Effects of immobilization on activity of catechol 2, 3 dioxygenase enzymes on Magnetic nanoparticles

Catechol 2,3-dioxygenase (C2, 3O) are a key enzyme in the meta-cleavage pathway of catechol metabolism. In the present study, the C2, 3O enzyme was immobilized on magnetic nanoparticles (MNPs). Firstly, the C2, 3O enzyme was purified of E. coli. Then magnetic nanoparticles were synthesized by sol-gel method. C2, 3O enzyme was conjugated with MNPs using1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 0.5 mg mL-1) and N-hydroxysuccinimide (NHS, 0.5 mg mL-1) at 25 ◦C for 12h. Subsequently, MNP-C2, 3O complex was washed three times with tris-base. The size, structure pure magnetic nanoparticles and MNP-C2, 3O complex were evaluated by SEM and DLS. The activity of MNP-C2, 3O complex was measured by adding catechol as substrate to mixture reaction using UV-Vis spectrophotometry. The result revealed that the structure and size of MNPs and MNP-C2, 3O complex were spherical and 21-43 nm, respectively. Also, Immobilization of C2, 3O enzyme on MNPs improved the stability toward the denaturation induced by pH, heat, and metal ions.