Catechol 1,2 dioxygenase: mutants production and immobilization in silica gel for bioremediation strategy

Protein engineering was applied on catechol 1,2-dioxygenase IsoB from A. radioresistens S13 (C1,2O) to produce a double mutant and a other 7 active mutants. The active site pocket mutations were suggested by analising a model based on the crystallographic structure of C1,2O from A. calcoaceticus ADP1 and the experimental data obtained for mutant catalysis rationalised in terms of structure-function interplay. The activity of the wt and mutants was assayed to determinate the functional properties (pH, temperature, Eatt measurement, demetallation) using the natural substrate, methylcatechols, chlorocatechols and other substituted diols, that are of environmental interest. In order to create bioreactors for bioremediation, C1,2O and selected mutants were encapsulated using as a precursor either tetramethyl orthosilicate or organically modified silicate (ormosil). Enzymatic assays indicate that encapsulation in ormosil dramatically enhances the activity of some of the mutants with respect to solution and leads to longer stability.