Microbiologia
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11840
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Resultados da Pesquisa
Item Isolation of recombinant strains with enhanced pectinase production by protoplast fusion between Penicillium expansum and Penicillium griseoroseum(Brazilian Journal of Microbiology, 2007-01) Queiroz, Marisa Vieira de; Lana, Taís Guimarães; Gonçalves, Daniel Bonoto; Araújo, Elza Fernandes de; Brito, Admilson Toscano Ribeiro de; Varavallo, Maurilio AntonioProtoplast fusion between complementary auxotrophic and morphological mutant strains of Penicillium griseoroseum and P. expansum was induced by polyethylene glycol and calcium ions (Ca2+). Fusant strains were obtained in minimal medium and a prototrophic strain, possibly diploid, was chosen for haplodization with the fungicide benomyl. Different recombinant strains were isolated and characterized for occurrence of auxotrophic mutations and pectinolytic enzyme production. The fusant prototrophic did not present higher pectinase production than the parental strains, but among 29 recombinants analyzed, four presented enhanced enzyme activities. The recombinant RGE27, which possesses the same auxotrophic and morphologic mutations as the P. griseoroseum parental strain, presented a considerable increase in polygalacturonase (3-fold) and pectin lyase production (1.2-fold).Item Development of a transformation system for Penicillium brevicompactum based on the Fusarium oxysporum nitrate reductase gene(Brazilian Journal of Microbiology, 2005-04) Queiroz, Marisa Vieira de; Pereira, Jorge Fernando; Ribeiro, Ronney Adriano; Soares, Marcos Antônio; Ribeiro, João Batista; Araújo, Elza Fernandes de; Varavallo, Maurílio AntônioPenicillium brevicompactum is a filamentous fungus that presents a potential for industrial use due its efficient pectinase production. A heterologous transformation system was developed for P. brevicompactum based on the complementation of a nitrate reductase mutant. Nitrate reductase mutants were obtained by resistance to chlorate in a rate of 23.24% when compared to other mutations that lead to the chlorate resistance. One mutant named 4457-18X was chosen for the transformation experiments with the pNH24 vector containing de Fusarium oxysporum nitrate reductase gene. A frequency of approximately 3 transformants/µg DNA was obtained using the circular vector pNH24. This frequency was multiplied about 10 fold using the linearized vector with the Xba I restriction enzyme. Southern analysis of the transformants showed a tendency of the linearized vector to diminish the number of integrations compared to the use of the circular vector. The integration was random and stable in the analyzed transformants. The establishment of a transformation system for P. brevicompactum is fundamental for genetic manipulation of this microorganism.Item Fermentation of sweet whey by recombinant Escherichia coli KO11(Brazilian Journal of Microbiology, 2000-07) Leite, Amarildo Ricardo; Guimarães, Walter Vieira; Araújo, Elza Fernandes de; Silva, Daison OlzanyThe production of ethanol from sweet whey using the recombinant Escherichia coli KO11, in batch fermentation, was tested. The maximum ethanol yield was reached after 96h, representing only 38% of the theoretical yield. The supplementation of whey with components of LB broth increased the maximum yield to 96% in 72h. The addition of 0.5% yeast extract to whey resulted in maximum yield of 74% at 36h and it increased to over 100% when yeast extract and trace metals solution (Fe++, Mn++ and Zn++) were added.Item Partial purification and characterization of xylanase produced by Penicillium expansum(Brazilian Archives of Biology and Technology, 2006-05) Querido, André Luiz de Souza; Coelho, Jorge Luiz Cavalcante; Araújo, Elza Fernandes de; Chaves-Alves, Virgínia MariaAn extracellular xylanase was found to be the major protein in the filtrate culture of Penicillium expansum when grown on 0.3 % wheat bran, which showed no xylanase multiplicity. The enzime was partial purified by.ammonium sulfate fractioning, molecular exclusion chromatography, ultrafiltration and anion exchange chromatography. The protein eluation profile showed only one form of xylanase that was partially characterized. The activity of purified xylanase was optimal at pH 5.5 and 40 ºC. The enzyme was stable at pH between 5.5 and 6.5 and temperatures between 20-40 ºC. The enzyme showed a Km of 3.03 mM and Vmax of 0.027 mmol min-1 mg -1 of protein. The enzymatic activity was increased 31 % by Mg2+ and 28 % by Al3+.Item Characterization, regulation, and phylogenetic analyses of the Penicillium griseoroseum nitrate reductase gene and its use as selection marker for homologous transformation(Canadian Journal Of Microbiology, 2004) Pereira, Jorge Fernando; Queiroz, Marisa Vieira de; Lopes, Francis Júlio Fagundes; Rocha, Rodrigo Barros; Daboussi, Marie-Josée; Araújo, Elza Fernandes dePenicillium griseoroseum has been studied because of its efficient pectinases production. In this work, the Penicillium griseoroseum nitrate reductase gene was characterized, transcriptionally analyzed in different nitrogen sources, and used to create a phylogenetic tree and to develop a homologous transformation system. The regulatory region contained consensus signals involved in nitrogen metabolism and the structural region was possibly interrupted by 6 introns coding for a deduced protein with 864 amino acids. RT-PCR analysis revealed high amounts of niaD transcript in the presence of nitrate. Transcription was repressed by ammonium, urea, and glutamine showing an efficient turnover of the niaD mRNA. Phylogenetics analysis showed distinct groups clearly separated in accordance with the classical taxonomy. A mutant with a 122-bp deletion was used in homologous transformation experiments and showed a transformation frequency of 14 transformants/microg DNA. All analyzed transformants showed that both single- and double-crossover recombination occurred at the niaD locus. The establishment of this homologous transformation system is an essential step for the improvement of pectinase production in Penicillium griseoroseum.Item Morphological and molecular characterization of mycorrhizal fungi isolated from neotropical orchids in Brazil(Canadian Journal of Botany, 2005-01) Pereira, Olinto Liparini; Kasuya, Maria Catarina Megumi; Borges, Arnaldo Chaer; Araújo, Elza Fernandes deTo initiate a conservation program of the Orchidaceae from the Brazilian Atlantic rain forest with the purpose of ex situ conservation or reintroduction in the State of Minas Gerais, seven mycorrhizal Rhizoctonia-like fungal strains were isolated from roots of seven neotropical orchid species from three different Atlantic rain forest fragments. Taxonomic studies revealed that the isolates belong to the genera Ceratorhiza and Epulorhiza. The Epulorhiza isolates were identified as Epulorhiza repens (N. Bernard) R.T. Moore and Epulorhiza epiphytica Pereira, Rollemberg et Kasuya. RAPD analysis indicated higher polymorphism between Epulorhiza epiphytica and Epulorhiza repens than found in the PCR RFLP analysis. RAPD and morphological analyses indicated a degree of relatedness among the Ceratorhiza isolates obtained from the roots of different Oncidium species. A combination of morphological and molecular characterizations permitted integration of fungal strain identification with genetic relatedness among the isolates, thus allowing some inferences to be made on specificity of these endosymbionts under field conditions.Key words: biodiversity, Ceratorhiza, Epulorhiza, orchid mycorrhiza, Rhizoctonia-like, symbiosis, specificity.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 Overexpression of the plg1 gene encoding pectin lyase in Penicillium griseoroseum(Journal of Industrial Microbiology & Biotechnology, 2007-11-12) Teixeira, Janaina Aparecida; Ribeiro, João Batista; Teixeira, Janaina Aparecida; Queiroz, Marisa Vieira de; Araújo, Elza Fernandes deThe pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml−1 respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data.Item Development of a transformation system for Crinipellis perniciosa, the causal agent of witches' broom in cocoa plants(Current Genetics, 2002-12-17) Lima, Juliana Oliveira; Santos, Jildete Karla dos; Pereira, Jorge Fernando; Resende, Mário Lúcio Vilela de; Araújo, Elza Fernandes de; Queiroz, Marisa Vieira deProtoplasts of the pathogenic plant fungus, Crinipellis perniciosa, were transformed to hygromycin B resistance using the pAN7-1 plasmid, which contains the Escherichia coli hph gene under the control of Aspergillus nidulans regulatory sequences. The pAN7-1 plasmid was introduced by PEG/CaCl2 treatment. Transformation frequencies of 1.6–2.5 transformants/μg of DNA were achieved. About 54% of the transformants were abortive and 40 analyzed transformants were mitotically stable and showed different hygromycin B resistance levels. The presence of the hph gene was checked by PCR in five transformants and the integration of multiple plasmid copies into different genome sites was observed by Southern analysis. This is the first report of a C. perniciosa transformation system and represents an important step for further research into genetic manipulation of this fungal plant pathogen.