Multi-immobilization of Cabohydrases and Proteases by Nanomagnetic Combi-CLEAs Method for Oil and Protein Hydrolysates Extraction from Oil Seeds in Aqueous Phase

Functionalized magnetic nanoparticles are effective enzyme carriers since they have several advantages such as easily separation under external magnetic fields as well as enhancing mass transfer and reusability of enzymes. In this article, three types of enzymes included Viscozyme L (carbohydrases), Alcalase ۲.۴ L (endo-peptidase), and Flavourzyme (Exo-peptidase) Simultaneously were immobilized by nanomagnetic cross-linked enzyme aggregates (CLEAs) method. Then assessment of the results obtained was performed. At first, Fe۳O۴ nanoparticles were synthesized by chemical co-precipitation then, surface coating procedure was used for functionalization of Fe۳O۴ by lysine amino acid. Thereafter, enzyme aggregation and cross-linking was achieved by using of enzymes with functionalized Fe۳O۴ and glutaraldehyde in saturated ammonium sulfate, and/or solvents such as, acetone, acetonitrile, tert-butanol, isopropanol, and ethanol at ۳-۴° C separately. Afterthat, for size reduction of formed NM-Combi-CLEAs, high speed homogenizer and then ultrasonic waves were applied. Then, produced NM-Combi-CLEAs was hold at ۳-۴° C for ۳-۲۴ hours and cross-linked enzymes were separated from liquid phase by centrifuge at ۱۵۰۰۰ rpm accurately. Finaly, activity assay, FE-SEM images and EDX, DLS and zeta potential analysis, FTIR, degree of hydrolysis (DH%), kinetic parameters (Km, Vmax, kcat, kcat/Km, t۱/۲, kd) and thermodynamic parameters (ΔG, ΔH, ΔS, Ea(in)) of immobilized enzymes compared to native enzymes mixtures was evaluated. The NM-Combi-CLEAs kept ۷۵-۸۰% of its original activity after ۱۰ cycles, which proposes strong operational stability. In conclusion, the NM-Combi-CLEAs are thermo-stable, reusable, and efficient nanobiocatalyst for enzymatic oil and protein hydrolysates extraction in aqueous phase.