Purification and partial characterization of a chymotrypsin-like serine fibrinolytic enzyme from Bacillus amyloliquefaciens FCF-11 using corn husk as a novel substrate
2014
Kotb, Essam
A non-toxic, direct-acting fibrinolytic enzyme, FCF-11, from a newly isolated Bacillus amyloliquefaciens FCF-11 was purified, characterized and assayed both in vitro and in vivo for its thrombolytic potential. Corn husk was used as for the first time as the sole carbon/nitrogen source for enzyme production. The molecular weight of the purified enzyme was 18.2 kDa and purification increased its specific activity 443.5-fold with a recovery of 17 %. Maximal activity was attained at a temperature of 40 °C and pH of 8.0. Additionally the isoelectric point of this protein was 10 ± 0.2. Tosyl lysine chloromethyl ketone, phenylmethylsulphonyl fluoride, soybean trypsin inhibitor, and aprotinin highly repressed this activity. The presence of ethylenediaminetetraacetic acid, and two metalloprotease inhibitors, 2,2′-bipyridine and o-phenanthroline, didn’t affect the enzymatic activity. Furthermore, it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like serine protease. Its apparent K ₘ and V ₘₐₓ for the synthetic substrate N-Suc-Phe-pNA were 0.45 mM and 8.26 μmoles/mg/min, respectively. FCF-11 showed direct action upon blood clots in vitro and prolonged the blood clotting time to 4.1-fold, suggesting this enzyme be a beneficial thrombolytic agent especially, with regard with low molecular weight and non specificity to other plasma proteins. FCF-11 could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, enzyme at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug.
Afficher plus [+] Moins [-]Mots clés AGROVOC
Informations bibliographiques
Cette notice bibliographique a été fournie par National Agricultural Library
Découvrez la collection de ce fournisseur de données dans AGRIS