Production and Partial Characterization of Fibrinolytic Enzyme From a Soil Isolate Aspergillus Carbonarius S-CSR-0007

Abstract

Objective: This work was undertaken with the aim of isolating and screening fungal soil isolates with fibrinolytic activity.Methods: Soil sample near slaughter house was collected and screened for fibrinolytic activity by using fibrin-agar. Enzyme production was optimized under various parameters like pH, temperature, substrate concentration and purified partially by ammonium sulphate precipitation. The stability of the partially purified enzyme was analyzed under the influence of a wide range of pH, temperature, and substrate concentrations.Results: Among the seven isolates screened, Aspergillus carbonarius S-CSR-0007 exhibited largest clear zone and was selected for further studies. Among the various substrates tested casein was found to support the highest caseinolytic activity of 816 U/ml and fibrinolytic activity of 510 U/ml. The culture supernatant of A. carbonarius S-CSR-0007 was fractionated by ammonium sulfate precipitation followed by dialysis, and maximum activity was obtained in the fraction with 80% ammonium sulfate, with an enzyme activity of 1200 U/ml using tyrosine as standard. The partially purified fibrinolytic enzyme showed optimal activity at 45 °C and pH 7.0. The enzyme was stable up to a temperature of 50 °C and pH 8.0, and the optimum substrate concentration was 4%. Conclusion:The crude enzyme showed high blood clot lysis activity, which may be a good candidate in the pharmaceutical industry. However, more studies need to be carried out to establish its clinical use.

Novel Fibrinolytic Enzyme by Scopulariopsis brevicaulis OS 3456: Production, Characterization, In vitro, and In vivo Activity

“…After the incubation period, the contents of the flasks were filtered through Whatman No.1 filter paper, and the filtrates were centrifuged at 5000rpm for 10min to obtain cell-free filtrates. The supernatant was used as the crude enzyme (Afini et al, 2016).…”

“…Fungal fibrinolytic enzymes may be better alternatives to those of bacterial origin due to their high stability, specificity, and ability to withstand extreme conditions. Thus, they are applied in the pharmaceutical industry as thrombolytic agents such as fibrinolytic proteases from Aspergillus brasiliensis AUMC 9735 ( Kotb et al, 2015), Aspergillus carbonarius S-CSR-0007 (Afini et al, 2016), Paecilomyces tenuipes (Kim et al, 2011), and Oidiodendron flavum (Tharwat, 2006).…”

Microbial Fibrinolytic Enzymes as Anti-Thrombotics: Production, Characterisation and Prodigious Biopharmaceutical Applications

“…Microorganisms exhibit a significant role in the mass-production of highly specific, low-cost fibrinolytic enzymes with feasibility of genetic modification through biotechnological approaches. During the past decades, numerous such fibrinolytic enzymes have been tested, specifically from genera Bacillus [2,5,14,15,18,[25][26][27][28][29] and Aspergillus [8,19,20,[30][31][32][33][34]. In addition, fibrinolytic enzymes with varied biochemical characteristics were obtained from bacterial species such as Streptococcus hemolyticus (Streptokinase, exudates of infected wounds) [35], Bacillus subtilis (Nattokinase, Fermented soybeans) [12], Staphylococcus aureus (Staphylokinase, human skin) [36], Bacillus sp.…”

“…Fungal enzymes, such as Aspergillus ustus 1, Arthrobotrys longa 1 and some others are able to hydrolyse fibrin at pH 6.0 [30,176]. Optimal temperature widely ranged from 20-70 • C [20,31,42,100,101,177,180], mostly approx. 50 • C for bacterial proteases [5,181] and 37-45 • C for some fungal species [20,30,62,101,104,107].…”

“…Some fibrinolytic metalloproteases require divalent ions Co 2+ , Ni 2+ , Zn 2+ for their activity [42,185]. Among inhibitors, Di-isopropyl fluorophosphate (DFP) and phenyl methyl sulfonyl fluoride (PMSF) are the frequently used irreversible serine protease inhibitors [5,19,20,27,[30][31][32]63,92]. Inhibitors such as ethylene diamine tetra acetic acid (EDTA), ethyleneglycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and 1, 10 phenanthroline strongly inhibit the fibrinolytic activity of metalloproteases [73,163,183].…”