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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 2(3) 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 (6000g/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 6000g/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%. SDS-PAGE and fibrin zymography showed that the purified protease has a molecular mass of 97kDa 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 150min 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.

Static magnetic field effects on proteases with fibrinolytic activity produced by Mucor subtilissimus.

The influence of a static magnetic field (SMF) on crude enzyme extracts with proteolytic activity is described and discussed. Proteolytic enzymes, which hydrolyze peptide bonds, and fibrinolytic enzymes, which dissolve fibrin clots, have industrial relevance, and applicability dependent on improvements of productivity and activity. We investigated whether a moderate SMF affects proteolysis in different in vitro tests: general proteolysis of azocasein substrate, and static and dynamic fibrinolytic processes (to compare fibrin gel configuration under exposure). Crude enzyme extracts, obtained from solid state fermentation of Mucor subtilissimus UCP (Universidade Católica de Pernambuco, Recife, Brazil) 1262, were used to carry out assays under slightly heterogeneous fields: a varied vertical SMF (for tests in Eppendorf tubes, from 0.100 to 0.170 T) and a varied horizontal SMF (for tests in Petri dishes, from 0.01 to 0.122 T), generated by two permanent magnets (NdFeB alloy). Results showed significant differences (P < 0.05) in static fibrinolysis assays after 24 h of exposure. The mean diameter of halos of fibrin degradation in the treated group increased by 21% compared to the control group; and the pixel number count of fibrin consumption (in a computational analysis of the area of each halo) enhanced by 30% with exposure. However, in dynamic fibrinolysis assays, no effects of SMF were observed. These results suggest a response of fibrin monomers to the SMF as a possible cause of the observed effects. Bioelectromagnetics. 38:109-120, 2017.

Produção e caracterização de protease fibrinolítica de Streptomyces parvulus DPUA 1573

As proteases fibrinoliticas sao capazes de degradar coagulos de fibrina formados dentro dos vasos sanguineos, evitando a trombose intravascular. Em animais, a tromboflebite, que acomete frequentemente os equinos, ocasiona, em seus casos graves, a obstrucao jugular e tambem um edema de laringe, derivando a obstrucao das vias aereas, o que possibilita um edema cerebral, ocorrendo o obito do animal. Devido ao fato de o tratamento ser de custo elevado, faz-se necessaria a investigacao de outras fontesde proteases fibrinoliticas com custos menores e com menos efeitos colaterais. Diante disso, este estudo tem como objetivo produzir e caracterizar proteases fibrinoliticas obtidas de Streptomyces parvulus DPUA 1573. Para producao da enzima, foi utilizado um planejamento fatorial 24 avaliando a concentracao da farinha de soja (0,5, 1,0 e 1,5%) e da glicose (0, 0,5 e 1,0g/L), temperatura (28, 32 e 37oC) e agitacao (150, 200 e 250rpm) sobre a biomassa e a atividade fibrinolitica. Pode-se verificar que a protease fibrinolitica apresentou atividade maxima (835U/mL) nas condicoes de concentracao de 1,5% de soja, 1g/L de glicose, 28°C e 150rpm com 48 horas de fermentacao. A protease fibrinolitica obtida teve temperatura e pH otimos de 55°C e pH 9,0, respectivamente. A atividade enzimatica foi inibida pelo EDTA, pelo ion Fe2+ e pelo SDS, o que indicou a enzima ser uma metaloprotease. A linhagem Streptomyces parvulus DPUA 1573 foi capaz de produzir protease fibrinolitica, possuindo caracteristicas bioquimicas favoraveis a aplicacao na medicina veterinaria e possivelmente humana.