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This work reports the immobilization of a fibrinolytic protease (FP) from Mucor subtilissimus UCP 1262 on Fe

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).

We have previously shown that increasing the hydrophobicity of PEG by adding a methyl group to every other monomer unit allowed the resulting polymer to alter protein folding and inhibit protein aggregation to amyloid fibrils. As a continuation of this work, we analyzed here the effects of this substitution on the structural properties of proteins capable of adopting multiple conformations (folded, and different partially folded states, e.g. a molten globule-like intermediate) at mild denaturing conditions. To this end, we have selected several proteins (α-lactalbumin, apomyoglobin, carbonic anhydrase, staphylococcal nuclease, and cytochrome c) and examined them at different conditions where they exist in different partially folded conformations (pH, temperature, salt concentrations, presence of cofactors). We were especially interested in the relative sensitivity of partially folded (e.g. molten globule) conformations of these proteins to the presence of polymers as these conformations are often the most sensitive to the environment. We used far-UV CD to test the changes in the protein secondary structure, near-UV CD to monitor changes in the tertiary structure, and quenching of intrinsic protein fluorescence by acrylamide to evaluate changes in the solvent accessibility of aromatic residues. We found that the complexity of the effect of polymers on protein structure cannot be ascribed solely to macromolecular crowding since the behavior of proteins in solutions containing polymers is dependent on protein and polymer structure. We also cannot exclude the possibility that the structures of both proteins and polymers determine the balance between attractive and repulsive forces that drive protein–polymer interactions.

We examined the effects of water-soluble polymers of various degrees of hydrophobicity on the folding and aggregation of proteins. The polymers we chose were polyethylene glycol (PEG) and UCON (1:1 copolymer of ethylene glycol and propylene glycol). The presence of additional methyl groups in UCON makes it more hydrophobic than PEG. Our earlier analysis revealed that similarly sized PEG and UCON produced different changes in the solvent properties of water in their solutions and induced morphologically different α-synuclein aggregates [Ferreira, L. A., et al. (2015) Role of solvent properties of aqueous media in macromolecular crowding effects. J. Biomol. Struct. Dyn., in press]. To improve our understanding of molecular mechanisms defining behavior of proteins in a crowded environment, we tested the effects of these polymers on secondary and tertiary structure and aromatic residue solvent accessibility of 10 proteins [five folded proteins, two hybrid proteins; i.e., protein containing ordered and disordered domains, and three intrinsically disordered proteins (IDPs)] and on the aggregation kinetics of insulin and α-synuclein. We found that effects of both polymers on secondary and tertiary structures of folded and hybrid proteins were rather limited with slight unfolding observed in some cases. Solvent accessibility of aromatic residues was significantly increased for the majority of the studied proteins in the presence of UCON but not PEG. PEG also accelerated the aggregation of protein into amyloid fibrils, whereas UCON promoted aggregation to amyloid oligomers instead. These results indicate that even a relatively small change in polymer structure leads to a significant change in the effect of this polymer on protein folding and aggregation. This is an indication that protein folding and especially aggregation are highly sensitive to the presence of other macromolecules, and an excluded volume effect is insufficient to describe their effect.

We analyzed the effect of a natural osmolyte, trimethylamine N-oxide (TMAO), on structural properties and conformational stabilities of several proteins under macromolecular crowding conditions by a set of biophysical techniques. We also used the solvent interaction analysis method to look at the peculiarities of the TMAO-protein interactions under crowded conditions. To this end, we analyzed the partitioning of these proteins in TMAO-free and TMAO-containing aqueous two-phase systems (ATPSs). These ATPSs had the same polymer composition of 6.0 wt.% PEG-8000 and 12.0 wt.% dextran-75, and same ionic composition of 0.01 M K/NaPB, pH 7.4. These analyses revealed that there is no direct interaction of TMAO with proteins, suggesting that the TMAO effects on the protein structure in crowded solutions occur via the effects of this osmolyte on solvent properties of aqueous media. The effects of TMAO on protein structure in the presence of polymers were rather complex and protein-specific. Curiously, our study revealed that in highly concentrated polymer solutions, TMAO does not always act to promote further protein folding.

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.

Fibrinolytic proteases are enzymes that degrade fibrin. They provide a promising alternative to existing drugs for thrombolytic therapy. A protease isolated from the filamentous fungus Mucor subtilissimus UCP 1262 was purified in three steps by ammonium sulfate fractionation, ion exchange, and molecular exclusion chromatographies, and characterized biochemically and structurally. The purified protease exhibited a molecular mass of 20 kDa, an apparent isoelectric point of 4.94 and a secondary structure composed mainly of α-helices. Selectivity for N-succinyl-Ala–Ala–Pro–Phe-p-nitroanilide as substrate suggests that this enzyme is a chymotrypsin-like serine protease, whose activity was enhanced by the addition of Cu2+, Mg2+, and Fe2+. The enzyme showed a fibrinolytic activity of 22.53 U/mL at 40 °C and its contact with polyethylene glycol did not lead to any significant alteration of its secondary structure. This protein represents an important example of a novel fibrinolytic enzyme with potential use in the treatment of thromboembolic disorders such as strokes, pulmonary emboli, and deep vein thrombosis.

The capacity of fibrinolytic enzymes to degrade blood clots makes them of high relevance in medicine and in the pharmaceutical industry. In this work, forty-three microorganisms of the genus Bacillus were evaluated for their potential to produce fibrinolytic proteases. Thirty bacteria were confirmed as producers of fibrinolytic enzymes, the best results obtained for the strain Bacillus amyloliquefaciens UFPEDA 485. The optimization of the enzyme production conditions was done by a central composite design (CCD) star 23 that allowed to define the optimal conditions for soybean flour and glucose concentrations and agitation rate. The highest fibrinolytic activity (FA) of 813 U mL-1 and a degradation of blood clot in vitro of 62% were obtained in a medium with 2% (w/v) of soybean flour and 1% (w/v) glucose at 200 rpm after 48 h of cultivation, at pH 7.2 and 37 °C. The obtained fibrinolytic enzyme was characterized biochemically. Fibrinolytic activity was inhibited by PMSF (fluoride methylphenylsulfonyl - C7H7FO2S) 91.52% and EDTA (ethylenediaminetetraacetic acid - C10H16N2O8) 89.4%, confirming to be a serine- metallo protease. The optimum pH and temperature were 7.0 and 37 oC, respectively, and the enzyme was stable for 12 h. The fibrinolytic activity at physiological conditions of this enzyme produced by Bacillus amyloliquefaciens UFPEDA 485, as well as its long term stability, demonstrate that it has suitable characteristics for human and veterinary applications, and promises to be a powerful drug for the treatment of vascular diseases., We express our thanks to Coordination for the Improvement of Higher Level Education Personnel (CAPES) - Doctoral Sandwich Program (PDSE) Nº 0259/ 12-8 and National Council for Scientific and Technological Development (CNPq) - Nº 202026/2011-6 for the financial support.

As bactérias são abundantes na microbiota do solo e são responsáveis por diversas transformações enzimáticas que auxiliam na decomposição e mineralização de diversos nutrientes. Lodo de esgoto é um resíduo gerado durante tratamento biológico de água residuária, rico em matéria orgânica e nutrientes, possuindo muitas vezes em sua composição bactérias patogênicas e metais pesados. Foram feitas coletas de lodo de esgoto na Estação de Tratamento Mangueira, Recife (PE) para isolamento, identificação, detecção enzimática de bactérias mesofílicas (amilase e urease) a 28 e a 37 oC e detecção de metais pesados. Os resultados obtidos demonstraram que 28 oC apresentou maior quantidade de isolados. Em ambas as temperaturas, encontraram-se bactérias patogênicas, e nos ensaios enzimáticos, melhores resultados foram obtidos a 28 oC. A detenção de urease foi observada nas duas temperaturas. Detectou-se a presença de metais pesados no lodo: cádmio, 2,2 ± 0,0004; zinco, 600 ± 0,01, e cobre, 909,8 ± 0,001 em mg/kg, respectivamente.

A novel protease with fibrinolytic activity was purified from MucorsubtilissimusUCP 1262usinga two-step purification protocol.The enzyme was pre-purified using cetonic precipitation and adsorbed by ion exchange chromatography on DEAE-sephadex G50. System two-dimensional electrophoresis (2DE) coupled to SDS-PAGE showed a single protein band of15.3kDa approximatelyand isoeletric focusing point of 3.9, exhibiting a nature as an acidic enzyme.The activity was suppressed by Co 2+ and HgCl2, but slightly enhanced byCa 2+ . Additionally, the activity was slightly inhibited by EDTA, but significantlyinhibited by PMSF. It exhibited fibrinolytic activity,which is weaker than that of plasmin, but also had a higher affinity for the N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAAPNA) and azocasein substrates, suggesting a chymotrypsin-like protease. These results demonstrated an economically viable protocol purification of a protease with fibrinolytic activity with high degree of purity that may represent a potential source of new therapeutic agents to treat thrombosis.

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, 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.