Ukr.Biochem.J. 2023; Volume 95, Issue 5, Sep-Oct, pp. 98-107


Isolation and characterization of Bacillus sp. IMV B-7883 proteases

O. V. Gudzenko*, L. D. Varbanets

Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv, Ukraine;

Received: 20 July 2023; Revised: 12 September 2023;
Accepted: 27 October 2023; Available on-line: 06 November 2023

The representatives of Bacillus are some of the best protease producers studied so far since they exhibit broad substrate specificity, significant activity, stability, simple downstream purification, short period of fermentation and low cost. Earlier, we showed that Bacillus sp. IMV B-7883 strain synthesizes an extracellular proteases, which exhibit elastolytic and fibrinogenolytic activity. The aim of the work was to isolate and purify these enzymes from the culture liquid of the Bacillus sp. IMV B-7883 strain, as well as to study their properties. Isolation and purification of proteases was carried out by precipitation of the culture liquid with ammonium sulfate, gel permeation and ion exchange chromatography and rechromatography on Sepharose 6B. As a result, proteases with elastolytic and fibrinogenolytic activity with a molecular weight of 23 and 20 kDa respectively were isolated with elastase activity increased by 63.6 and fibrinogenolytic activity by 44.1 times. The enzyme with elastase activity had a pH-optimum of 7.0 and hydrolyzed only elastin, while the enzyme with fibrinogenolytic activity was an alkaline protease with a pH-optimum of 8.0 and in addition to fibrinogen, showed specificity for fibrin and, in trace amounts, for collagen.

Keywords: , , , ,


  1. Solanki P, Putatunda C, Kumar A, Bhatia R, Walia A. Microbial proteases: ubiquitous enzymes with innumerable uses. 3 Biotech. 2021;11(10):428. PubMed, PubMedCentral, CrossRef
  2. Razzaq A, Shamsi S, Ali A, Ali Q, Sajjad M, Malik A, Ashraf M. Microbial Proteases Applications. Front Bioeng Biotechnol. 2019;7:110. PubMed, PubMedCentral, CrossRef
  3. AlShaikh-Mubarak GA, Kotb E, Alabdalall AH, Aldayel MF. A survey of elastase-producing bacteria and characteristics of the most potent producer, Priestia megaterium gasm32. PLoS One. 2023;18(3):e0282963. PubMed, PubMedCentral, CrossRef
  4. Utility Model Patent No 145188. Strain Bacillus sp. – producer of extracellular elastase. Gudzenko OV, Varbanets LD, Butsenko LM, Pasichnyk LA. Bulletin No 22. 25.11.2020. (In Ukrainian).
  5. Utility Model Patent No 145577. Strain Bacillus sp. IMV B-7883 is a producer of extracellular proteinase with fibrinogenolytic activity. Gudzenko OV, Varbanets LD, Butsenko LM, Pasichnyk LA, Chernyshenko VO, Stohnii YM. Bulletin № 24. 28.12.2020 (In Ukrainian).
  6. Varbanets LD, Matseliukh EV. Peptidases of microorganisms and methods of their investigations. K: Naukova Dumka, 2014. 323 p.
  7. Chernyshenko V, Platonova T, Makogonenko Y, Rebriev A, Mikhalovska L, Chernyshenko T, Komisarenko S. Fibrin(ogen)olytic and platelet modulating activity of a novel protease from the Echis multisquamatis snake venom. Biochimie. 2014;105:76-83. PubMed, CrossRef
  8. Chernyshenko VO, Lugovska NE. Molecular mechanisms of intravascular inhibition and stimulation of extravascular thrombosis. Biotechnol Acta. 2021;14(6):5-22. CrossRef
  9. Gardiner EE, Andrews RK. The cut of the clot(h): snake venom fibrinogenases as therapeutic agents. J Thromb Haemost. 2008;6(8):1360-1362. PubMed, CrossRef
  10. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265-275. PubMed
  11. Anson ML. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J Gen Physiol. 1938;22(1):79-89. PubMed, PubMedCentral, CrossRef
  12. Nidialkova NA, Matseliukh OV, Varbanets LD. Physico-chemical properties of Bacillus thuringiensis IMV B-7324 fibrinolytic peptidase. Mikrobiol Zh. 2013;75(4):3-7. (In Ukrainian). PubMed
  13. Moore S, Stein WH. Photometric ninhydrin method for use in the chromatography of amino acids. J Biol Chem. 1948;176(1):367-388. PubMed
  14. Masada M. Determination of the thrombolytic activity of Natto extract. Food Style. 2004;8(1):92-95.
  15. Santhi R. Microbial production of protease by Bacillus cereus using cassava waste water. Eur J Exp Biol. 2014;4(2):19-24.
  16. Craik CS, Page MJ, Madison EL. Proteases as therapeutics. Biochem J. 2011;435(1):1-16. PubMed, PubMedCentral, CrossRef
  17. Lei Y, Zhao P, Li C, Zhao H, Shan Z, Wu Q. Isolation, identification and characterization of a novel elastase from Chryseobacterium indologenes. Appl Biol Chem. 2018;61(3):365-372. CrossRef
  18. Komori Y, Nonogaki T, Nikai T. Hemorrhagic activity and muscle damaging effect of Pseudomonas aeruginosa metalloproteinase (elastase). Toxicon. 2001;39(9):1327-1332. PubMed, CrossRef
  19. Teufel P, Götz F. Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis. J Bacteriol. 1993;175(13):4218-4224. PubMed, PubMedCentral, CrossRef
  20. Chen Q, Guoqing H, Jinling W. Acid shock of elastase-producing Bacillus licheniformis ZJUEL31410 and its elastase characterization evaluation. J Food Eng. 2007;80(2):490-496. CrossRef
  21. Abd EI-Aziz AB, Hassan AA. Optimization of microbial elastase production. J Radiat Res Appl Sci. 2010;3(4B):1237-1257.
  22. Sharma C, Osmolovskiy A, Singh R. Microbial Fibrinolytic Enzymes as Anti-Thrombotics: Production, Characterisation and Prodigious Biopharmaceutical Applications. Pharmaceutics. 2021;13(11):1880. PubMed, PubMedCentral, CrossRef
  23. Baggio LM, Panagio LA, Gasparin FGM, Sartori D, Celligoi MAPC, Baldo C. Production of fibrinogenolytic and fibrinolytic enzymes by a strain of Penicillium sp. isolated from contaminated soil with industrial effluent. Acta Scientiarum. Health Sci. 2019;41(1):40606. CrossRef
  24. Chiou SH, Chen YS. Characterization of two major families of fibrinogenolytic proteases from the venom of Taiwan habu with special reference to their medical applications. Toxin Rev. 2005;24(1):43-61. CrossRef
  25. Xiao P, Yao S, Rizwan-Ur-Rehman, Kang R, Wang Y. A fibrinolytic enzyme produced by Bacillus subtilis using Chickpea (Cicer arietinum L.) as substrate. Adv J Food Sci Technol. 2014;6(12):1294-1300. CrossRef

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