Tyrosinase immobilization on aminated magnetic nanoparticles by physical adsorption combined with covalent crosslinking with improved catalytic activity, reusability and storage stability

Abstract

In the present study, the immobilization method of physical adsorption combined with covalent crosslinking was developed to avoid the shortcomings of both the noncovalent and covalent coupling methods. For the first time, tyrosinase (TYR) was immobilized on the surface of aminated magnetic nanoparticles (Fe3O4-NH2) by the developed method. TYR was firstly adsorbed on the surface of Fe3O4-NH2 by electrostatic interaction, and then by covalent crosslinking with glutaraldehyde (GA), TYR was firmly immobilized on the supports. The immobilized TYR showed enhanced pH and temperature endurances at the optimum pH of 7.0 and temperature of 35 °C. TYR reusability was significantly improved after immobilization and it retained 61.4 ± 2.3% of its initial activity after 5 repeated cycles. Immobilized TYR also showed improved storage stability (73.2 ± 1.1% after 30 days of storage at 4 °C). In addition, the immobilized TYR showed a higher biological affinity to substrate owing to the stabilization of TYR in its active conformation by electrostatic interaction prior to covalent crosslinking. Finally, the immobilized TYR was used to screen of enzyme inhibitors from 11 traditional Chinese medicines (TCMs) to validate whether this method can be used for enzyme inhibitor screening or not.