Integrated process production and extraction of the fibrinolytic protease from Bacillus sp. UFPEDA 485

Fibrinolytic proteases are enzymes that degrade fibrin; these enzymes are a promising alternative for thrombolytic therapy, and microorganisms produce them. The aim of this study was to evaluate the optimum conditions for the integrated production and purification of fibrinolytic protease from Bacillus sp. UFPEDA 485. Extractive fermentation was carried out in a culture medium containing soybean flour and by adding polyethylene glycol (PEG) and Na2SO4 according to a 23 experimental design. In all assays, the enzyme preferentially partitioned to the bottom phase (K < 1), with an optimum activity of 835 U ml−1 in the bottom phase (salt-rich phase). The best conditions for extractive fermentation were obtained with 18 % PEG 8000 and 13 % Na2SO4. Characterization showed that it is a metalloprotease, as a strong inhibition—residual activity of 3.13 %—occurred in the presence of ethylenediaminetetraacetic acid. It was also observed that enzymatic activity was stimulated in the presence of ions: CaCl2 (440 %), MgCl2 (440 %), FeSO4 (268 %), and KCl (268 %). The obtained results indicate that the use of a low-cost substrate and the integration of fermentation with an aqueous two-phase system extraction may be an interesting alternative for the production of fibrinolytic protease.The authors thank CAPES (National Council for the Improvement of Higher Education) for the scholarship and CNPq (National Counsel of Technological and Scientific Development) and RENORBIO for the financial support

Production and characterization of new fibrinolytic protease from Mucor subtillissimus UCP 1262 in solid-state fermentation

Fibrinolytic enzymes have received attention regarding their medicinal potential for thrombolytic diseases, a leading cause of morbidity and mortality worldwide. Various natural enzymes purified from animal, plant and microbial sources have been extensively studied. The aim of this work was to produce fibrinolytic protease by solid state fermentation using agro industrial substrates. Rhizopus arrhizus var. arrhizus UCP 1295 and Mucor subtillissimus UCP 1262 filamentous fungi species isolated from soil of Caatinga-PE, Brasil, were used as producer microorganisms. Wheat bran was shown to be the best substrate for the production of the enzyme and by using a 23 full factorial design the main effects and interactions of the quantity of the substrate wheat bran, moisture and temperature on the fibrinolytic enzyme production and protease were evaluated. The best results for fibrinolytic and protease activities, 144.58 U/mL and 48.33 U/mL, respectively, were obtained with Mucor subtillissimus UCP 1262 using as culture medium 3 g wheat bran, 50% moisture at a temperature of 25˚C for 72 hours. The optimum temperature for the produced enzyme was 45˚C and most of its original activity was retained after being subjected to 80˚C for 120 min. The protease activity was enhanced by K+, Ca+ and Mn+; but with Cu+ there was an inhibition. The specificity to chromogenic substrate and the inhibition by PMSF indicates that it is a chymotrypsin-like serine protease. Presented results suggest that this enzyme produced by solid-state fermentation is an interesting alternative as a candidate for thrombolytic therapy

Evaluation the best condition of Fibrinolytic protease production using factorial design by Streptomyces sp DPUA 1573

XI Reunião Regional Nordeste da SBBq | 4th International Symposium in Biochemistry of Macromolecules and BiotechnologyFibrinolytic enzymes have the ability to degrade fibrin clots formed for avoiding intravascular thrombosis. In the pharmaceutical industry there is a search for new fibrinolytic agent that reduces the production cost and increasing productivity. The use of microorganism for enzyme production, such as the genus Streptomyces has been reported. Streptomyces is a Gram-positive bacteria, responsible for producing many bioactive compounds and extracellular enzymes of pharmaceutical interest. This study aimed to evaluated the production of fibrinolytic protease by Streptomyces sp DPUA 1573. Microbial cells were cultivated in the ISP2 for 48 hours, after this period the strains were inoculated in MS2 (soybean medium) that according with factorial design 24 (concentrations of soybean 0.5; 1.0 and 1.5%, glucose 0; 0.5 and 1.0% and different speeds 150 rpm; 200 rpm and 250 rpm and temperature 28C; 30C and 32C). The factorial design was analyzed by variance analysis (anova) and the glucose concentration showed a positive and significative effect. The results showed that the variable interaction had significant effect. that the best condition was composed 1.5% soybean, 1% glucose, 28 ºC and 150 speed in 48 hours, with production fibrinolytic 1391.66 U/mL. These values were higher than those reported in the literature. However these results show the biggest potencial in production fibrinolytic enzyme by Streptomyces.info:eu-repo/semantics/publishedVersio

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P &lt; 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Protease fibrinolítica de Mucor subtilissimus UCP 1262 : produção, purificação, caracterização bioquímica e estrutural

Proteases fibrinolíticas são enzimas que degradam a fibrina, o principal componente dos coágulos sanguíneos. O acúmulo da fibrina nos vasos sanguíneos leva a trombose, fenômeno responsável por doenças cardiovasculares. Uma alternativa promissora para a terapia trombolítica tem sido a produção dessas enzimas por micro-organismos que promovem baixo custo, alta eficiência e capacidade de produção em larga escala. Produzir proteases fibrinolíticas por linhagens de fungos filamentosos por fermentação submersa e desenvolver o processo de purificação utilizando Sistemas de Duas Fases Aquosas (SDFA) e cromatografia líquida, além de caracterizar bioquímico e estruturalmente a enzima. Dentre as 36 espécies estudadas, 58% apresentaram atividade fibrinolítica acima de 100 U/mL A espécie com maior atividade foi Mucor subtilissimus UCP 1262 com 415 U/mL. Foram realizados processos fermentativos que resultaram na produção de 1075 U/mL de atividade fibrinolítica, com capacidade de degradação do coágulo sanguíneo de 16,7% in vitro. A extração da protease fibrinolítica produzida por fermentação submersa foi realizada utilizando o sistema de duas fases aquosas (SDFA) com Polietileno glicol (PEG) e sulfato de amônio. O PEG 8000 (g/mol) a 15% e sulfato de amônio a 25% foi selecionado como a condição mais eficiente para a extração da enzima na fase do sal, apresentando 345 U/mL de atividade, coeficiente de partição K=0,65; Recuperação Y=253,1% e Fator de purificação FP=8,8. A protease fibrinolítica produzida por Mucor subitilissimus UCP 1262 foi também pré-purificada utilizando fermentação extrativa com SDFA (PEG e sulfato de amônio), onde a espécie fúngica foi capaz de crescer mesmo em altas concentrações de sal, produzir e extrair simultaneamente para a fase do PEG do sistema. A protease fibrinolítica foi purificada também através de métodos cromatográficos utilizando um protocolo de purificação com dois passos. O extrato bruto inicial com a enzima foi pré-clarificado utilizando precipitação com acetona e adsorção em cromatografia de troca-iônica em DEAE-sephadex G50, o qual foi capaz de aumentar a pureza em 5,30 vezes com a recuperação de 36, 31%. O sistema de eletroforese bidimensional 2DE acoplado ao SDS-PAGE mostrou uma banda única de aproximadamente 15,3 kDa e a focalização isoelétrica apresentou o ponto isoelétrico no pH 3,9, exibindo uma natureza de enzima ácida. Adicionalmente a enzima foi significativamente inibida por PMSF e alta afinidade catalítica para o substrato sintético amidolítico N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAApNA) e azocaseína. Sugerindo ser uma serino-protease semelhante à quimotripsina. Desdobramento proteico induzido por pH e temperatura foram aplicados para estudar as mudanças conformacionais da enzima e mostraram através da curva de desnaturação térmica, mudança da elipticidade a 222 nm, indicando um Tm (Temperatura de desnaturação) da proteína de 58,14°C. O dicroísmo circular no UV distante (far UV CD) da protease fibrinolítica mostrou a estrutura secundária da proteína com maior teor de α-hélix. Estes resultados demonstram um protocolo de purificação de enzimas eficiente. E o estudo da enzima purificada estabeleceu bases para elucidar mecanismos responsáveis pelas mudanças de conformação de uma nova enzima fibrinolítica sob a variação de condições variadas de temperatura e pH. Esta enzima fibrinolítica pode representar uma nova fonte de agente terapêutico no tratamento de doenças trombolíticas.Fibrinolytic proteases are enzymes that degrade fibrin, the main component of blood clots. The accumulation of this protein leads to thrombosis responsible for cardiovascular disease including myocardial infarction. A promising alternative to thrombolytic therapy has been the production of these enzymes by microorganisms which promotes low cost, high efficiency and capacity for large scale production. This study aimed to select species of filamentous fungi isolated from Caatinga soil samples - Pernambuco - Brazil and assess their potential for production of proteases with fibrinolytic activity. Among the 36 isolates studied, 58% showed fibrinolytic activity above 100 U/mL. The microorganism with the higher activity in terms of enzyme production was Mucor subtilissimus UCP 1262 with 415 U/mL. Further optimization of the fermentation process resulted in the production of 1075 U/mL of enzymatic activity. The fibrinolytic enzyme had a capacity of enzymatic degradation of the blood clot of 16.7 % in vitro. Extraction of fibrinolytic protease produced at submerged fermentation was carried out using a PEG/ammonium sulphate aqueous two-phase system (ATPS). PEG 8000 15% and 25% ammonium sulphate were selected as the most appropriate components for extraction with Fibrinolytic Activity in salt phase: 345 U/mL; K: 0.65; Y: 253.1 % and FP: 8.8. The fibrinolytic enzyme from Mucor subitilissimus UCP 1262 was pre-purified using extractive fermentation in PEG and ammonium sulphate ATPS, in which the fungal strain was able to grown even in high salt concentration, produced and extracted simultaneously to the PEG phase. A novel protease with fibrinolytic activity was purified also by chromatographic methods using a two-step purification protocol. Compared to the crude enzyme extract, the specific activity of the enzyme increased 5.30 fold with a recovery of 36.31%. The initial crude extract with the enzyme was pre-purified using acetone precipitation and adsorbed by ion exchange chromatography on DEAE-sephadex G50. The two-dimensional electrophoresis system (2DE) coupled with SDS-PAGE showed a single protein band of approximately 15.3 kDa and isoelectric focusing point of 3.9, exhibiting a nature as an acidic enzyme. Additionally, the activity was slightly inhibited by EDTA, but significantly inhibited by PMSF and also had a higher affinity for the N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAApNA) and azocasein substrates, suggesting to be a chymotrypsin-like protease. Protein unfolding induced by pH and temperature were applied to study the protein conformational changes and showed from the thermal denaturation curve, change in ellipticity at 222 nm, indicated Tm (Melting temperature) of the protein to be 58.14°C. The far UV circular dichroism (CD) of the fibrinolytic protease showed the secondary structure with most content percentage of α-helix. These results demonstrate an economical, viable enzyme purification protocol. And studying the purified fibrinolytic enzyme have established basis for elucidating mechanisms responsible for the changes in conformation of the new fibrinolytic enzyme under varying conditions of temperature and pH. This novel fibrinolytic enzyme may represent a new source of therapeutic agents to treat thrombosis diseases.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPE

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate)

A fibrinolytic protease from M. subtilissimus UCP 1262 was recovered and partially purified by polyethylene glycol (PEG)/sodium sulfate aqueous two-phase systems (ATPS). The simultaneous influence of PEG molar mass, PEG concentration and sulfate concentration on the enzyme recovery was first investigated using a 23 full factorial design, and the Response Surface Methodology used to identify the optimum conditions for enzyme extraction by ATPS. Once the best PEG molar mass for the process had been selected (6000 g/mol), a two-factor central composite rotary design was applied to better evaluate the effects of the other two independent variables. The fibrinolytic enzyme was shown to preferentially partition to the bottom phase with a partition coefficient (K) ranging from 0.2 to 0.7. The best results in terms of enzyme purification were obtained with the system formed by 30.0% (w/w) PEG 6000 g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10.0, K of 0.2 and activity yield of 102.0%. SDSPAGE and fibrin zymography showed that the purified protease has a molecular mass of 97 kDa and an apparent isoelectric point of 5.4. When submitted to assays with different substrates and inhibitors, it showed selectivity for succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and was almost completely inhibited by phenylmethylsulfonyl fluoride, behaving as a chymotrypsin-like protease. At the optimum temperature of 37° C, the enzyme residual activity was 94 and 68% of the initial one after 120 and 150 min of incubation, respectively. This study demonstrated that M. subtilissimus protease has potent fibrinolytic activity compared with similar enzymes produced by solid-state fermentation, therefore it may be used as an agent for the prevention and therapy of thrombosis. Furthermore, it appears to have the advantages of low cost production and simple purification.The authors acknowledge the financial support of the Brazilian Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Fundacao de Amparo a Ciencia e Tecnologia do Estado de Pernambuco (FACEPE), and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq). The authors also thank the project approved in the REN-NORFUN network (MCT/CNPq/MMA/MEC/CAPES/FNDCT, Acao Transversal/FAPs, No.47/2010, Sistema Nacional de Pesquisa em Biodiversidade - SISBIOTA/Brazil)