Characterization Of Fibrinolytic Enzyme Research Articles

Isolation, Extraction, Purification, and Characterization of Fibrinolytic Enzyme from Pseudomonas aeruginosa and Estimation of the Molecular Weight of the Enzyme.

Pseudomonas aeruginosa was isolated from injuries of patients' wounds and burns, and to ensure that the isolate was belonging to P. aeruginosa, several tests were performed, such as staining techniques, a biochemical test, morphological test, Vitek 2 system, and sensitivity test. The results of the gram stain test showed rod pink gram-negative bacteria, demonstrating that the isolate belonged to P. aeruginosa. Growth optimization of bacterial was performed by assessing different combinations of pH and temperatures. It is revealed that the best conditions for increasing the number of bacteria were achieved at 37°C with the bacterial number of 5.53×108 and pH 6 with the bacterial number of 5.87×108. Fibrinolytic enzyme is an agent that lysis fibrin clots. This fibrinolytic factor has prospective use to treat cardiovascular diseases, such as stroke and heart attack. Cardiovascular diseases have attracted worldwide attention for their elevation morbidity and mortality. Fibrinolytic enzyme was extracted by centrifugation at 10000 × g at 4°C for 10 min, the supernatant was kept and the pellet having bacterial cells was discarded. Purification of the fibrinolytic enzyme was achieved using salt precipitation, ion exchange, and gel filtration chromatographic techniques. The results showed that the gel filtration chromatography had optimal specific activity and purification fold at 562.6 U/ml, and the final specific activity of the purified enzyme increased 4.1 times. The molecular weight of the fibrinolytic enzyme was determined at26 kDa by gel filtration chromatography. The purified fibrinolytic enzyme had optimum activity atpH 7 and40°C.The pH stability for the enzyme activity was found in pH 6-7 and the range of 10-40°C.

Purification and Characterization of Fibrinolytic Enzyme Produced by Bacillus subtilis Egy.

A novel extracellular enzyme with strong fibrinolytic activity, produced by Bacillus subtilis Egy, which was isolated from the Egyptian soil was purified and characterized. The enzyme was secreted by cultured B. subtilis Egy under solid state fermentation and purified 21.5 purification fold using precipitation by ammonium sulphate followed by ion exchange chromatography, and gel filtration on Sephadex G- 100 chromatography. Purified enzyme showed optimum activity at 50oCreaction temperature and pH 8. Enzyme stability studies revealed that the enzymeis stable up to 30 °C and it retained 80% of its activity at 40oC.In addition, the purified enzyme is stable at pH 8-9. The fibrinolytic activity was enhanced by Mn2+,Cu2+, Ca2+, Na+, and Ba2+ and inhibited by, Zn2+, and Hg2+. Moreover, the activity was decreased by EDTA and PMSF at 2 mM final concentration.

Purification and characterization of fibrinolytic enzyme from a bacterium isolated from soil.

A novel extracellular enzyme with strong fibrinolytic activity, produced by Bacillus tequilensis, which was isolated from the soil of Zhuhai City (China) was purified and characterized. The enzyme was secreted by cultured B. tequilensis in solid state and purified at a high efficiency using the combination of salting out, ion exchange chromatography, and size exclusion chromatography. The enzyme was estimated to have a molecular weight of approximately 27kDa, pI of 8.9±0.1, to stable at pH 5.0-12.0 and up to 50°C; the optimum pH and temperature are 10.5 and 45°C (2373.59±54.81 U/mg), respectively. The fibrinolytic activity was enhanced by K+, Na+, Mg2+, Mn2+, Ca2+, and Ba2+ and inhibited by Cu2+, Zn2+, and Fe3+. Moreover, the activity was slightly enhanced by PMSF and EDTA at low concentrations and inhibited by β-mercaptoethanol. The N-terminal amino acid sequence is AQSVPYGISQI. The enzyme has a higher enzymatic activity than most other fibrinolytic enzymes. The high thermal stability indicated that it is easy to preserve and could be activated under high-temperature conditions.

Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media.

Intravascular thrombosis is one of the major causes of variety of cardiovascular disorders leading to high mortality worldwide. Fibrinolytic enzymes from microbial sources possess ability to dissolve these clots and help to circumvent these problems in more efficient and safer way. In the present study, fibrinolytic protease with higher fibrinolytic activity than plasmin was obtained from Serratia sp. KG-2-1 isolated from garbage dump soil. Response surface methodology was used to study the interactive effect of concentration of maltose, yeast extract+peptone (1:1), incubation time, and pH on enzyme production and biomass. Maximum enzyme production was achieved at 33°C after 24h at neutral pH in media containing 1.5% Maltose, 4.0% yeast extract+peptone and other trace elements resulting in 1.82 folds increased production. The enzyme was purified from crude extract using ammonium sulfate precipitation and DEAE-Sephadex chromatography resulting in 12.9 fold purification with 14.9% yield. The purified enzyme belongs to metalloprotease class and had optimal activity in conditions similar to physiological environment with temperature optima of 40°C and pH optima of 8. The enzyme was found to be stable in various solvents and its activity was enhanced in presence of Na+, K+, Ba2+, Cu2+, Mn2+, Hg2+ but inhibited by Ca2+ and Fe3+. Hence, the obtained enzyme may be used as potential therapeutic agent in combating various thrombolytic disorders.

Purification and Characterization of Fibrinolytic Enzyme from Pseudoalteromonas sp., IND11 and its in vitro Activity on Blood Clot

Purification and Characterization of Fibrinolytic Enzyme from Pseudoalteromonas sp., IND11 and its in vitro Activity on Blood Clot

Production, optimization and characterization of fibrinolytic enzyme by Bacillus subtilis RJAS19.

The present study aimed at the production, purification and characterization of fibrinolytic nattokinase enzyme from the bacteria isolated from natto food. For the purpose, a fibrinolytic bacterium was isolated and identified as Bacillus subtilis based on 16S rDNA sequence analysis. The strain was employed for the production and optimization of fibrinolytic enzyme. The strain showed better enzyme production during 72nd h of incubation time with 50 degrees C at the pH 9. The lactose and peptone were found to be increasing the enzyme production rate. The enzyme produced was purified and also characterized with the help of SDS-PAGE analysis. The activity and stability profile of the purified enzyme was tested against different temperature and pH. The observations suggesting that the potential of fibrinolytic enzyme produced by Bacillus subtilis RJAS 19 for its applications in preventive medicines.

Partial Purification and Characterization of Fibrinolytic Enzymes from Yellow Mealworm

Two proteins with fibrinolytic activity were partially purified from yellow mealworm (Tenebrio molitor) by ammonium sulfate precipitation between 30 and 70% saturation, gel filtration on Sephacryl-S200-HR, ion exchange chromatography on DEAE-Sepharose-FF and metal chelate on Cu–HiTrap–IMAC–FF, but the enzymes had not been completely separated from each other. The two partially purified fibrinolytic enzymes were designated as TMFE-I and TMFE-II (Tenebrio molitor fibrinolytic enzyme) with molecular weights of 27.5 and 24.9 kDa by SDS-PAGE individually. The partially purified solution of TMFE-I and TMFE-II was considerably stable in the range of pH 5–10 and characterized by pH optimum of the enzymatic activity at 8.0. Thermal stability of TMFE was excellent at 45°C and below. The K M value was 0.26 mM for amidolysis of Bz–Arg–pNA. According to inhibitor analysis by fibrin plate method, phenylmethylsulfonyl fluoride and tosyl-lysine chloromethyl ketone inactivated TMFE almost completely, but trans-(epoxysuccinyl)-l-leucylamino-4-guanidinobutane (E-64) and EDTA had little effect on their fibrinolytic activity. According to metal ion analysis by fibrin plate method, the effect of metal ions on activity of TMFE showed a great difference. Na+, K+ and Zn2+ had little effect on the activity of TMFE. Mg2+ and Cu2+ showed inhibition effect on the fibrinolytic activity of TMFE, but Ca2+ increased the fibrinolytic activity of TMFE at final concentration varying from 0 to 30 mM.

Isolation, production, purification, assay and characterization of fibrinolytic enzymes (Nattokinase, Streptokinase and Urokinase) from bacterial sources

Nattokinase, Streptokinase and Urokinase are novel fibrinolytic enzymes which are isolated from Bacillus subtilis, β-haemolytic Streptococci and urine sample . The fibrinolytic enzyme Nattokinase, Streptokinase and Urokinase was purified from supernatant of Bacillus subtilis, β-haemolytic Streptococci and recombinant E.coli containing short fragment genomic DNA of Pseudomonas sp. Culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH of Nattokinase, Streptokinase and Urokinase were 37-55°C and 9, 27-37°C and 7 and 55°C and 9, respectively. The molecular weight of Nattokinase, Streptokinase and Urokinase was approximately 28 kDa, 47 kDa and 34 kDa, respectively, as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The caseinolytic activity of Nattokinase, Streptokinase and Urokinase were 576.73 U, 467.73 U and 785.73 U, respectively, while fibrinolytic activity achieved by fibrin plate method were 10 U, 5 U and 15 U, respectively.

Purification and characterization of fibrinolytic enzyme from Bacillus subtilis-fermented red beans

Purification and characterization of fibrinolytic enzyme from Bacillus subtilis-fermented red beans

Purification and characterization of fibrinolytic enzyme from cultured mycelia of Armillaria mellea

A fibrinolytic enzyme was purified from the cultured mycelia of Armillaria mellea by ion-exchange chromatography followed by gel filtration, and was designated A. mellea metalloprotease (AMMP). The purification protocol resulted in a 627-fold purification of the enzyme, with a final yield of 6.05%. The apparent molecular mass of the purified enzyme was estimated to be 21 kDa by SDS–PAGE, fibrin-zymography and gel filtration chromatography, which revealed a monomeric form of the enzyme. The optimal reaction pH value and temperature were, pH 6.0, and 33 °C, respectively. This protease effectively hydrolyzed fibrinogen, preferentially digesting the Aα-chain over the Bβ- and r-chains. Enzyme activity was inhibited by Cu 2+ and Co 2+, but enhanced by the addition of Ca 2+ and Mg 2+ ions. Furthermore, AMMP activity was potently inhibited by EDTA, and was found to exhibit a higher specificity for the substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 24 amino acid residues of the N-terminal sequence were MFSLSSRFFLYTLCL SAVAVSAAP, which is extremely similar to the 24 amino acid residues of the N-terminal sequence of the fruiting body of A. mellea. These data suggest that the fibrinolytic enzyme AMMP, obtained from the A. mellea exhibits a profound fibrinolytic activity. The mycelia of A. mellea may thus represent a potential source of new therapeutic agents to treat thrombosis.