Tarallo, Vincenzo (2017) New lipases for industrial applications. [Tesi di dottorato]

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Lipases are enzymes widely used in the Industry. There is a strong interest in searching for new enzymes endowed with peculiar characteristics. In this framework, two approaches were carried out: Culture and Genome Mining. At first, a new Geotrichum candidum lipase producing strain was isolated from marine polluted water. The effect of calcium and carbon and nitrogen sources on lipase secretion was analysed. The maximum lipase activity was reached after 58 h of cultivation in a bioreactor, in an optimized medium, at 28°C, and was found to be 9,000 U/L. The presence of lipase activity was assessed both in the culture broth and associated with the mycelium pellet. The two enzymatic pools (secreted and cell-associated), were both characterized testing the effect of pH and temperature on their activity and stability. Furthermore, substrate specificity was also assessed. Four different samples from the composting centre CESCO were used to screen for new lipase producing microorganisms. The samples were suspended in buffer at pH 8 and incubated at 60°C. When the growth reached a significantly optical density, serial dilutions were carried out in order to select individual colonies. Lipase producing microorganisms were then identified and subjected to a genomic DNA extraction. The amplification of the putative 16s rRNA/ITS region led then to the identification of Alicyclobacillus acidocaldarius strain. Several growth media were tested in order to obtain an extracellular lipase activity of about 700 U/L. Nine putative lipolytic enzyme sequence were annotated in the online genome available for this strain. Following a bioinformatics analysis, one sequence was chosen for the recombinant expression in the heterologous host Pichia pastoris, that reached a maximum of extracellular lipase activity of 300 U/L. In the second approach, an edible fungus was taken in analysis. This is the first report on the characterization of lipases from Pleurotus ostreatus. The database of the annotated genome reports 44 lipase putative coding-genes. For this reason P.ostreatus represent a potential source of lipases. Different growth media were tested to induce the extracellular lipase activity. The media with the best activity values were used to carried out a zymogram analysis to visualize positive bands. Following the hydrolysis and mass spectrometric analysis of the positive bands, five lipases were found in a culture media of P. ostreatus. Two sequences were chosen for recombinant expression in the heterologous host P. pastoris. The two lipase coding sequences were synthesised and cloned in a P. pastoris expression vector. The selected clone PleoLip241 reached a maximum of activity of 4,000 U/L after 9 days at 28°C, whereas, in the same conditions, the other selected clone, PleoLip369, reached a maximum of activity of 700 U/L. The enzymes were purified, characterised and the KM and Kcat values were determined using different substrates. These enzymes showed a high affinity towards medium and long chains esters. Pleolip241 was tested in the treatment of the wool.

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