Navegando por Autor "Passos, Flávia M. L."
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Item Construction of a Kluyveromyces lactis ku80 − Host strain for recombinant protein production: extracellular secretion of Pectin Lyase and a Streptavidin–Pectin Lyase Chimera(Molecular Biotechnology, 2014-01-01) Colombo, Lívia T.; Rosa, Júlio César C.; Bragança, Caio R. S.; Ignacchiti, Raphael P.; Alvim, Mariana C. T.; Silveira, Wendel B.; Queiroz, Marisa V. de; Bazzolli, Denise M. S.; Passos, Flávia M. L.In several organisms used for recombinant protein production, integration of the expression cassette into the genome depends on site-specific recombination. In general, the yeast Kluyveromyces lactis shows low gene-targeting efficiency. In this work, two K. lactis ku80 − strains defective in the non-homologous end-joining pathway (NHEJ) were constructed using a split-marker strategy and tested as hosts for heterologous gene expression. The NHEJ pathway mediates random integration of exogenous DNA into the genome, and its function depends on the KU80 gene. KU80-defective mutants were constructed using a split-marker strategy. The vectors pKLAC1/Plg1 and pKLAC1/cStpPlg1 were used to evaluate the recovered mutants as hosts for expression of pectin lyase (PNL) and the fusion protein streptavidin–PNL, respectively. The transformation efficiency of the ku80 − mutants was higher than the respective parental strains (HP108 and JA6). In addition, PNL secretion was detected by PNL assay in both of the K. lactis ku80 − strains. In HP108ku80−/cStpPlg1 and JA6ku80−/Plg1 cultures, the PNL extracellular specific activity was 551.48 (±38.66) and 369.04 (±66.33) U/mg protein. This study shows that disruption of the KU80 gene is an effective strategy to increase the efficiency of homologous recombination with pKLAC1 vectors and the production and secretion of recombinant proteins in K. lactis transformants.Item Extracellular α-Galactosidase from Debaryomyces hansenii UFV-1 and Its use in the hydrolysis of raffinose oligosaccharides(Journal of Agricultural and Food Chemistry, 2006-02-17) Rezende, Sebastião T. de; Marques, Virgínia M.; Trevizano, Larissa M.; Passos, Flávia M. L.; Oliveira, Maria G. A.; Bemquerer, Marcelo P.; Oliveira, Jamil S.; Guimarães, Valéria M.; Viana, Pollyanna A.Raffinose oligosaccharides (RO) are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. ROs are hydrolyzed by α-galactosidases that cleave α-1,6-linkages of α-galactoside residues. The objectives of this study were the purification and characterization of extracellular α-galactosidase from Debaryomyces hansenii UFV-1. The enzyme purified by gel filtration and anion exchange chromatographies presented an Mr value of 60 kDa and the N-terminal amino acid sequence YENGLNLVPQMGWN. The Km values for hydrolysis of pNPαGal, melibiose, stachyose, and raffinose were 0.30, 2.01, 9.66, and 16 mM, respectively. The α-galactosidase presented absolute specificity for galactose in the α-position, hydrolyzing pNPGal, stachyose, raffinose, melibiose, and polymers. The enzyme was noncompetitively inhibited by galactose (Ki = 2.7 mM) and melibiose (Ki = 1.2 mM). Enzyme treatments of soy milk for 4 h at 60 °C reduced the amounts of stachyose and raffinose by 100%.Item Genomic sequence of the yeast Kluyveromyces marxianus CCT 7735 (UFV-3), a highly lactose-fermenting yeast isolated from the Brazilian dairy industry(Genome Announcements, 2014-11-06) Silveira, Wendel B.; Diniz, Raphael H. S.; Cerdán, M. Esperanza; González-Siso, María I.; Souza, Robson de A; Vidigal, Pedro M. P.; Brustolini, Otávio J. B.; Prata, Emille R. B. de Almeida; Medeiros, Alexsandra C.; Paiva, Lílian C.; Nascimento, Moysés; Ferreira, Éder G.; Santos, Valdilene C. dos; Bragança, Caio R. S.; Fernandes, Tatiana A. R.; Colombo, Lívia T.; Passos, Flávia M. L.Here, we present the draft genome sequence of Kluyveromyces marxianus CCT 7735 (UFV-3), including the eight chromosomes and the mitochondrial genomic sequences.Item The high fermentative metabolism of Kluyveromyces marxianus UFV-3 relies on the increased expression of key lactose metabolic enzymes(Antonie van Leeuwenhoek, 2001-11-09) Diniz, Raphael H. S.; Silveira, Wendel B.; Fietto, Luciano G.; Passos, Flávia M. L.The aim of this work was to obtain insights about the factors that determine the lactose fermentative metabolism of Kluyveromyces marxianus UFV-3. K. marxianus UFV-3 and Kluyveromyces lactis JA6 were cultured in a minimal medium containing different lactose concentrations (ranging from 0.25 to 64 mmol l−1) under aerobic and hypoxic conditions to evaluate their growth kinetics, gene expression and enzymatic activity. The increase in lactose concentration and the decrease in oxygen level favoured ethanol yield for both yeasts but in K. marxianus UFV-3 the effect was more pronounced. Under hypoxic conditions, the activities of β-galactosidase and pyruvate decarboxylase from K. marxianus UFV-3 were significantly higher than those in K. lactis JA6. The expression of the LAC4 (β-galactosidase), RAG6 (pyruvate decarboxylase), GAL7 (galactose-1-phosphate uridylyltransferase) and GAL10 (epimerase) genes in K. marxianus UFV-3 was higher under hypoxic conditions than under aerobic conditions. The high expression of genes of the Leloir pathway, LAC4 and RAG6, associated with the high activity of β-galactosidase and pyruvate decarboxylase contribute to the high fermentative flux in K. marxianus UFV-3. These data on the fermentative metabolism of K. marxianus UFV-3 will be useful for optimising the conversion of cheese whey lactose to ethanol.Item Optimizing and validating the production of ethanol from cheese whey permeate by Kluyveromyces marxianus UFV-3(Biocatalysis and Agricultural Biotechnology, 2014-04) Diniz, Raphael H. S.; Rodrigues, Marina Q. R. B.; Fietto, Luciano G.; Passos, Flávia M. L.; Silveira, Wendel B.The purpose of this study was to optimize the production of ethanol from cheese whey permeate using Kluyveromyces marxianus UFV-3. We used the response surface methodology (RSM) with a central composite rotational design (CCRD) to evaluate the effects of pH (4.5–6.5), temperature (30–45 °C), lactose concentration (50–250 g l^−1), and cell biomass concentration (A600 2–4). We performed 29 fermentations under hypoxia in cheese whey permeate and seven fermentations for the validation of the equation obtained via RSM. Temperature was the most significant factor in optimizing ethanol production, followed by pH, cell biomass concentration and lactose concentration. The conditions for producing ethanol at yields above 90% were as follows: temperature between 33.3 and 38.5 °C, pH between 4.7 and 5.7, cell biomass concentration between A600 2.4 and 3.3, and lactose concentration between 50 and 108 g l^−1. The equation generated from the optimization process was validated and exhibited excellent bias and accuracy values for the future use of this model in scaling up the fermentation process.Item Production and Characterization of β-Glucanase Secreted by the Yeast Kluyveromyces marxianus(Applied Biochemistry and Biotechnology, 2014-01-04) Lopes, Mariana R.; Souza, Carlos J. A. de; Rodrigues, Marina Q. R. B.; Costa, Daniela A.; Santos, Ancély F. dos; Oliveira, Leandro L. de; Ramos, Humberto J. O.; Guimarães, Valéria M.; Silveira, Wendel B.; Passos, Flávia M. L.; Fietto, Luciano G.An extracellular β-glucanase secreted by Kluyveromyces marxianus was identified for the first time. The optimal conditions for the production of this enzyme were evaluated by response surface methodology. The optimal conditions to produce β-glucanase were a glucose concentration of 4 % (w/v), a pH of 5.5, and an incubation temperature of 35 °C. Response surface methodology was also used to determine the pH and temperature required for the optimal enzymatic activity. The highest enzyme activity was obtained at a pH of 5.5 and a temperature of 55 °C. Furthermore, the enzyme was partially purified and sequenced, and its specificity for different substrates was evaluated. The results suggest that the enzyme is an endo-β-1,3(4)-glucanase. After optimizing the conditions for β-glucanase production, the culture supernatant was found to be effective in digesting the cell wall of the yeast Saccharomyces cerevisiae, showing the great potential of β-glucanase in the biotechnological production of soluble β-glucan.