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

Descoloração de efluente de uma lavanderia de beneficiamento têxtil localizada em Toritama/PE por fungo filamentoso/ Effluent decoloration of a textile benefiting laundry located in Toritama / PE by filament fungus

Durante a atividade do setor industrial têxtil grandes quantidades de efluentes são gerados, configurados por sua forte coloração, condição que tem levantado preocupações devido os impactos causados por esses efluentes como, processo de eutrofização e redução da taxa fotossintética nos corpos hídricos, além de apresentarem potencial tóxicos bioacumulativo. Dessa maneira, o presente trabalho teve por objetivo analisar a potencialidade do fungo Aspergillus sp. URM 5741 no biotratamento de efluente têxtil, especialmente na descoloração do efluente de acordo com a variação da massa do adsorvente. Foram utilizadas 1g e 4g da biomassa do fungo no processo de tratamento do efluente têxtil real sob condições de agitação (120 RPM) durante 48 horas de ensaio, analisados em cinética de 120 min a cada 15 min, e posteriormente a cada 24 horas. Verificou-se que a biomassa foi capaz de descolorir 81% e 92% com 1g e 4g, respectivamente, durante 24 horas de experimento. Após 48 horas houve um aumento na absorbância observada para o tratamento com 4g, indicando que além de descolorir, outros compostos do efluente estão sendo particulados. Em síntese, o Aspergillus utilizado se mostrou como microrganismo com potencial capacidade de aplicação para o tratamento biológico de ambientes contaminados com efluentes do setor têxtil

Purification of a lectin from Cratylia mollis crude extract seed by a single step PEG/phosphate aqueous two-phase system

The partitioning and purification of lectins from the crude extract of Cratylia mollis seeds (Cramoll 1,4) was investigated in aqueous two-phase systems (ATPS). A factorial design model (24) was used to evaluate the influence of polyethylene glycol (PEG) molar mass (15008000g/mol), PEG concentration (12.517.5% w/w), phosphate (1015% w/w) concentration, and pH (68) on the differential partitioning, purification factor, and yield of the lectin. Polymer and salt concentration were the most important variables affecting partition of lectin and used to find optimum purification factor by experimental BoxBehnken design together with the response surface methodology (RSM). ATPS showed best conditions composed by 13.9% PEG1500, 15.3% phosphate buffer at pH 6, which ensured purification factor of 4.70. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis showed a single band of protein with 26.1kDa. Furthermore, results demonstrated a thermostable lectin presenting activity until 60°C and lost hemagglutinating activity at 80°C. According to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of lectins.We are grateful to the following bodies for the grants awarded: CAPES (Coordination for the Improvement of Level Personnel Superior); FACEPE (Pernambuco Science and Technology Foundation): Researcher's scholarship grant: BFP-0017-5.05/18 CNPq (National Council for Scientific Development and Technological) process: 427153/2016-6 and we also thank the reviewers for their valuable comments and suggestions as these helped us to improve the manuscript.info:eu-repo/semantics/publishedVersio

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)