Tyrosinase-Catalyzed Phenol-Mediated Immobilization of β-Agarase on l-Lysine-Coated Magnetic Particles for the Production of Neoagarooligosaccharides from Gelidium amansii

Cross-linked enzyme aggregates of β-Agarase (Aga2) were immobilized on l-lysine-coated magnetic nanoparticles (Lys@Fe₃O₄) for the production of neoagarooligosaccharides (NAOSs) from Gelidium amansii. The support Lys@Fe₃O₄ was prepared in a one-pot aqueous medium, whereas Aga2 was engineered to carry five tyrosine residues at its N-Terminus (Aga2Y5N). Cross-linked immobilization of Aga2Y5N on Lys@Fe₃O₄ was effectively catalyzed by tyrosinase but its efficiency was further improved with phenol addition, affording the best biocatalyst, Aga2Y5N-TP@Lys@Fe₃O₄. Although its kinetic properties were slightly reduced, the overall results show that Aga2Y5N-TP@Lys@Fe₃O₄ performed remarkably better than free Aga2Y5N, as reflected by its excellent stability at wider pH and temperature ranges. Moreover, Aga2Y5N-TP@Lys@Fe₃O₄ can be effectively collected and recycled through magnetization, unlike the free Aga2Y5N, which cannot be easily retrieved from the reaction mixture after use. Hydrolysis results demonstrate the capability of Aga2Y5N-TP@Lys@Fe₃O₄ to effectively and selectively produce NAOS as a mixture of neoagarotetraose and-hexaose. The proposed route could be useful for the immobilization of other enzymes in various biotechnological applications.