Immobilisation and kinetic study of tyrosinase for biosensor construction

The catalytic properties and stability of soluble and immobilised in nylon- 6,6 mesh tyrosinase were studied with the help of an oxygen sensor. A varie ty of methods were examined for the immobilisation of tyrosinase, although active immobilised enzyme was obtained only with the help of benzidine and dicyclohexylcarbodiimide. The immobilisation caused an increase in the K-m value for catechol almost 2 times in comparison with that found for soluble enzyme (0.39 and 0.22 mM, respectively). The immobilised tyrosinase retain ed sufficient activity for several months. Due to its characteristic suicid e inactivation induced by catechol, it is only of single use for analytical purposes. Obtained data were also used for evaluation of the model of oximeter-based biosensors. This model allows the calculation of steady-state parameters fr om transient state data, excluding the influence of accompanying side proce sses; for tyrosinase-bound biosensors it gave very reproducible results for automatic data processing.