Del Vecchio, Claudia (2010) DEVELOPMENT OF MICROBIAL AND ENZYMATIC BIOSYSTEMS FOR THE TREATMENT OF COLOURED WASTEWATERS. [Tesi di dottorato] (Unpublished)

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This research project - that is part of a broader research project, carried out in collaboration with the University of Turin (Department of Vegetal Biology, Dr. Cristina Varese and financed by the “Compagnia di S. Paolo”) - aims at developing bio-systems for the treatment of industrial coloured wastewaters, based on microbial and enzymatic catalysts. In the present research program two kind of biosystems have been specifically analysed: Streptomyces spp. bacteria and laccase isoenzymes produced by the white rot fungus Pleurotus ostreatus. Their effectiveness was tested on four textile wastewater models (Acid Bath, Optional Acid, Direct, Reactive) and single dyes composing them, by assessing and comparing their performances in terms of decolourisation ability of the treated effluent. In particular, the decolourization ability of eleven Streptomyces spp. strains has been tested towards single dyes and the four wastewater models. Since the analysed strains originate from soil contaminated by gasoil their expanded metabolic capabilities can be considered a promising starting point for the selection of new dye degraders. The best performing Streptomyces spp. strains have shown good decolourization abilities (percentage up to 50%), although the process is mainly ascribable to absorption processes instead of degradation ones. However, according to the obtained data, these biomasses may stand out as good candidates for biosorption treatment of industrial wastewaters. As far as the enzymatic system, decolouration ability of recombinant laccases from Pleurotus ostreatus has been analyzed. In order to ascertain the contribution of single laccase isoenzyme to the decolourization process, and to identify the most efficient laccase based biosystem, the decolourization ability of the single P. ostraetus laccase isoenzymes POXA1b, POXC and POXA3, heterologously expressed in the yeast Kluyveromyces lactis, has been tested towards the four wastewater models. The data indicate laccases preference in decolourizing anthraquinonic type dye and aniline mono-azo dye, characteristics of Acid wastewater models. In particular recombinant POXC laccase displays a slightly higher decolourization ability against Acid Bath, reaching up to 22% decolourization after 24h, in comparison with POXA3 and POXA1b. The differences in decolourization efficiency can be ascribed to structural differences of the dyes and to substrate specificity of the laccase isoenzymes. A parallel aspect of this section has been focused on selecting laccase variants that are better suited to the conditions of industrial wastewaters (high pHs, heterogeneous composition), i.e able to operate on a wider range of substrates, or stable, and especially active, in extreme conditions of pH. For this reason, seven POXA1b variants, previously selected from a collection of mutants on the basis of different criteria (increased activity toward different substrates and higher stability at pHs and temperature), have been tested for their decolourization ability towards the four wastewater models. Selected POXA1b variants show an increased decolourization ability than POXA1b wild-type toward the Acid Bath model. As a fact, for all the mutants, a two-fold increase in decolourization percentage compared to that of POXA1b has been obtained. Another section of the work has been focused on the understanding of the structure/function relationships of laccases with the aim to characterize molecular determinants of the activity of these enzymes, and to develop and characterize new laccases obtained from rational design using POXA1b P. ostreatus isoenzyme. Rational mutants of POXA1b laccase allowed to demonstrate a role of the C-terminal tail of POXA1b in affecting its catalytic and stability properties. Moreover, site-directed mutagenesis experiments allowed to demonstrate that introducing Arg205 mutation, instead of Asp 205, in a highly conserved region perturbs the structural local environment in POXA1b, leading to a large rearrangement of the enzyme structure. Hence, a single substitution in the binding site introduces a local conformational change that not only leads to very different catalytic properties, but can also significantly destabilize the protein. Finally, in order to improve industrially useful enzymatic biosystems, suitable for decolourization processes of coloured wastewaters, the last session of the project was aimed at producing new P. ostreatus dikaryotic strains with improved efficiencies in laccase expression, by classical breeding approach. In particular, starting from two different P. ostreatus variants, three laccase higher-producing dikaryotic strains have been obtained by crossing compatible characterized monokaryons. The three selected strains reached expression levels of 100,000 U/L, increasing the trite of parental strains up to four folds. Moreover, a new laccase isoenzyme, POX1, has been produced and identified in these culture conditions.

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