De Santi, Concetta (2014) Metagenomics of polar marine sediments: study of microbial biodiversity and isolation of new biocatalysts for use in industrial processes. [Tesi di dottorato]

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Marine bioprospecting is a highly topical research subject since the marine ecosystem is a relatively unexplored source of enzymes with potential biocatalytic activity. Development of new biocatalysts from marine extreme environments, such as Polar Regions, can be considered value-added. In fact, there is an increasing demand in bio discovering powerful biocatalysts for biotechnological applications in terms of esterase/lipases from cold environments. This is due to their salt tolerance, hyperthermostability, barophilicity, cold adaptivity, substrate specificity and affinity. The first session of the research project was focused on functional-based screening method to screen a collection of 100 marine bacteria isolated from Svalbard and Lofoten islands for their ability to produce a broad spectrum of cold-active enzymes.The isolated bacteria were classified by 16S rRNA sequencing and the phylogenetic distribution of the detected activities was evaluated with the interest for finding new cold-active biocatalysts. In the second session of the project, a gene encoding for an esterase was amplified by PCR from a bacterial sequenced genome belonging to Thalassospira sp. GB04J01. After cloning and gene heterologous expression in E. coli, the recombinant protein was obtained as high level in soluble form and then purified to homogeneity. A full structural and functional protein characterization was carried on and in addition, the enzyme was able to form diamond crystals which diffracted at 1.7 Å. This was the first biochemical characterization and structural analysis of a cold-active esterase isolated from the genus Thalassospira. A parallel session of the project was aimed at identifying and at characterizing a new cold-active and salt tolerant esterase isolated from Arctic metagenomic libraries. Taking advantage of an “omic” technique, such as Metagenomics, it was possible to access to unculturable microbes in such extreme environment, the Arctic. A fosmid containing an ORF encoding a gene with potential esterase/lipase activity was detected by functional screening on tributyrin agar plates. The positive clones employing the largest halo size were sequenced and a gene encoding a putative lipase was found. Gene cloning and expression were followed by a production of the recombinant protein in a soluble form. A biochemical approach highlighted protein features with a functional characterization methodology. To achieve the summarized results several expertise in different research fields were required. The research activity was realized through short exchanges of researchers between Italy and Norway. This research project and the data obtained strongly represent a contribution to enhance the market of cold-active lipolytic enzymes of white biotechnology field as powerful biocatalysts.

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