ABSTRACT
This work reports the immobilization of a fibrinolytic protease (FP) from Mucor subtilissimus UCP 1262 on Fe3O4 magnetic nanoparticles (MNPs) produced by precipitation of FeCl3·6H2O and FeCl2·4H2O, coated with polyaniline and activated with glutaraldehyde. The FP was obtained by solid state fermentation, precipitated with 40-60% ammonium sulfate, and purified by DEAE-Sephadex A50 ion exchange chromatography. The FP immobilization procedure allowed for an enzyme retention of 52.13%. The fibrinolytic protease immobilized on magnetic nanoparticles (MNPs/FP) maintained more than 60% of activity at a temperature of 40 to 60 °C and at pH 7 to 10, when compared to the non-immobilized enzyme. MNPs and MNPs/FP did not show any cytotoxicity against HEK-293 and J774A.1 cells. MNPs/FP was not hemolytic and reduced the hemolysis induced by MNPs from 2.07% to 1.37%. Thrombus degradation by MNPs/FP demonstrated that the immobilization process guaranteed the thrombolytic activity of the enzyme. MNPs/FP showed a total degradation of the γ chain of human fibrinogen within 90 min. These results suggest that MNPs/FP may be used as an alternative strategy to treat cardiovascular diseases with a targeted release through an external magnetic field.
Subject(s)
Fibrinolytic Agents/chemistry , Magnetite Nanoparticles/chemistry , Mucor/enzymology , Peptide Hydrolases/chemistry , Peptide Hydrolases/isolation & purification , Chromatography, Ion Exchange , Enzyme Stability , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Enzymes, Immobilized/pharmacology , Fibrinogen/chemistry , Fibrinogen/metabolism , Fibrinolytic Agents/isolation & purification , Fibrinolytic Agents/pharmacology , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , Humans , Hydrogen-Ion Concentration , Mucor/chemistry , Mucor/genetics , Peptide Hydrolases/pharmacology , TemperatureABSTRACT
The partitioning and purification of lectins from the crude extract of Cratylia mollis seeds (Cramoll 1,4) was investigated in aqueous two-phase systems (ATPS). A factorial design model (24) was used to evaluate the influence of polyethylene glycol (PEG) molar mass (1500-8000 g/mol), PEG concentration (12.5-17.5% w/w), phosphate (10-15% w/w) concentration, and pH (6-8) on the differential partitioning, purification factor, and yield of the lectin. Polymer and salt concentration were the most important variables affecting partition of lectin and used to find optimum purification factor by experimental Box-Behnken design together with the response surface methodology (RSM). ATPS showed best conditions composed by 13.9% PEG1500, 15.3% phosphate buffer at pH 6, which ensured purification factor of 4.70. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of protein with 26.1 kDa. Furthermore, results demonstrated a thermostable lectin presenting activity until 60 °C and lost hemagglutinating activity at 80 °C. According to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of lectins.
