Purification, biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262

• Published in: Bioprocess and biosystems engineering Vol. 40; no. 8; pp. 1209 - 1219
• Main 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
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-08-2017; Springer Nature B.V
• Subjects: Amino Acid Sequence; Ammonium; Ammonium sulfate; Biochemistry; Bioengineering; Biotechnology; Chemistry; Chemistry and Materials Science; Chymotrypsin; Copper; Dipeptides; Disorders; Environmental Engineering/Biotechnology; Enzymes; Fibrin; Food Science; Fractionation; Fungi; Helices; Hydrogen-Ion Concentration; Industrial and Production Engineering; Industrial Chemistry/Chemical Engineering; Ion exchange; Iron; Magnesium; Molecular structure; Molecular Weight; Mucor; Peptide Hydrolases; Polyethylene glycol; Polyethylenes; Protease; Protein structure; Proteinase; Research Paper; Secondary structure; Selectivity; Serine; Serine proteinase; Stroke; Structural analysis; Temperature; Thromboembolism; Thrombolysis; Thrombosis; Enzyme activity; Fibrinolytic enzyme; Polyethylene glycol; Circular dichroism; Mucor subtilissimus
• ISSN: 1615-7591; 1615-7605
• DOI: 10.1007/s00449-017-1781-3

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 Cu
2+
, Mg
2+
, and Fe
2+
. 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.