title: Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate).
abstract: 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.
authors.affiliation: Laboratory of Immunopathology Keizo Asami (LIKA), Federal University of Pernambuco-UFPE, Av. Prof. Moraes Lins do Rego, 50670-901 Recife, Pernambuco, Brazil.
authors.forename: Thiago Pajeú
authors.initials: TP
authors.lastname: Nascimento
authors.affiliation: Department of Morphology and Animal Physiology, Federal Rural University of Pernambuco-UFRPE, Rua Dom Manoel de Medeiros s/n, 52171-900 Recife, Pernambuco, Brazil.
authors.forename: Amanda Emmanuelle
authors.initials: AE
authors.lastname: Sales
authors.affiliation: Unit of Penedo, Federal University of Alagoas-UFAL, Av. Duque de Caxias, 1074, 57200-000 Penedo, Alagoas, Brazil.
authors.forename: Camila Souza
authors.initials: CS
authors.lastname: Porto
authors.affiliation: Department of Morphology and Animal Physiology, Federal Rural University of Pernambuco-UFRPE, Rua Dom Manoel de Medeiros s/n, 52171-900 Recife, Pernambuco, Brazil.
authors.forename: Romero Marcos Pedrosa
authors.initials: RM
authors.lastname: Brandão
authors.affiliation: Center for Research in Environmental Sciences, Catholic University of Pernambuco-UNICAP, Rua do Príncipe, 526, 50050-900 Recife, Pernambuco, Brazil.
authors.forename: Galba Maria
authors.initials: GM
authors.lastname: de Campos-Takaki
authors.affiliation: Center of Biological Engineering, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
authors.forename: José Antônio Couto
authors.initials: JA
authors.lastname: Teixeira
authors.affiliation: Department of Morphology and Animal Physiology, Federal Rural University of Pernambuco-UFRPE, Rua Dom Manoel de Medeiros s/n, 52171-900 Recife, Pernambuco, Brazil; Academic Unit of Garanhuns, Federal Rural University of Pernambuco-UFRPE, Avenida Bom Pastor, s/n, 55296-901 Garanhuns, Pernambuco, Brazil.
authors.forename: Tatiana Souza
authors.initials: TS
authors.lastname: Porto
authors.affiliation: Department of Morphology and Animal Physiology, Federal Rural University of Pernambuco-UFRPE, Rua Dom Manoel de Medeiros s/n, 52171-900 Recife, Pernambuco, Brazil.
authors.forename: Ana Lúcia Figueiredo
authors.initials: AL
authors.lastname: Porto
authors.affiliation: Department of Civil, Chemical and Environmental Engineering, Pole of Chemical Engineering, University of Genoa-UNIGE, via Opera Pia 15, 16145 Genoa, Italy. Electronic address: converti@unige.it.
authors.forename: Attilio
authors.initials: A
authors.lastname: Converti
mesh_terms: D004795:Enzyme Stability; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009089:Mucor / Q000737:chemistry / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011092:Polyethylene Glycols; D013431:Sulfates; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D013431:Sulfates; C012036:sodium sulfate; D011092:Polyethylene Glycols; D010447:Peptide Hydrolases
keywords: ATPS; Fibrinolytic protease; Mucor subtilissimus