01. Universidade Federal Rural de Pernambuco - UFRPE (Sede)
URI permanente desta comunidadehttps://arandu.ufrpe.br/handle/123456789/1
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Resultados da Pesquisa
Item Investigação do extrato de cladódios de Cereus jamacaru quanto à composição química, potencial antimicrobiano contra Staphylococcus aureus e efeito larvicida para Aedes aegypti(2023-09-21) Guimarães, Júlia Maria Rodrigues; Pontual, Emmanuel Viana; Alves, João Victor de Oliveira; http://lattes.cnpq.br/0882174483226946; http://lattes.cnpq.br/1777060469196142; http://lattes.cnpq.br/3701748857935689Cereus jamacaru (Cactaceae), mandacaru, is a plant from the Brazilian semi-arid region that has economic importance for livestock farming and use in folk medicine. The use of currently commercialized antibiotics leads to many unwanted effects and has led to the emergence of resistant bacteria, while synthetic insecticides generally have high persistence in the environment and strong non-target toxicity. In this sense, the search for new antimicrobial agents and insecticides has grown. The aim of this work was to investigate the cladode extract of C. jamacaru regarding its chemical composition (presence of lectins, protease inhibitors and secondary metabolites), antimicrobial potential against pathogenic bacteria and larvicidal effect against Aedes aegypti. Cladodes of C. jamacaru were collected in Recife, Pernambuco. The spines were removed and the cladodes were cut into slices and air-dried (28°C, 4 days). Then, the cladodes were crushed and the powder (10 g) was homogenized (28°C, 16 h) with 0.15 M NaCl solution (100 mL) in water, using a magnetic stirrer. The mixture was filtered and centrifuged (3,000 g, 15 min) and the clear supernatant corresponded to cladode extract (CjCE) which was investigated for the presence of lectins using rabbit erythrocytes, protease inhibitor using bovine trypsin (0.1 mg/ mL) and the chromogenic and peptidomimetic substrate BApNA (8 mM), and secondary metabolites by thin layer chromatography. The content of phenolic compounds in CjCE was determined using the Folin-Ciocalteu reagent (10%, v/v) and a gallic acid standard curve, while flavonoids were quantified using the aluminum chloride reagent (20%, m/v) and quercetin as standard. Then, the extract was investigated for antioxidant activity using the DPPH, ABTS and Phosphomolybdenum methods. The antibacterial potential of CjCE was determined using bacterial strains sensitive or resistant to antibiotics by the plate microdilution method. The minimum inhibitory concentration (MIC, lowest concentration of CjCE capable of inhibiting bacterial growth by 50%) was determined. Hemolysis assay by S. aureus was performed using human erythrocytes and the effect of CjCE (128, 64 and 32 µg/mL) on hemolysis was investigated. The larvicidal potential of CjCE (0.40 to 3.5%, m/v) was evaluated by treatment (48 h) of A. aegypti larvae in the third instar (L3). CjCE caused agglutination of rabbit erythrocytes (8 UAH), suggesting the presence of lectins. The extract reduced the hydrolysis of the BApNA substrate by trypsin, indicating the presence of a protease inhibitor. CjCE thin layer chromatography revealed the presence of reducing sugars and qualitative-quantitative analysis showed 40.20±0.97 mgEAG/g of total phenols, among which, 3.36±0.07 mgEAG/g (8, 36%) were flavonoids. CjCE showed relevant oxidant activity, with the ability to scavenge ABTS and DPPH radicals (IC50 of 3,735 µg/mL and 2704.50 µg/mL, respectively), but was not able to cause reduction of phosphomolybdenum. CjCE was toxic only to the Staphylococcus aureus strain (UFPEDA 02), revealing a strong bacteriostatic effect (MIC of 199.09±0.85 µg/mL), and reduced erythrocyte lysis caused by the bacteria by more than 90%, compared to to control. The treatment of L3 of Ae. aegypti with CjCE resulted in dose-dependent mortality (LC50 = 0.68%, m/v). On the other hand, when larvae were treated with CjCE at a concentration 10 times higher than the LC50, the intestinal contents covered by the peritrophic membrane were eliminated in an attempt to eliminate the toxic components of the extract. In conclusion, C. jamacaru cladode extract is a novel antimicrobial agent capable of strongly inhibiting the growth of S. aureus and reducing bacterial toxicity to human erythrocytes. Furthermore, the toxicity to A. aegypti larvae shown here points to the C. jamacaru cladode extract as an interesting starting material for obtaining larvicidal formulations. The antibacterial and insecticidal effects of the extract may be linked to the presence of lectins, protease inhibitors and phenolic compounds.Item Análise de colinearidade gênica do operon aprX-lipA em isolados de Pseudomonas fluorescens(2023-09-15) Silva, Israel Santos da; Freitas, Nara Suzy Aguiar de; Souza, Paulo Roberto Eleutério de; http://lattes.cnpq.br/1971832245117283; http://lattes.cnpq.br/6891650997818766; http://lattes.cnpq.br/9803828236017805Pseudomonas fluorescens are Gram-negative bacilli that are motile and found in terrestrial and aquatic ecosystems. Due to their psychrotrophic characteristics, these bacteria are often associated with the contamination of unpasteurized milk and its derivatives, such as cheese and butter. The proteases and lipases produced by P. fluorescens are the primary factors in the prevalence of dairy product contamination. These enzymes are encoded by the aprX and lipA genes, which are present in the aprX-lipA operon. In this regard, we evaluated the genomic components surrounding the aprX-lipA operon of P. fluorescens from different sources, aiming to detect genetic patterns inherent to these organisms and their correlation with proteolytic activity. We used the NCBI database, the String platform, and Interpro for comparative evaluation of the selected genomes. In the four isolates analyzed, the aprX-lipA operon is highly variable structurally, with unique configurations for each genome. The gene co-expression relationships of the genes surrounding the aprX protease also show qualitative and quantitative variations, both intra- and inter-species within the Pseudomonas genus. We detected components of the CRISPR type 1 system, previously unrelated to the operon, which can amplify, move, and modify genes related to the defense mechanism, pathogenic or not, of the genus. The patterns related to pathogenicity indicate that new biomarkers can be used for genomic surveillance.Item Estudo in silico de derivados naftoquinônicos potenciais inibidores da principal protease do SARS-CoV-2(2020-11-05) Alencar, Natanael Ferreira de; Camara, Celso de Amorim; http://lattes.cnpq.br/4500025814149366; http://lattes.cnpq.br/7715134143959483Item Produção de proteases por Aspergillus ochraceus URM 604 obtidas por fermentação em estado sólido utilizando farelo de trigo e resíduo de café como substrato(2021-03-03) Santos, Amanda Lucena dos; Porto, Ana Lúcia Figueiredo; Cardoso, Kethylen Barbara Barbosa; http://lattes.cnpq.br/4784303425329040; http://lattes.cnpq.br/4989617783837981; http://lattes.cnpq.br/3697084385877618Proteases are enzymes of great commercial interest, since it has several industrial applications, such as the pharmaceutical, food, beverages and cleaning products. Among the organisms capable of producing these enzymes are filamentous fungi, having as advantages the possibility of secreting enzymes in the extracellular medium and growing on low-cost substrates. The Solid State Fermentation (SSF) is one of the recommended techniques in the cultivation of filamentous fungi, especially because it simulates its natural habitat, favoring their growth. Aiming the importance of proteases and the growing demand of the global market needs, it is necessary to search for new sources and better production methods. Thus, the objective of this research was to produce proteases by the filamentous fungus Aspergillus ochraceus URM 604 under SSF using substrates derived from the agribusiness, in 2³ factorial design. It was investigated the influence of the type of substrate (coffee residue, wheat bran and 1: 1 coffee + wheat bran), amount of substrate (3g, 5g and 7g ) and humidity (20, 40 and 60%) to determine the ideal conditions for protease production. Fermentation took place for 7 days at 30 °C and the metabolic extract was used for further analysis. For biochemical and physical analysis the protein activity, total proteins, temperature and optimal pH of the obtained enzyme were determined. When analyzing the influence of the variables adopted in the 2³ factorial design, only the type of substrate was a significant parameter. The best substrate was wheat bran, which showed a specific enzymatic activity of 218.27 U / mg under 3g of substrate and 60% humidity. The other conditions also showed high results when compared to the literature. The optimum temperature of the enzyme produced was 50°C and the optimum pH was pH 8-9 (alkaline protease). Thus, this research shows that the fungus A. ochraceus URM 604 has biotechnological potential for protease production under SSF using low-cost substrates such as coffee grounds and wheat bran, this being the first report for the species.Item Recuperação e purificação parcial de proteases colagenolíticas de tainha (Mugil liza) usando precipitação e particionamento em sistemas de fases(2020-02-03) Costa, Beatriz de Aquino Marques da; Porto, Ana Lúcia Figueiredo; http://lattes.cnpq.br/4989617783837981; http://lattes.cnpq.br/2650705679652460Proteases play an important role in the field of biotechnology studies, which is why the research for alternative sources is highly desirable. Thus, combining extraction, recovery and purification techniques to alternative sources, such as by-products of the fishery production chain, in order to generate the least possible damage to the aquatic environment is profitable for the global enzyme market. Therefore, the objective of the present work is to partially recover and purify collagenolytic proteases from Mullet (Mugil liza) digestive viscera with potential biotechnological application. For this, the digestive viscera extract (intestine, liverand a mixture of various viscera) was precipitated using ammonium sulfate ((NH4) 2SO4). In addition, the intestinal viscera were submitted to partitioning by an Aqueous Two-Phase System (ATPS) and a Three-Phase Partitioning system (TPP), to evaluate the most beneficial conditions for the purification of collagenolytic proteases. The results obtained demonstrate that, for ammonium sulphate precipitation, the best results of Purification Factor occurred in concentrations of 30-60% for all assessed extracts (intestine, liver and mix); the aqueous two-phase system (PEG/Citrate) carried out with Mullet intestinal viscera extract demonstrates that the conditions: PEG of 8000 g/mol molar mass; 20% concentration of PEG; 15% citrate concentration lead to the highest purification factor. It is also observed that the collagenases tend to migrate to the PEG-rich phase; for the three-phase system (t-butanol / (NH4) 2SO4) the highest recovery rate of collagenolytic protease was observed in the proportion of 1:0.5, obtaining recovery of 240.01% and a purification factorof 2,55. Thus, it is concluded that the tests can be used in the recovery of biomolecules from neglected organic solid waste from the fishing industry.Item Produção, extração, purificação e caracterização de proteases fibrinolíticas produzidas por Streptomyces parvulus DPUA 1573(2021-11-29) Nascimento, Maria Clara do; Bezerra, Raquel Pedrosa; Batista, Juanize Matias da Silva; http://lattes.cnpq.br/6699725036732885; http://lattes.cnpq.br/1466206759539320; http://lattes.cnpq.br/5929405825655717Due to their fibrin degradation potential, fibrinolytic proteases are a promising alternative in the pharmaceutical industry for the treatment of cardiovascular diseases, especially thrombosis. There are several sources of fibrinolytic proteases, however, the microbial sources are the ones that stand out in terms of low cost and high production rates. From their production to application, enzymes need to go through several processes, which sounds negative, making the steps more costly and late. A method capable of overcoming these problems is the aqueous two-phase system (SDFA), a process capable of reducing downstream steps. The objective of this work was to produce, purify and biochemically characterize the fibrinolytic protease produced by Streptomyces parvulus DPUA 1573. The protease was produced by submerged fermentation using agro-industrial waste or co-products. The crude extract that showed the highest enzymatic activity (passion fruit peel flour) was subjected to extraction by SDFA consisting of polyethylene glycol (PEG) and phosphate salts (potassium and sodium), following a 24 plan. After extraction by SDFA, the protease was subjected to purification by gel filtration chromatography, and already purified had its biochemical characterization performed. The protease produced by S. parvulus DPUA 1573 showed fibrinolytic activity of 15.46 U/mL and was able to form a halo of 317.31 mm2 acting on fibrin degradation. In SDFA, the fibrinolytic protease partitioned preferentially to the PEG-rich phase. The best assay selected according to the combination of the highest specific activity index, purification factor and activity yield was 16, composed of PEG 8,000 gmol-1, 17.5 v/v PEG, pH 8.0 and 15 v/v of phosphate salts. The protease activity of the enzyme was highly stimulated in the presence of iron, reaching a 55% increase in activity, and drastically decreased in the presence of the protease inhibitor 2-mercaptoethanol (91%). The optimum temperature and pH for the enzymatic activity were 40ºC and pH 7.0, respectively, keeping the enzyme activity stable between 30ºC and 60ºC and in the pH range from 7.0 to 8.5. Based on the analyzed results, it was seen that S. parvulus DPUA 1573 proved to be a good producer of fibrinolytic proteases, and the PEG/Phosphate aqueous two-phase system proved to be a great alternative for the extraction and pre-purification of fibrinolytic proteases.