title: Purification and partial characterization of a chymotrypsin-like serine fibrinolytic enzyme from Bacillus amyloliquefaciens FCF-11 using corn husk as a novel substrate.
abstract: 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.
authors.affiliation: Research Laboratory of Bacteriology, Department of Microbiology, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt, essamkottb@yahoo.com.
authors.forename: Essam
authors.initials: E
authors.lastname: Kotb
mesh_terms: D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000818:Animals; D001407:Bacillus / Q000201:enzymology*; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D051379:Mice; D008970:Molecular Weight; D010618:Phenanthrolines / Q000494:pharmacology; D013379:Substrate Specificity; D003313:Zea mays / Q000737:chemistry*
chemical_list: D005343:Fibrinolytic Agents; D010618:Phenanthrolines; D015082:2,2'-Dipyridyl; D004492:Edetic Acid; D002918:Chymotrypsin; C025205:1,10-phenanthroline