Guarino, Lucia (2015) New biosystems: from applied research to technology transfer. [Tesi di dottorato]

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In the frame of white biotechnology, enzymes are attracting an enormous attention not only because of their potential but also for the recent social and economical interest in green chemistry. Microorganisms constitute a huge reserve of genetic diversity, representing an important resource reservoir to undertake bioprospecting for new biocatalysts (such as lipases and laccases) with applications spanning across all major industries. Bioprospecting is defined as "the exploration of biodiversity for commercially valuable genetic and biochemical resources". Bioprospecting efforts are currently potentiated by the integration of high-throughput approaches, where robotized technologies are employed to systematically screen through the biodiversity from specific environments. Unimaginable habitats, and in particular the marine one represent interesting source of new bio-systems. Bioprospecting can be conducted by culture-dependent (a traditional approach based on cultivation of natural isolates that can be screened for activities of interest) or culture-independent methods (metagenomic approach). Considering the interest towards lipases and laccases in many industrial applications, this study has been focused on the identification of new biocatalysts using different bioprospecting strategy. In detail this study allowed: i) the isolation of a new lipolytic organism (Geotrichum candidum) from the polluted fisherman‟s mole in Putueoli, trough an enrichment culture approach, and the optimization of lipase activity production, formulating the most inductive growth medium; ii) the recombinant expression in the yeast Pichia pastoris of a putative lipase annotated in the genome of the fungus Pleurotus ostreatus showing the potentiality to be a bifuctional enzyme; iii) the recombinant production of a bacterial laccase from the arctic Pseudoalteromonas haloplanktis bacterium in the soluble intracellular fraction of Escherichia coli and its application for the oxidation of dye precursors (resorcinol and 2,5 DABSA), iv) the identification of 40 lipase positive clones from a marine metagenomic library of 30,000 fosmidic clones. Moreover, because of the interest in marine bioprospecting, a comparative philogenetic analysis of marine biome in two close sites has been performed. Obtained results showed, surprisingly, a clear divergence in the two biome.

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