Ukr.Biochem.J. 2025; Volume 97, Issue 6, Nov-Dec, pp. 33-45

doi: https://doi.org/10.15407/ubj97.06.033

Purification and physico-chemical properties of Bacillus sp. L9 protease with fibrin(ogen)olytic activity

16.12.2025   Uncategorized

O. V. Gudzenko1*, L. D. Varbanets1, V. O. Chernyshenko2, E. M. Stognii2

1D.K. Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine, Kyiv;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
*e-mail: alena.gudzenko81@gmail.com

Received: 18 September 2025; Revised: 13 October 2025;
Accepted: 28 November 2025; Available on-line: 23 December 2025

Previously, we isolated a number of Bacillus sp. strains from the dry grass of the coastal zone of the Kinburn Spit, which may be promising for further research as producers of proteases with fibrinolytic and fibrinogenolytic activity. The aim of the work was to isolate, purify and study the properties of fibrin(ogen)ase from the Bacillus sp. L9 strain. The enzyme preparation was isolated from the supernatant of the Bacillus sp. L9 culture liquid. The yield of the purified enzyme was 1.8%, the specific fibrinogenolytic and fibrinolytic activities were 483 and 383 U/mg protein, respectively, the molecular weight of the enzyme was about 40 kDa, the optimum pH was 8.0, and the thermooptimum was 40°C. Bacillus sp. L9 fibrin(ogen)ase is a serine protease, in the active center of which is the carboxyl group of the C-terminal (aspartic or glutamic) amino acid. At some distance from the active site are localized sulfhydryl groups that do not participate in catalysis, but play an important role in maintaining the catalytically active conformation of the protein molecule. The enzyme from Bacillus sp. L9 hydrolyzed fibrin molecules much more slowly than fibrinogen, and showed the greatest specificity in the hydrolysis of bonds formed by the Aα-chain of fibrinogen. According to the specifici­ty of action on fibrinogen, the enzyme was identified as α-fibrinogen(ogen)ase.

Keywords: , , , , ,

References:

  1. Hazare C, Bhagwat P, Singh S, Pillai S. Diverse origins of fibrinolytic enzymes: A comprehensive review. Heliyon. 2024;10(5):e26668. PubMed, PubMedCentral, CrossRef
  2. Diwan D, Usmani Z, Sharma M, Nelson JW, Thakur VK, Christie G, Molina G, Gupta VK. Thrombolytic Enzymes of Microbial Origin: A Review. Int J Mol Sci. 2021;22(19):10468. PubMed, PubMedCentral, CrossRef
  3. Barzkar N, Jahromi ST, Vianello F. Marine Microbial Fibrinolytic Enzymes: An Overview of Source, Production, Biochemical Properties and Thrombolytic Activity. Mar Drugs. 2022;20(1):46. PubMed, PubMedCentral, CrossRef
  4. Seo MJ. Editorial for the Special Issue: Environment Microorganisms and Their Enzymes with Biotechnological Application. Microorganisms. 2024;12(1):204. PubMed, PubMedCentral, CrossRef
  5. Gudzenko OV, Varbanets LD. Ivanytsia VO, Shtenikov MD. Representatives of Bacillus from deep-water bottom sediments of the Black Sea – producers elastase, fibrin(ogen)ase and collagenases. Mikrobiol J. 2024 86(3):51-57. CrossRef
  6. Devaraj Y, Rajender SK, Halami PM. Purification and characterization of fibrinolytic protease from Bacillus amyloliquefaciens MCC2606 and analysis of fibrin degradation product by MS/MS. Prep Biochem Biotechnol. 2018;48(2):172-180. PubMed, CrossRef
  7. Wu J, Lan G, He N, He L, Li C, Wang X, Zeng X. Purification of fibrinolytic enzyme from Bacillus amyloliquefaciens GUTU06 and properties of the enzyme. Food Chem X. 2023;20:100896.
    PubMed, PubMedCentral, CrossRef
  8. Gudzenko OV, Varbanets LD, Ivanytsia VO. Effect of Cultivation Temperature on Elastic, Fibrinogenolitic, and Collagenase Activity of Marine Bacteria Bacillus atrophaeus 08, Bacillus licheniformis 043, and Bacillus subtilis 248. Mikrobiol Zh. 2025;87(1): 27-35. CrossRef
  9. 9. Gudzenko OV, Varbanets LD. Screening of protease producers among representatives of the genus Bacillus isolated from the coastal zone of the Kinburn split. Mikrobiol Zh. 2024;86(2):3-9.
    CrossRef
  10. 10. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-685. PubMed, CrossRef
  11. Ostapchenko L, Savchuk O, Burlova-Vasilieva N. Enzyme electrophoresis method in analysis of active components of haemostasis system. Adv Biosci Biotechnol. 2011;2(1):20-26. CrossRef
  12. 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
  13. Masada M. Determination of the thrombolytic activity of Natto extract. Food Style. 2004;8(1):92-95.
  14. Trowbridge JO, Moon HD. Purification of human elastase. Proc Soc Exp Biol Med. 1972;141(3):928-931. PubMed, CrossRef
  15. Anson ML. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J Gen Physiol. 1938 22(1):79-89. PubMed, PubMedCentral, CrossRef
  16. Moore S, Stein WH. Photometric ninhydrin method for use in the chromatography of amino acids. J Biol Chem. 1948;176(1):367-388. PubMed, CrossRef
  17. Iskandarov E, Gryshchuk V, Platonov O, Kucheriavyi Y, Slominskyi O, Stohnii Y, Vartanov V, Chernyshenko V. Fractionation of Vipera berus berus snake venom and detection of bioactive compounds targeted to blood coagulation system. Southeastern Eur Med J. 2022;6(2):20-31. CrossRef
  18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1): 265-275. PubMed, CrossRef
  19. Ma Z, Elango J, Hao J, Wu W. Purification and Characterization of a Novel Fibrinolytic Enzyme from Marine Bacterium Bacillus sp. S-3685 Isolated from the South China Sea. Mar Drugs. 2024;22(6):267. PubMed, PubMedCentral, CrossRef
  20. Shettar SS, Bagewadi ZK, Alasmary M, Mannasaheb BA, Shaikh IA, Khan AA. Comprehensive biochemical, molecular and structural characterization of subtilisin with fibrinolytic potential in bioprocessing. Bioresour Bioprocess. 2025;12(1):21. PubMed, PubMedCentral, CrossRef
  21. 21. 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
  22. Wang J, Liu X, Jing Y, Zheng X. Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Culture Supernatant of Coprinus comatus. Foods. 2024;13(9):1292. PubMed, PubMedCentral, CrossRef
  23. Varol A, Albayrak S, Ozkan H, Demir Y, Taskin M, Adiguzel A. Production, purification and characterization of novel fibrinolytic enzyme from Bacillus atrophaeus V4. Biologia. 2023;78:591-600. CrossRef
  24. Syahbanu F, Giriwono PE, Tjandrawinata RR, Suhartono MT. Molecular analysis of a fibrin-degrading enzyme from Bacillus subtilis K2 isolated from the Indonesian soybean-based fermented food moromi. Mol Biol Rep. 2020;47(11):8553-8563. PubMed, CrossRef
  25. Vignesh M Iyer, Sreelakshmi R Nair, Sanjeev K Ganesh, Mohanasrinivasan V, Subathra Devi C. Production, optimization and partial purification of nattokinase from Bacillus cereus; a strain isolated from grape wine. J Microbiol Biotechnol Food Sci. 2024;13(5):e10355. CrossRef
  26. Hu Y, Yu D, Wang Z, Hou J, Tyagi R, Liang Y, Hu Y. Purification and characterization of a novel, highly potent fibrinolytic enzyme from Bacillus subtilis DC27 screened from Douchi, a traditional Chinese fermented soybean food. Sci Rep. 2019;9(1):9235. PubMed, PubMedCentral, CrossRef
  27. Singh R, Gautam P, Sharma C, Osmolovskiy A. Fibrin and Fibrinolytic Enzyme Cascade in Thrombosis: Unravelling the Role. Life (Basel). 2023;13(11):2196. PubMed, PubMedCentral, CrossRef
  28. Al Sharif MA, Mathews N, Tasneem S, Moffat KA, Carlino SA, Mithoowani S, Hayward CPM. Measurement of factor XIII for the diagnosis and management of deficiencies: insights from a retrospective review of 10 years of data on consecutive samples and patients. Res Pract Thromb Haemost. 2025;9(1):102689. PubMed, PubMedCentral, CrossRef
  29. Moroi M, Induruwa I, Farndale RW, Jung SM. Factor XIII is a newly identified binding partner for platelet collagen receptor GPVI-dimer-An interaction that may modulate fibrin crosslinking. Res Pract Thromb Haemost. 2022;6(3):e12697. PubMed, PubMedCentral, CrossRef
  30. Hwang KJ, Choi KH, Kim MJ, Park CS, Cha J. Purification and characterization of a new fibrinolytic enzyme of Bacillus licheniformis KJ-31, isolated from Korean traditional Jeot-gal. J Microbiol Biotechnol. 2007;17(9):1469-1476. PubMed
  31. Dhamodharan D, Jemimah Naine S, Merlyn Keziah S, Subathra Devi C. Novel Fibrinolytic Protease Producing Streptomyces radiopugnans VITSD8 from Marine Sponges. Mar Drugs. 2019;17(3):164.
    PubMed, PubMedCentral, CrossRef
  32. Zhou Y, Chen H, Yu B, Chen G, Liang Z. Purification and Characterization of a Fibrinolytic Enzyme from Marine Bacillus velezensis Z01 and Assessment of Its Therapeutic Efficacy In Vivo. Microorganisms. 2022;10(5):843. PubMed, PubMedCentral, CrossRef
  33. Krishnamurthy A, Belur PD. A novel fibrinolytic serine metalloprotease from the marine Serratia marcescens subsp. sakuensis: Purification and characterization. Int J Biol Macromol. 2018;112:110-118. PubMed, CrossRef
  34. Silva PEDCE, Barros RC, Albuquerque WWC, Brandão RMP, Bezerra RP, Porto ALF. In vitro thrombolytic activity of a purified fibrinolytic enzyme from Chlorella vulgaris. J Chromatogr B Analyt Technol Biomed Life Sci. 2018;1092:524-529. PubMed, CrossRef
  35. 35. Barros PDS, Silva PECE, Nascimento TP, Costa RMPB, Bezerra RP, Porto ALF. Fibrinolytic enzyme from Arthrospira platensis cultivated in medium culture supplemented with corn steep liquor. Int J Biol Macromol. 2020;164:3446-3453. PubMed, CrossRef
  36. Yao Z, Kim JA, Kim JH. Gene Cloning, Expression, and Properties of a Fibrinolytic Enzyme Secreted by Bacillus pumilus BS15 Isolated from Gul (Oyster) Jeotgal. Biotechnol Bioproc E. 2018;23:293-301. CrossRef
  37. Taneja K, Bajaj BK, Kumar S, Dilbaghi N. Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media. 3 Biotech. 2017;7(3):184. PubMed, PubMedCentral, CrossRef
  38. Yao M, Ma C, Bian X, Yang Y, Xu Y, Wu Q, Xu X, Li L, Zhang N, Tian Y. Isolation and optimal fermentation conditions of Bacillus licheniformis SFD-Y5 for a new douchi fibrinolytic enzyme producer. Fermentation. 2023;9(7):668. CrossRef
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