Microbiologia
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11840
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Item Production of pectin lyase by Penicillium griseoroseum in bioreactors in the absence of inducer(Brazilian Journal of Microbiology, 2001-04) Passos, Flávia Maria Lopes; Passos, Frederico José Vieira; Silva, Daison Olzany; Piccoli-Valle, Roberta HilsdorfPenicillium griseoroseum was grown in bioreactors on mineral medium supplemented with yeast extract and sucrose. The influence of inoculum and carbon source concentrations, aeration and pH on pectin lyase (PL) production, as well as the capacity of P. griseoroseum to produce PL when grown on sugar cane syrup as carbon source were evaluated. Inoculum concentration did not influence PL production. Production was higher in non-aerated than in aerated medium. The best results were obtained using 60 mM sucrose at pH 6.3-7.2. Production using cane syrup 25% (v/v), without yeast extract supplement, was equal to that obtained under the conditions cited above.Item Molecular characterization and expression profile of pectin-lyase-encoding genes from Penicillium griseoroseum(Canadian Journal of Microbiology, 2006) Bazzolli, Denise S.; Ribon, Andrea O. B.; Queiroz, Marisa V. de; Araujo, Elza F. dePenicillium griseoroseum has been studied by our group because of its good pectinase production. Attempts have been done to clone pectinolytic genes, aiming to obtain pectinase-overproducing strains for industrial purposes. Here, two genes coding for pectin lyase were isolated from the P. griseoroseum genome. The plg1 gene has an open reading frame of 1341 bp coding for a putative protein of 374 amino acids with a calculated molecular mass of 40.1 kDa. The plg2 gene is characterized by an open reading frame of 1400 nucleotides and codes for a polypeptide of 383 amino acids. The plg1 gene 5'-flanking region contains putative binding sites for the transcription factors involved in regulation by ambient pH and catabolite repression. The primary structure of Plg1 and Plg2 proteins showed a relatively high homology (varying between 32.4% and 74.8%) to fungal pectin lyases characterized to date. Southern blotting analysis revealed that both genes are present as single copies in the fungus genome. Expression studies revealed a differing pattern of gene expression of plg1 and plg2 when mycelium was cultivated on medium containing different pectic components. Citric pectin followed by apple pectin were the carbon sources that best induced plg1 expression, and transcripts were detected from 24 to 76 h. The expression of the plg2 gene was monitored by reverse transcriptase--polymerase chain reaction, since Northern analysis failed to detect hybridization signals. The differential expression of these genes may provide means for the fungus to adapt to various growth conditions.Item Pectin lyase production by recombinant Penicillium griseoroseum strain 105(Canadian Journal of Microbiology, 2010) Teixeira, Janaina Aparecida; Queiroz, Marisa Vieira de; Araújo, Elza Fernandes de; Cardoso, Patrícia GomesRecombinant Penicillium griseoroseum strain 105 overproduces an extracellular pectin lyase (PL) under the transcriptional control of the strong gpdA promoter of Aspergillus nidulans. Our aim was to evaluate PL production by recombinant P. griseoroseum strain 105 in submerged fermentation system bioreactors BioFloIII and BioFloIV using 2 or 10 L working volumes under different growth conditions and to analyze the production of cellulase, polygalacturonase, pectin methylesterase, and protease. PL overproduction by recombinant P. griseoroseum strain 105 was 112 times higher than that of P. griseoroseum PG63 grown in sugarcane juice. Cellulases and proteases were not detected in the culture filtrate, and evaluation for extracellular proteins in the culture medium by SDS–PAGE showed the presence of a 36 kDa predominant band, similar to the molecular mass estimated from the nucleotide sequence of plg1 gene for PL of P. griseoroseum strain 105. This recombinant strain provides the advantage of PL production, which predominates over other extracellular proteins usually present in most commercial pectinase preparations, using sugarcane juice as a substrate of low cost.Item Molecular characterization and evaluation of pectinase and cellulase production of Penicillium spp.(Biotechnology Letters, 2002-05) Pereira, Jorge Fernando; Queiroz, Marisa Vieira de; Gomes, Eliane Aparecida; Muro-Abad, Júpiter Israel; Araújo, Elza Fernandes dePenicillium species were analyzed with molecular markers and for pectinase and cellulase production. RAPD and PCR-RFLP analysis indicated high polymorphism among at least 5 of 10 Penicillium species. Five species were chosen for pectinase and cellulase production in liquid medium and four of which appeared similar based on molecular analyses. P. brevicompactum and P. griseoroseum gave the highest pectinase production and were highly divergent by molecular techniques.Item The minimal regulatory region necessary for the expression of the Penicillium griseoroseum plg1 gene(Annals of Microbiology, 2015-06) Bazzolli, Denise Mara Soares; Reis, Klédna Constância Portes; Teixeira, Janaina Aparecida; Ribon, Andréa Oliveira Barros; Queiroz, Marisa Vieira de; Araújo, Elza Fernandes deThe expression of the Penicillium griseoroseum plg1 gene is induced by citric pectin and repressed by glucose. In this work, the minimal region of the plg1 gene promoter essential for expression in pectin and sucrose plus yeast extract was identified by using constructs containing the gfp ORF under control of the plg1 gene promoter. The fragment A (283 bp) is essential for plg1 expression in sucrose plus yeast extract. Fragment B (309 bp plus 184; core promoter) was critical for expression in pectin and abolished the catabolic repression by glucose. Therefore, the fragment of 776 bp (fragment A and B) is essential for the expression of the plg1 gene in natural inducing conditions (pectin as carbon source) and in sucrose plus yeast extract. The fragment B is a promising minimal promoter usable for heterologous expression in filamentous fungi, since genes that contain it could be activated by the presence of peel from citric fruits (which contains citric pectin) and are not affected by glucose in these agricultural by-products.Item Improved pectinase production in Penicillium griseoroseum recombinant strains(Journal of Applied Microbiology, 2011-06-27) Teixeira, J.A.; Gonçalves, D.B.; Queiroz, M.V. de; Araújo, E.F. deTo obtain recombinant strains of Penicillium griseoroseum that produce high levels of pectin lyase (PL) and polygalacturonase (PG) simultaneously. A strain with high production of PL was transformed with the plasmid pAN52pgg2, containing the gene encoding PG of P. griseoroseum, under control of the gpd promoter gene from Aspergillus nidulans. Southern blot analysis demonstrated that all strain had at least one copy of pAN52pgg2 integrated into the genome. The recombinant strain P. griseoroseum T20 produced levels of PL and PG that were 266‐ and 27‐fold greater, respectively, than the wild‐type strain. Furthermore, the extracellular protein profile of recombinant T20 showed two protein bands of c. 36 and 38 kDa, associated with PL and PG, respectively. This recombinant strain T20 produces PL and PG using carbon sources of low costs, and an enzyme preparation that is free of cellulolytic and proteolytic activities. PL and PG play an important role in the degradation of pectin. Owing to their use in the juice and wines industries, there is a growing interest in the inexpensive production of these enzymes. This work describes an efficient system of protein expression and secretion using the fungus P. griseoroseum.Item Differential expression of plg genes from Penicillium griseoroseum: plg1 a pectinolytic gene is expressed in sucrose and yeast extract(Journal of Applied Microbiology, 2008-04-22) Bazzolli, D.M.S.; Ribon, A. de O.B.; Reis, K.C.P.; Queiroz, M.V. de; Araújo, E.F. deTo study the regulation of the plg1 and plg2 genes of Penicillium griseoroseum, in order to identify the industrial potential of their products in alternative carbon sources that are cheaper and widely available in Brazil. RT-PCR and Northern blot were used to investigate if plg1 and plg2 expression is under influence of catabolic repression, ambient pH and cAMP. Results demonstrated that the genes were differentially regulated depending on the carbon sources in the culture medium and pH. Sucrose, a noninducing carbon source of the pectinolytic system, was able to promote plg1 transcription but only when yeast extract was added into the culture medium. The plg genes are differentially expressed. The plg1 gene is more attractive for industrial use due to its expression in alternative carbon sources like sucrose and yeast extract. In recent years, industries have been trying to replace the toxic conventional treatments employed in these processes by more eco-friendly enzyme treatment. Alternative carbon sources will be tested with the aim to reduce the costs associated to pectin lyase production in Brazil.