Immobilization of catechol 1,2-dioxygenase in silica gel as a potential bioremediation device

Dioxygenases are responsible for the aerobic catabolism of several toxic aromatic compounds released in the environment as detergents, oil, solvents, paints, pesticides, fungicides and insecticides as a result of decades of industrialization and ntensive farming. As a consequence, dioxygenases might be of interest in the development of bioremediation devices. Catechol 1,2-dioxygenase (C 1,2O) Iso B from Acinetobacter radioresistens S13 [1] is a homodimer containing one non-heme ferric ion for monomer (αFe3+)2 that catalyzes the intradiol cleavage reaction of the catechol to yield cis,cis muconic acid. In order to create bioreactors for bioremediation, the enzyme was immobilized in silica gel and its catalytic properties were compared to those in solution. C 1,2O encapsulation in wet nanoporous silica gel was obtained through a sol-gel process, consisting of an acid catalyzed hydrolysis of silica alkoxides, such as tetramethylortosylane (TMOS) and tetraethylortosylane (TEOS) and a following polycondensation at alkaline pH. The enzyme was also immobilized using an Ormosil (organically modified silicate), consisting in a mixture of TMOS and dimethyldimethoxysylane (DMDMS). The Ormosil was chosen to increase the hydrophobicity of the gel, since C 1,2O is specific for hydrophobic substrates and has an hydrophobic tunnel binding two phospholipidis, that could modulate the enzyme activity [2]. Enzymatic assays carried out on wild type C 1,2O point out encapsulation does not stabilize and enhance the activity of the enzyme. Some mutants, such as L69A, A72G and A72S, were designed in order to increase the specificity towards chlorocatechols, which are more toxic than catechol and potentially cancerogenic. The immobilization in silica gel, especially in the Ormosil-derived gels, seems to stabilize these mutants and to enhance their activity. This is possibly due to their larger and more hydrophobic ligand binding pocket as to the wild type. Further studies are ongoing on other matrices, in the attempt to discover the best condition to encapsulate the enzyme and develop bioremediation strategies. As well, important information will be also obtained from crystallization studies which are in progress.