Tyrosinase immobilized enzyme reactor: Development and evaluation

• Immobilized enzyme reactors of tyrosinase were prepared using different conditions.
• The reactors were fully evaluated and compared.
• Tests with known inhibitors confirmed the usefulness for inhibitors screening.
• Compatibility tests with organic solvents were performed.

Immobilized enzyme reactors of tyrosinase (tyr-IMERs) for use on-line in HPLC system were prepared by different procedures and then compared. The enzyme, obtained from Agaricus bisporus, was immobilized on epoxy-silica which was prepared using different conditions. Enzyme immobilization was conducted by both in situ and in batch techniques. The different procedures were compared in terms of protein and activity retention, IMERs activity, kinetics and stability. The influence of immobilization procedure on enzyme activity and the behavior of the IMERs against a standard inhibitor were also investigated. In situ immobilization on epoxy-silica, synthesized using microwave assistance, provided the best conditions to prepare tyrosinase IMERs. The tyr-IMERs were successfully tested with known and potential inhibitors of tyrosinase, and the results showed that they can be used for the screening of inhibitors of that enzyme.