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

Abstract

Enzymes entrapped in wet, nanoporous silica gel have great potential as bioreactors for bioremediation because of 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 toward the environmental pollutant 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 toward chlorocatechols. These gels also showed a fivefold increase in relative reactivity toward chlorocatechols with respect to the natural substrate catechol, thus compensating for their relatively low activity for these substrates in solution. The encapsulated enzyme, unlike the enzyme in solution, proved resilient in assays carried out in urban wastewater and bacteria-contaminated solutions mimicking environmentally relevant conditions. Overall, the combination of a structure-based rational design of enzyme mutants, and the selection of a suitable encapsulation material, proved to be a powerful approach for the production and optimization of a potential bioremediation device, with increased activity and resistance toward bacterial degradation.

From protein structure to function via single crystal optical spectroscopy

“…As in solution, the unproductive binding of catechol was observed under anaerobic conditions. Spectra were also correlated with the three dimensional structures of the wild type and two mutants exhibiting higher specificity for chloro cathecols and designed as the basis for bioreactors to be used in bioremediation (Micalella et al, 2014 ). Overall, the consistency of the spectroscopic properties of Ar-1,2-CTD in solution and in the crystalline state indicated that Fe(III) coordination and ligand binding observed through X-ray crystallography reflected those of the enzyme in solution.…”