilization enhanced the stability of papain compared to free form. Efficiency of immobilized papain was demonstrated by apple juice clarification study as an industrial use of the enzyme. Phenolic compound, protein, total soluble solid contents, and viscosity of apple juice before and after clarification were determined.