Obtenção de linhagens recombinantes de Penicillium griseoroseum para produção de poligalacturonase

• Autor(a) principal: Ribeiro, João Batista
• Data de Publicação: 2005
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1530
• topic: Linhagens recombinantes; Penicillium griseoroseum; Poligalacturonase; Recombinant strains; Penicillium griseoroseum; Polygalacturonase; CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS

Analysis of a 170 bp DNA fragment of the promoter region of the pgg2 gene containing a putative CAAT box at - 270 bp from ATG code by electrophoretic mobility shift assay with nuclear extracts prepared from mycelia grown under pectin- containing medium revealed a high mobility complex that was subsequently confirmed when analysis was conducted employing a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied. A 23 bp sinthetic double- stranded oligonucleotide containing a cis-element resembling the cAMP response element (CTACAGTG) observed in yeasts and mammals promoters was also used as probe in EMSA assays. Band shifts were not seen when nuclear extracts prepared from mycelia cultured on pectin and glucose- containing medium supplemented with yeast extract were used in the reactions. This result suggests that the CTACAGTG element tested is not a target site for regulatory activators. In order to inactivate the P. griseoroseum pgg2 gene, a disruption vector, named pPG15pgg2Δniafo was constructed by inserting a 4 Kb HindIII DNA fragment, containing the nia D gene from Fusarium oxysporum in the coding region of pgg2. The disruption of pgg2 resulted in transformant strains producing at most 12 % of PG activity of the host strain (PG63), indicating that this gene responds for almost 90% of PG total activity in P. griseoroseum. To overexpress extracellular polygalacturonase in P. griseoroseum, an expression cassete was constructed, designated pAN52-pgg2 that contained the pgg2 gene under the control of the strong constitutive promoter gpdA from Aspergillus nidulans. The transformation of the PG63 mutant strain using this vector and the selective plasmid (pNPG1) resulted in the isolation of a recombinant strain (146) that, when cultivated in glucose, sucrose or sugar cane juice as sole carbon source, produced a PG activity 12 times higher than that produced by the mutant strain cultivated in pectin and yeast extract. The PG produced by the recombinant strain possesses an optimum pH range from 3,5 to 5,0 and is inactivated at the extrem pHs 2,5 and 8,0. It was demonstrated that this enzyme acts efficiently hydrolising polygalacturonic acid in a broad range of temperature (30 to 60 oC) with the optimal temperature ranging from 30 to 45 oC. In addition, storage of the recombinant strain culture supernatants at temperatures of - 20, 4, and 30 oC for five weeks did not affect enzyme activity. On the other hand, the enzyme exhibited low thermostability, being inactivated when pre-incubated for 1 hour at temperatures of 40, 50, and 60oC.