Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification and Characterization of a Thrombolytic Enzyme Produced by a New Strain of Bacillus subtilis

  • Frias, Jorge (CBA - Biotechnology Centre of Azores, Faculty of Sciences and Technology, University of Azores) ;
  • Toubarro, Duarte (CBA - Biotechnology Centre of Azores, Faculty of Sciences and Technology, University of Azores) ;
  • Fraga, Alexandra (ICVS - Life and Health Research Institute, University of Minho) ;
  • Botelho, Claudia (CEB - Centre of Biological Engineering, University of Minho) ;
  • Teixeira, Jose (CEB - Centre of Biological Engineering, University of Minho) ;
  • Pedrosa, Jorge (ICVS - Life and Health Research Institute, University of Minho) ;
  • Simoes, Nelson (CBA - Biotechnology Centre of Azores, Faculty of Sciences and Technology, University of Azores)
  • Received : 2020.08.06
  • Accepted : 2020.11.02
  • Published : 2021.02.28
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Abstract

Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alternative to other thrombolytic enzymes known to cause undesired side effects. Twenty-four Bacillus isolates were screened for production of fibrinolytic enzymes using a fibrin agar plate. Based on produced activity, isolate S127e was selected and identified as B. subtilis using the 16S rDNA gene sequence. This strain is of biotechnological interest for producing high fibrinolytic yield and consequently has potential in the industrial field. The purified fibrinolytic enzyme has a molecular mass of 27.3 kDa, a predicted pI of 6.6, and a maximal affinity for Ala-Ala-Pro-Phe. This enzyme was almost completely inhibited by chymostatin with optimal activity at 48℃ and pH 7. Specific subtilisin features were found in the gene sequence, indicating that this enzyme belongs to the BPN group of the S8 subtilisin family and was assigned as AprE127. This subtilisin increased thromboplastin time by 3.7% (37.6 to 39 s) and prothrombin time by 3.2% (12.6 to 13 s), both within normal ranges. In a whole blood euglobulin assay, this enzyme did not impair coagulation but reduced lysis time significantly. Moreover, in an in vitro assay, AprE127 completely dissolved a thrombus of about 1 cc within 50 min and, in vivo, reduced a thrombus prompted in a rat tail by 11.4% in 24 h compared to non-treated animals.

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