DEFAR - Departamento de Farmácia
URI permanente desta comunidadehttp://www.hml.repositorio.ufop.br/handle/123456789/530
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Item High‑affinity transport, cyanide‑resistant respiration, and ethanol production under aerobiosis underlying efficient high glycerol consumption by Wickerhamomyces anomalus.(2019) Cunha, Aureliano Claret da; Gomes, Lorena Soares; Santos, Fernanda Godoy; Oliveira, Fábio Faria; Teixeira, Janaina Aparecida; Sampaio, Geraldo Magela Santos; Trópia, Maria José Magalhães; Castro, Ieso de Miranda; Lucas, Cândida Manuel Ribeiro Simões; Brandão, Rogélio LopesWickerhamomyces anomalus strain LBCM1105 was originally isolated from the wort of cachaça (the Brazilian fermented sugarcane juice-derived Brazilian spirit) and has been shown to grow exceptionally well at high amounts of glycerol. This paramount residue from the biodiesel industry is a promising cheap carbon source for yeast biotechnology. The assessment of the physiological traits underlying the W. anomalus glycerol consumption ability in opposition to Saccharomyces cerevisiae is presented. A new WaStl1 concentrative glycerol-H+ symporter with twice the affinity of S. cerevisiae was identified. As in this yeast, WaSTL1 is repressed by glucose and derepressed/induced by glycerol but much more highly expressed. Moreover, LBCM1105 aerobically growing on glycerol was found to produce ethanol, providing a redox escape to compensate the redox imbalance at the level of cyanide-resistant respiration (CRR) and glycerol 3P shuttle. This work is critical for understanding the utilization of glycerol by non-Saccharomyces yeasts being indispensable to consider their industrial application feeding on biodiesel residue.Item Sugar transport systems in Kluyveromyces marxianus CCT 7735.(2019) Silveira, Fernando Augusto da; Diniz, Raphael Hermano Santos; Sampaio, Geraldo Magela Santos; Brandão, Rogélio Lopes; Silveira, Wendel Batista da; Castro, Ieso de MirandaThe pattern of glucose repression in most Kluyveromyces marxianus strains does not correlate with fermentative behaviour; however, glucose repression and fermentative metabolism appear to be linked to the kinetics of sugar uptake. In this work, we show that lactose transport in K. marxianus CCT 7735 by lactose-grown cells is mediated by a low-affinity H+-sugar symporter. This system is glucose repressed and able to transport galactose with low affinity. We also observed the activity of a distinct lactose transporter in response to raffinose. Regarding glucose uptake, specificities of at least three low-affinity systems rely on the carbon source available in a given growth medium. Interestingly, it was observed only one high-affinity system is able to transport both glucose and galactose. We also showed that K. marxianus CCT 7735 regulates the expression of sugar transport systems in response to glucose availability.Item Cachaça yeast strains : alternative starters to produce beer and bioethanol.(2018) Araújo, Thalita Macedo; Souza, Magalhaes Teixeira de; Diniz, Raphael Hermano Santos; Yamakawa, Celina Kiyomi; Soares, Lauren Bergmann; Lenczak, Jaciane Lutz; Oliveira, Juliana Velasco de Castro; Goldman, Gustavo Henrique; Barbosa, Edilene Alves; Campos, Anna Clara Silva; Castro, Ieso de Miranda; Brandão, Rogélio LopesThis work was performed to verify the potential of yeast strains isolated from cachaça distilleries for two specific biotechnological applications: beer and bioethanol production. In the beer production, the strains were tested for characteristics required in brewery practices, such as: capacity to ferment maltose and maltotriose, ability to grow at lowest temperatures, low H2S production, and flocculation profile. Among the strains tested, two of them showed appropriate characteristics to produce two different beer styles: lager and ale. Moreover, both strains were tested for cachaça production and the results confirmed the capacity of these strains to improve the quality of cachaça. In the bioethanol production, the fermentation process was performed similarly to that used by bioethanol industries: recycling of yeast biomass in the fermentative process with sulfuric acid washings (pH 2.0). The production of ethanol, glycerol, organic acids, dry cell weight, carbohydrate consumption, and cellular viability were analyzed. One strain presented fermentative parameters similar to PE2, industrial/commercial strain, with equivalent ethanol yields and cellular viability during all fermentative cycles. This work demonstrates that cachaça distilleries seem to be an interesting environment to select new yeast strains to be used in biotechnology applications as beer and bioethanol production.Item Lpx1p links glucose-induced calcium signaling and plasma membrane H+-ATPase activation in Saccharomyces cerevisiae cells.(2017) Castanheira, Diogo Dias; Santana, Eduardo Perovano; Santos, Fernanda Godoy; Diniz, Raphael Hermano Santos; Oliveira, Fábio Faria; Pereira, Renata Rebeca; Trópia, Maria José Magalhães; Castro, Ieso de Miranda; Brandão, Rogélio LopesIn yeast, as in other eukaryotes, calcium plays an essential role in signaling transduction to regulate different processes. Many pieces of evidence suggest that glucose-induced activation of plasma membrane H+-ATPase, essential for yeast physiology, is related to calcium signaling. Until now, it was not identified any protein that could be regulated by calcium in this context. Lpx1p, a serine-protease that is also involved in the glucose-induced activation of the plasma membrane H+-ATPase activation, could be a candidate to respond to intracellular calcium signaling involved in this process. In this work, and by using different approaches, we showed many pieces of evidence suggesting that the requirement of calcium signaling for activation of the plasma membrane H+-ATPase is due to its requirement for activation of Lpx1p. According to the current model, activation of Lpx1p would cause hydrolysis of an acetylated tubulin that keeps the plasma membrane H+-ATPase in an inactive state. Therefore, after its activation, Lpx1p would hydrolyze the acetylated tubulin making the plasma membrane H+-ATPase phosphorylation accessible for at least one protein kinase.Item New lager brewery strains obtained by crossing techniques using cachaça (Brazilian Spirit) yeasts.(2017) Figueiredo, Bruna Inez Carvalho de; Saraiva, Margarete Alice Fontes; Pimenta, Paloma Patrick de Souza; Testasicca, Miriam Conceição de Souza; Sampaio, Geraldo Magela Santos; Cunha, Aureliano Claret da; Afonso, Luís Carlos Crocco; Queiroz, Marisa Vieira de; Castro, Ieso de Miranda; Brandão, Rogélio LopesThe development of hybrids has been an effective approach to generate novel yeast strains with optimal technological profile for use in beer production. This study describes the generation of a new yeast strain for lager beer production by direct mating between two Saccharomyces cerevisiae strains isolated from cachaça distilleries: one that was strongly flocculent, and the other with higher production of acetate esters. The first step in this procedure was to analyze the sporulation ability and reproductive cycle of strains belonging to a specific collection of yeasts isolated from cachaça fermentation vats. Most strains showed high rates of sporulation, spore viability, and homothallic behavior. In order to obtain new yeast strains with desirable properties useful for lager beer production, we compare haploid-to-haploid and diploid-to-diploid mating procedures. Moreover, an assessment of parental phenotype traits showed that the segregant diploid C2-1d generated from a diploid-to-diploid mating experiment showed good fermentation performance at low temperature, high flocculation capacity, and desirable production of acetate esters that was significantly better than that of one type lager strain. Therefore, strain C2-1d might be an important candidate for the production of lager beer, with distinct fruit traces and originating using a non-genetically modified organism (GMO) approach.Item Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lin.(2002) Barata, Ricardo Andrade; Andrade, Milton Hércules Guerra de; Rodrigues, Roberta Dias; Castro, Ieso de MirandaAn alkaline serineprotease, capable of hydrolyzing Nu-benzoyl-DL arginine p-nitroanilide, was secreted by Fusurium oxysporum var. hi grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, aMnity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45OC and showed a rapid decrease of activity at 48OC. The optimum pH was around 8.0. Characterization of the protease showed that Ca*+ and MgZ+ cations increased the activity, which was not inhibited by EDTA or l,lO-phenanthroline. The enzyme activity on Nubenzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride,p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and IVtosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5x lo-4 M. The protease had a Michaelis-Menten constant of 0.16 mM and a V,, of 0.60 pm01 released product .min-‘.mg’ enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nu-benzoyl-DL arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A Ki value of 0.04 mM was obtained.Item Quality improvement and geographical indication of cachaça (Brazilian spirit) by using local selected yeast strains.(2016) Barbosa, Edilene Alves; Souza, Magalhães Teixeira de; Diniz, Raphael Hermano Santos; Santos, Fernanda Godoy; Oliveira, Fábio Faria; Correa, Lygia Fátima da Mata; Alvarez, Florencia; Coutrim, Maurício Xavier; Afonso, Robson José de Cássia Franco; Castro, Ieso de Miranda; Brandão, Rogélio LopesAims: In order to improve the quality and to create a biological basis for obtainment of the protected denomination of origin (PDO), indigenous yeast were isolated and characterized for use in Salinas city (the Brazilian region of quality cachac a production). Material and methods: Seven thousand and two hundred yeast colonies from 15 Salinas city distilleries were screened based on their fermentative behaviour and the physicochemical composition of cachac a. Molecular polymorphic analyses were performed to characterize these isolates. Results: Two Saccharomyces cerevisiae strains (nos. 678 and 680) showed appropriate characteristics to use in the cachac a production: low levels of acetaldehyde and methanol, and high ethyl lactate/ethyl acetate ratio respectively. They also presented polymorphic characteristics more closely related between themselves even when compared to other strains from Salinas. Conclusions: The application of selected yeast to cachac a production can contribute for the improvement of the quality product as well as be used as a natural marker for PDO. Significance and Impact of the Study: This study suggests that the use of selected yeast strains could contribute to obtain a cachac a similar to those produced traditionally, while getting wide acceptation in the market, yet presenting more homogeneous organoleptic characteristics, and thus contributing to the PDO implementation.Item Genetic ancestry is associated with systolic blood pressure and glucose in Brazilian children and adolescents.(2016) Queiroz, Erica Maria de; Barbosa, Priscila Oliveira; Candido, Ana Paula; Castro, Ieso de Miranda; Coelho, George Luiz Lins Machado; Leite, Tailce Moura; Pereira, Rinaldo Wellerson; Freitas, Renata Nascimento deBackground: Studies in admixed populations show that the prevalence of obesity and related diseases, such as type 2 diabetes and hypertension, may vary by ethnic group. The aim of this study was to investigate the relationship of genetic ancestry with phenotypes associated with obesity in a sample of school children and adolescents from Ouro Preto, Minas Gerais. Methods: We used data from genetic ancestry of 189 individuals previously determined by 15 ancestry informative markers (AIMs), and segregated individuals into three ancestral groups (predominantly African (PAFR), predominantly mixed (PMIX), and predominantly European (PEUR)) using the proportion of ancestry. The ancestral groups were compared with mean values of anthropometric, clinical, biochemical, and demographic variables. The simple linear regression analysis was used to test whether differences in mean values of the dependent variables (blood pressure and glucose) between the ancestral groups were dependent on the other variables. Results: Our results show that the proportions of African (F = 144.2, P < 0.001), Amerindian (F = 15.5, P < 0.001) and European (F = 184.9, P < 0.001) ancestry differed significantly (P < 0.001) among the three ancestral groups. PAFR individuals had higher mean blood pressure (P ≤ 0.029) and glucose (P = 0.025) as compared to PEUR. In the linear regression model, the difference in systolic blood pressure (SBP) values remained significant in all models tested and independent of confounding variables (P ≤ 0.041). The difference in diastolic blood pressure values observed in PAFR and PEUR groups did not remain significant when the metabolic profile was included in the tested model (P = 0.097). The difference in glucose values was significant only between PMIX and PEUR groups and independent of the settings (P ≤ 0.037). Conclusion: The positive correlation between genetic ancestry and SBP and glucose in Brazilian children and adolescents suggests the need for special care in the subgroups of this population.Item Adhesion on yeast cell surface as a trapping mechanism of pathogenic bacteria by Saccharomyces probiotics.(2012) Tiago, Fabiana da Conceição Pereira; Martins, Flaviano dos Santos; Souza, Éricka Lorenna de Sales e; Pimenta, Paulo Filemon Paolucci; Araújo, Helena Rocha Corrêa de; Castro, Ieso de Miranda; Brandão, Rogélio Lopes; Nicoli, Jacques RobertRecently, much attention has been given to the use of probiotics as an adjuvant for the prevention or treatment of gastrointestinal pathology. The great advantage of therapy with probiotics is that they have few side effects such as selection of resistant bacteria or disturbance of the intestinal microbiota, which occur when antibiotics are used. Adhesion of pathogenic bacteria onto the surface of probiotics instead of onto intestinal receptors could explain part of the probiotic effect. Thus, this study evaluated the adhesion of pathogenic bacteria onto the cell wall of Saccharomyces boulardii and Saccharomyces cerevisiae strains UFMG 905, W303 and BY4741. To understand the mechanism of adhesion of pathogens to yeast, cell-wall mutants of the parental strain of Saccharomyces cerevisiae BY4741 were used because of the difficulty of mutating polyploid yeast, as is the case for Saccharomyces cerevisiae and Saccharomyces boulardii. The tests of adhesion showed that, among 11 enteropathogenic bacteria tested, only Escherichia coli, Salmonella Typhimurium and Salmonella Typhi adhered to the surface of Saccharomyces boulardii, Saccharomyces cerevisiae UFMG 905 and Saccharomyces cerevisiae BY4741. The presence of mannose, and to some extent bile salts, inhibited this adhesion, which was not dependent on yeast viability. Among 44 cell-wall mutants of Saccharomyces cerevisiae BY4741, five lost the ability to fix the bacteria. Electron microscopy showed that the phenomenon of yeast–bacteria adhesion occurred both in vitro and in vivo (in the digestive tract of dixenic mice). In conclusion, some pathogenic bacteria were captured on the surface of Saccharomyces boulardii, Saccharomyces cerevisiae UFMG 905 and Saccharomyces cerevisiae BY4741, thus preventing their adhesion to specific receptors on the intestinal epithelium and their subsequent invasion of the host.Item Detailed search for protein kinase(s) involved in plasma membrane H+-ATPase activity regulation of yeast cells.(2015) Pereira, Renata Rebeca; Castanheira, Diogo Dias; Teixeira, Janaina Aparecida; Bouillet, Leoneide Érica Maduro; Ribeiro, Erica Milena de Castro; Trópia, Maria José Magalhães; Alvarez, Florencia; Correa, Lygia Fátima da Mata; Mota, Bruno Eduardo Fernandes; Conceição, Luís Eduardo Fernandes Rodrigues da; Castro, Ieso de Miranda; Brandão, Rogélio LopesThis study displays a screening using yeast strains deficient in protein kinases known to exist in Saccharomyces cerevisiae. From 95 viable single mutants, 20 mutants appear to be affected in the glucose-induced extracellular acidification. The mutants that are unaffected in calcium signaling were tested for their sensitivity to hygromycin B. Furthermore, we verified whether the remaining mutants produced enzymes that are appropriately incorporated at plasma membrane. Finally, we measure the kinetic properties of the enzyme in purified plasma membranes from glucose-starved as well as glucose-fermenting cells. We confirmed the kinase Ptk2 involvement in H+−ATPase regulation (increase of affinity for ATP). However, the identification of the kinase(s) responsible for phosphorylation that leads to an increase in Vmax appears to be more complex. Complementary experiments were performed to check how those protein kinases could be related to the control of the plasma membrane H+−ATPase and/or the potential membrane. In summary, our results did not permit us to identify the protein kinase(s) involved in regulating the catalytic efficiency of the plasma membrane H+−ATPase. Therefore, our results indicate that the current regulatory model based on the phosphorylation of two different sites located in the C-terminus tail of the enzyme could be inappropriate.