Covalent immobilization of tyrosinase onto cyanuric chloride crosslinked amine-functionalized superparamagnetic nanoparticles: Synthesis and characterization of the recyclable nanobiocatalyst.

Magnetic nanoparticles (MNPs) were synthesized using the chemical co-precipitation method. Then the nanoparticles were coated with silica via hydrolysis of tetraethyl orthosilicate using the sol-gel process. The silica coated magnetic nanoparticles were amine-functionalized with 3-aminopropyltriethoxysilane/ethanol solution. Subsequently, the nanoparticles were added to a solution of cyanuric chloride in tetrahydrofuran to synthesize cyanuric chloride-functionalized magnetic nanoparticles (Cy-MNPs). For covalent immobilization of tyrosinase, Cy-MNPs were added to a freshly prepared tyrosinase solution and the mixture was shaken. The FTIR spectra, as well as EDX, analysis proved the covalent immobilization of tyrosinase on the nanoparticles. The magnetic properties of tyrosinase-immobilized magnetic nanoparticles (tyrosinase-MNPs) were specified by VSM analysis. TEM images indicated that the most of the tyrosinase-MNPs had a semi-spherical shape with an average size of 17nm. The synthesized nanoparticles had a high loading capacity of 194mg tyrosinase/g nanoparticles with an immobilization yield of 69%. The optimum condition for both free and immobilized tyrosinase was found at pH 7.0 and 35°C. The immobilized enzyme was active after treatment of the particles at various pHs and temperatures for 100min. In addition, reusability of the immobilized enzyme was investigated and it was proved its suitability to be used for more than 7 cycles. Also, tyrosinase-MNPs remained about 70% of its initial activity after storing at 4°C for 40days. This nanobiocatalyst with interesting properties is promising for practical application in wastewater treatment and biosensor development.