Piscitelli, Alessandra (2005) Recombinant expression of fungal oxidases for industrial application. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||Laccase, Yeasts, Heterologous expression, Glycosylation, Evolution|
|Date Deposited:||04 Aug 2008|
|Last Modified:||30 Apr 2014 19:22|
Laccases catalyse the oxidation of a range of organic substrates coupled to the reduction of molecular oxygen to water. They are members of the ubiquitous blue multi-copper oxidase family. These enzymes are implicated in a wide variety of biological activities. Most of the laccases studied thus far are of fungal origin. Large variety of potential substrates has raised interest in the use of laccases in several industrial applications, such as pulp delignification, textile dye bleaching, effluent detoxification, biopolymer modification and bioremediation. Cloning of the laccase genes followed by heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (different substrate specificities and improved stabilities) for industrial applications. Heterologous expression of Pleurotus ostreatus laccases POXC and POXA1b in two yeasts and a first approach of directed evolution experiments are reported. The yeasts of choice were Saccharomyces cerevisiae, proven to be success-full in recombinant laccase expression and directed-evolution experiments, and Kluyveromyces lactis, a non-conventional yeast offering significant advantages, such as high-level secretion of non-hyperglycosylated recombinant proteins. Expression vectors were set up cloning the cDNAs under the control of different promoters. Furthermore, the laccase leader peptides (poxc and poxa1b), as well as the yeast derived signal peptides (S. cerevisiae invertase and K. lactis killer toxin), were alternatively used to direct the secretion of active laccase into the culture medium. The laccase signals proved to be more effective to drive the secretion of recombinant proteins in both hosts. Levels of laccase secreted activity were markedly different: rPOXA1b transformants always gave much higher activity than rPOXC transformants, and production of both laccases in S. cerevisiae was significantly lower than that in K. lactis. Recombinant laccases from K. lactis were purified to electrophoretic homogeneity and characterized. rPOXA1b specific activity was similar to that of the native protein, whilst rPOXC specific activity was much lower than that of the native POXC. Mass spectrometry analyses of the recombinant proteins allowed to verify their primary structures and to identify post-translational modifications. Our data confirm that K. lactis has a lower tendency, respect to S. cerevisiae, to hyperglycosylate recombinant proteins. The S. cerevisiae laccase expression systems were further used to set off directed evolution experiments. Mutated cDNAs libraries with different mutation rate were created, and homologous recombination experiments were performed, giving rise to libraries of mutated laccase secreting yeasts. Moreover a screening procedure to isolate clones exhibiting desired property was realized. As a result, this work allowed obtaining the heterologous expression of two P. ostreatus laccases in yeasts, and their purification and characterisation. Moreover, this research work broadened the potentiality of the developed expression system addressing enzymes to such large markets and different industrial application such as pulp and textile bleaching, and enzymatic remediation of waste streams. A new laccase host (K. lactis) has been built on, and its promising performances will lead to further investigate its utilization for further structure-activities studies, as well as for directed evolution. Results obtained demonstrate the potential of the recombinant expression for the study of potential industrial interest.
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