Miele, Annalisa (2009) New oxidative enzimes for industrial application. [Tesi di dottorato] (Unpublished)

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Abstract

Fungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. The white-rot fungus Pleurotus ostreatus is able to express multiple laccase genes encoding isoenzymes with different and particularly interesting physico-chemical characteristics: POXC, POXA1w, POXA1b, POXA3a and POXA3b. Several P. ostreatus laccases have been successfully expressed in yeasts and the availability of established heterologous recombinant expression systems has allowed the construction of mutated, “better performing” enzymes through molecular evolution techniques. The cDNAs encoding POXA1b and POXC have been selected as “parent molecules” to guide the evolution of laccases with higher specific activity and different substrate specificities. Genetic variants were created by random mutagenesis through error prone PCR (EP-PCR) and DNA shuffling. After two round of mutations, four POXA1b mutants (1M9B,1L2B, 1M10B and 3M7C) were selected for their improved activity against nonphenolic substrates. New criteria of selection were applied in a further screening of the already avalaible 2300 mutants library, using different substrates (e.g. 2,6 dimethoxyphenol, DMP), assaying the enzyme stability and activity at different operating conditions (different pHs). Three new mutants were selected for their improved performances and characterized from a structural and functional point of view. They showed stability at pH 5 and at 60°C higher than that of the wild-type enzyme. A new mutant was rationally designed and constructed. This new mutant, R4, contains the mutations of the two parental enzymes 1M10B and 3M7C. Catalytic and kinetic properties of R4 mutants were analyzed and compared with those of the wildtype enzyme. It showed stability at pH10 2 fold higher than that of the wild-type enzyme. This new clone was used as template for producing a new collection of 1100 mutants. Two new mutants (4M10G and 1H6C) were selected for their improved activity against ABTS. The isolated mutants were partially purified and fully characterized. Amino acid substitutions of each mutant were identified and located in POXA1b laccase model. Analysis of possible molecular mechanisms responsible of better performances was carried out. A parallel session of the project was aimed at overproducing POXA1b laccase and its best variant 1H6C using the filamentous fungi Aspergillus niger as host. The necessity to produce large amounts of enzymes for biotechnological applications and the low yields of laccases secreted from wild-type fungal organisms claims for an efficient heterologous expression of the enzymes of interest. The two laccases have been expressed in the filamentous fungi A. niger, obtaining an improvement of 100- fold in the enzyme production. These preliminary results indicate that A. niger could be a promising expression system for P. ostreatus laccases if compared to other expression systems.

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