Lacases de actinobatérias: caracterização, obtenção de novos primers e expressão heteróloga na levedura Kluyveromyces marxianus UFV-3

• Autor(a) principal: Fernandes, Tatiana Alves Rigamonte
• Data de Publicação: 2014
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1564
• topic: Actinobactérias; Lacase; Enzimas ligninolíticas; Multicobre oxidases; Actinobacteria; Laccase; Ligninolytic enzymes; Multicobre oxidases; CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOSSANIDADE::MICROBIOLOGIA AGRICOLA

The great diversity in substrate range oxidized by laccase enzymes has attracted attention in both areas of research: basic, and applied to industrial and environmental activities. Among microbial laccases, little is known about the ones produced by bacteria. In this work, it is highlighted the phylum Actinobacteria, order Actinomycetales, due to its recognized biotechnological importance and capacity of degrading recalcitrant compounds. Owing to the large amounts of enzyme required for industrial application, summed to the difficulty in manipulation of native producers, heterologous expression is here suggested using the yeast Kluyveromyces marxianus as host, which presents the industrial-relevant properties such as fast growth, thermotolerance and status generally regarded as safe (GRAS). The objective of this work was to obtain a strain of Kluyveromyces marxianus expressing and secreting a laccase from actinobacteria; and, in order to accomplish the main objective, we searched for isolating laccase-producing actinobacteria from soil, characterizing the enzyme, isolating a laccase gene, cloning into expression vector and, finally, transforming K. marxianus UFV-3. In this work, an isolation from soil samples provided laccase-producer colonies, having one of the selected been characterized. From literature data, a critical review was produced, and available gene sequences that codify characterized proteins were employed for designing oligonucleotide primers. The new primers proved to be effective for detection of laccase genes in actinobacteria, and, furthermore, allowed distinction of genes that corresponded to two structurally different laccases. After selection of a complete laccase gene, it was cloned into an expression cassette for integration into K. marxianus UFV-3 genome, and data suggest successful integration.