Ormosil gels doped with engineered catechol 1,2 dioxygenases for chlorocatechols bioremediation

Enzymes entrapped in wet, nanoporous silica gel have a great potential as bioreactors for bioremediation due to their improved thermal, chemical and mechanical stability with respect to enzymes in solution. The B isozyme of catechol 1,2 dioxygenase from Acinetobacter radioresistens and its mutants of Leu69 and Ala72, designed for an increased reactivity towards the environmental pollutants chlorocatechols, were encapsulated using alkoxysilanes and alkyl alkoxysilanes as precursors in varying proportions. Encapsulation of the mutants in a hydrophobic tetramethoxysilane/dimethoxydimethylsilane-based matrix yielded a remarkable 10 to 12-fold enhancement in reactivity towards chlorocatechols. These gels also showed a 5-fold increase in relative reactivity towards chlorocatechols with respect to the natural substrate catechol, thus compensating for their relative low activity for these substrates in solution. The encapsulated enzyme, unlike the enzyme in solution, proved resilient in assays carried out in urban wastewaters and bacteria-contaminated solutions mimicking environmentally relevant conditions. Overall, the combination of a structure-based rational design of enzyme mutants and the selection of the suitable encapsulation material proved to be a powerful approach for the production and optimization of a potential bioremediation device, with increased activity and resistance towards bacterial degradation.