Metagenomics of polar marine sediments: study of microbial biodiversity and isolation of new biocatalysts for use in industrial processes
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, th...
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| Format: | Doctoral or Postdoctoral Thesis |
| Language: | Italian English |
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2014
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| Online Access: | http://www.fedoa.unina.it/9671/ http://www.fedoa.unina.it/9671/1/Tesi%20dottorato%20De%20Santi%20Concetta.pdf |
| Summary: | 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 ... |
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