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Purification,biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262 |
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| Authors: | Nascimento Thiago Pajeú Sales Amanda Emmanuelle Porto Tatiana Souza Costa Romero Marcos Pedrosa Brandão Breydo Leonid Uversky Vladimir N Porto Ana Lúcia Figueiredo Converti Attilio |
| Institution: | 1.Laboratory of Immunopathology Keizo Asami (LIKA), Federal University of Pernambuco, Av. Prof. Moraes Lins do Rego, Recife, PE, 50670-901, Brazil ;2.Department of Morphology and Animal Physiology, Federal Rural University of Pernambuco, Rua Dom Manoel de Medeiros, s/n, Dois Irmãos, Recife, PE, 52171-900, Brazil ;3.Department of Molecular Medicine and Byrd Alzheimer’s Institute, Morsani College of Medicine, University of South Florida, 12901 Bruce B Downs Blvd., Tampa, FL, 33612, USA ;4.Academic Unit of Garanhuns, Federal Rural University of Pernambuco, AvenidaBom Pastor, s/n, Boa Vista, Garanhuns, PE, 55296-901, Brazil ;5.Laboratory of New Methods in Biology, Institute for Biological Instrumentation, Russian Academy of Sciences, Institutskaya str., 7, Pushchino, Moscow Region, 142290, Russia ;6.Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Ave., Saint Petersburg, 194064, Russia ;7.Department of Civil, Chemical and Environmental Engineering, Pole of Chemical Engineering, University of Genoa, via Opera Pia 15, 16145, Genoa, Italy ; |
| Abstract: | 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. |
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