Microbiologia

URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11840

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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 Antonio
Protoplast 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).
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 Antônio
Penicillium 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.
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 Olzany
The 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.
Restriction enzyme improves the efficiency of genetic transformations in Moniliophthora perniciosa, the causal agent of witches’ broom disease in Theobroma cacao
(Brazilian Archives of Biology and Technology, 2008-01) Lopes, Francis Julio Fagundes; Queiroz, Marisa Vieira de; Lima, Juliana Oliveira; Silva, Viviane Aline Oliveira; Araújo, Elza Fernandes de
The presence of restriction enzymes in the transformation mixture improved the efficiency of transformation in Moniliophthora perniciosa. The influence of the vector shape (linear or circular), the patterns of plasmid integration in genomic sites and the influence of the promoter used to express the gene marker were also analyzed. The addition of BamHI or NotI increased the number of transformants by 3-10-fold and 3-fold, respectively, over the control without added enzyme. The use of pre-linearized plasmid did not increase the transformation efficiency in comparison with the circular plasmid. However, the frequency of multi-copy transformants increased significantly. The transformation procedure here reported resulted in better production of protoplasts and transformation efficiency. In addition, the time necessary for the detection of the first transformants and the number of insertions were reduced.
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 Maria
An 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+.
Production and regeneration of protoplasts from orchid Mycorrhizal Fungi Epulorhiza repens and Ceratorhiza sp.
(Brazilian Archives of Biology and Technology, 2010-01) Coelho, Irene da Silva; Queiroz, Marisa Vieira de; Costa, Maurício Dutra; Kasuya, Maria Catarina Megumi; Araújo, Elza Fernandes de
The aim of this work was to study the standardization of conditions to obtain and regenerate Epulorhiza repens and Ceratorhiza sp. protoplasts. For E. repens, the largest number of protoplasts (8.0 × 106 protoplasts/mL) was obtained in 0.6 M KCl, using 15 mg/mL of Lysing Enzymes, and 2-day-old fungal mycelium. When 0.5 M sucrose was used as osmotic stabilizer, the highest frequency of regeneration was achieved (8.5 %); 80.0 % of protoplasts were nucleated, and 20.0 % anucleated. For Ceratorhiza sp., the largest number of protoplasts (4.0 × 107 protoplasts/mL) was achieved in 0.6 M NaCl, when 15 mg/mL of Lysing Enzymes and 15mg/mL of Glucanex, with 2-day-old fungal mycelium were used. The highest frequency of regeneration was 6.7 % using 0.5 M sucrose as osmotic stabilizer; 88.8 % of protoplasts were nucleated, and 11.2 % anucleated.
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 de
Penicillium 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.
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 de
To 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.
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 Gomes
Recombinant 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.
Development of molecular markers based on retrotransposons for the analysis of genetic variability in Moniliophthora perniciosa
(European Journal of Plant Pathology, 2012-11) Santana, Mateus Ferreira; Araújo, Elza Fernandes de; Queiroz, Marisa Vieira de; Souza, Jorge Teodoro de; Mizubuti, Eduardo Seiti Gomide
Moniliophthora perniciosa is a fungus that causes witches’ broom disease (WBD) in the cacao tree (Theobroma cacao). The M. perniciosa genome contains different transposable elements; this prompted an evaluation of the use of its retrotransposons as molecular markers for population studies. The inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) techniques were used to study the variability of 70 M. perniciosa isolates from different geographic origins and biotypes. A total of 43 loci was amplified. Cluster analysis of different geographical regions of C biotype revealed two large groups in the state of Bahia, Brazil. Techniques using retrotransposon-based molecular markers showed advantages over previously used molecular techniques for the study of genetic variability in M. perniciosa.

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