Article Search

Microbiology and Biotechnology Letters


View PDF

Food Microbiology (FM)  |  Bioactive Compounds or Metabolites: Function and Application

Microbiol. Biotechnol. Lett. 2021; 49(3): 289-297

Received: April 21, 2021; Revised: May 7, 2021; Accepted: May 14, 2021

Cloning and Expression of a Fibrinolytic Enzyme Gene, aprECJ1, from Bacillus velezensis CJ1 Isolated from Myeolchi Jeotgal

Ji Yeon Yoo1, Zhuang Yao1, Se Jin Lee1, Hye Sung Jeon1, and Jeong Hwan Kim1,2*

1Division of Applied Life Science (BK21 Four), Graduate School, 2Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea

Correspondence to :
Jeong Hwan Kim,

Bacillus velezensis CJ1, showing significant fibrinolytic activity, was isolated from Myeolchi Jeotgal, a popular Korean fermented seafood. When B. velezensis CJ1 was grown on four different culture media, the culture on the Luria-Bertani (LB) broth showed the highest fibrinolytic activity (102.94 mU/μl) at 48 h. LB was also the best medium for growth. SDS-PAGE of culture supernatant showed four major bands, 38, 35, 27, and 22 kDa in size. Fibrin zymography showed four active bands, 50, 47, 40, and 30 kDa in size. A gene homologous to aprE of the Bacillus species was cloned by PCR. DNA sequencing showed that aprECJ1 can encode a protease consisting of 382 amino acids. The translated amino acid sequence of AprECJ1 showed high identity values with those of B. velezensis strains and other Bacillus species. The aprECJ1 gene was introduced into B. subtilis WB600 using an E. coli-Bacillus shuttle vector, pHY300PLK, and overexpressed. A 27 kDa band corresponding to the mature form of AprECJ1 was produced and confirmed by SDS-PAGE and fibrin zymography. B. subtilis WB600 [pHYaprECJ1] showed 1.8-fold higher fibrinolytic activity than B. velezensis CJ1 at 48 h.

Keywords: Fibrinolytic enzyme, Bacillus velezensis, Myeolchi Jeotgal, gene expression

Graphical Abstract

  1. Kho CW, Park SG, Cho S, Lee DH, Myung PK, Park BC. 2005. Confirmation of Vpr as a fbrinolytic enzyme present in extracellular proteins of Bacillus subtilis. Protein Expr. Purif. 39: 1-7.
  2. Chen H, McGowan EM, Ren N, Lal S, Nassif N, Shad-Kaneez F, et al. 2018. Nattokinase: a promising alternative in prevention and treatment of cardiovascular diseases. Biomark. Insights 13:1177271918785130.
    Pubmed KoreaMed
  3. Uesugi Y, Usuki H, Iwabuchi M, Hatanaka T. 2011. Highly potent fibrinolytic serine protease from Streptomyces. Enzyme Microb. Technol. 48: 7-12.
  4. Danilova L, Sharipova M. 2020. The practical potential of bacilli and their enzymes for industrial production. Front. Microbiol. 11:1782.
    Pubmed KoreaMed
  5. Weng Y, Yao J, Sparks S, Wang KY. 2017. Nattokinase: an oral antithrombotic agent for the prevention of cardiovascular disease. Int. J. Mol. Sci. 18: 523.
    Pubmed KoreaMed
  6. Kurosawa Y, Nirengi S, Homma T, Esaki K, Ohta M, Clark JF, et al. 2015. A single-dose of oral nattokinase potentiates thrombolysis and anti-coagulation profiles. Sci. Rep. 5: 11601.
    Pubmed KoreaMed
  7. Mine Y, Wong AHK, Jiang B. 2005. Fibrinolytic enzymes in Asian traditional fermented foods. Food Res. Int. 38: 243-250.
  8. Jeong SJ, Kwon GH, Chun JY, Kim JS, Park CS, Kwon DY, et al. 2007. Cloning of fibrinolytic enzyme gene from Bacillus subtilis isolated from Cheonggukjang and its expression in protease-deficient Bacillus subtilis strains. J. Microbiol. Biotechnol 17: 1018-1023.
  9. Kim SH, Choi NS. 2000. Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. strain DJ-4 screened from Doen-Jang. Biosci. Biotechnol. Biochem. 64: 1722-1725.
  10. Fujita M, Nomura K, Hong K, Ito Y, Asada A, Nishimuro S. 1993. Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. Biochem. Biophys. Res. Commun. 197:1340-1347.
  11. Peng Y, Huang Q, Zhang R, Zhang YZ. 2003. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. Comp. Biochem. Physiol. Biochem. Mol. Biol. 134:45-52.
  12. Rabbee MF, Ali MS, Choi J, Hwang BS, Jeong SC, Baek KH. 2019. Bacillus velezensis: a valuable member of bioactive molecules within plant microbiomes. Molecules 24: 1046.
    Pubmed KoreaMed
  13. Koo OK, Lee SJ, Chung KR, Jang DJ, Yang HJ, Kwon DY. 2016. Korean traditional fermented fish products: jeotgal. J. Ethn. Foods 3: 107-116.
  14. Astrup T, Mullertz S. 1952. The fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. Biophys. 40: 346-351.
  15. Kwon GH, Lee HA, Park JY, Kim JS, Lim J, Park CS, et al. 2009. Development of a RAPD-PCR method for identification of Bacillus species isolated from cheonggukjang. Int. J. Food Microbiol. 129:282-287.
  16. Kim GM, Lee AR, Lee KW, Park JY, Chun J, Cha J, et al. 2009. Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from cheonggukjang. J. Microbiol. Biotechnol. 19: 997-1004.
  17. Sambrook J, Russell DW. 2001. Molecular cloning a laboratory manual, chapter 5. 3rd Ed. pp. 1-90. Cold Spring Harbor laboratory Press, Cold Spring harbor, New York.
  18. Choi NS, Chung DM, Park CS, Ahn KH, Kim JS, Song JJ, et al. 2010. Expression and identification of a minor extracellular fibrinolytic enzyme (Vpr) from Bacillus subtilis KCTC 3014. Biotechnol. Bioprocess Eng. 15: 446-452.
  19. Choi NS, Chung DM, Ryu CH, Yoon KS, Maeng PJ, Kim SH. 2006. Identification of three extracellular proteases from Bacillus subtilis KCTC 3014. J. Microbiol. Biotechnol. 16: 457-464.
  20. Yao Z, Meng Y, Le HG, Lee SJ, Jeon HS, Yoo JY, et al. 2020. Cloning of a novel vpr gene encoding a minor fibrinolytic enzyme from Bacillus subtilis SJ4 and the properties of Vpr. J. Microbiol. Biotechnol. 30: 1720-1728.
  21. Wu XC, Lee W, Tran L, Wong SL. 1991. Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J. Bacteriol. 173: 4952-4958.
    Pubmed KoreaMed
  22. Dabbagh F, Negahdaripour M, Berenjian A, Behfar A, Mohammadi F, Zamani M, et al. 2014. Nattokinase: production and application. Appl. Microbiol. Biotechnol. 98: 9199-9206.
  23. Peng Y, Yang X, Zhang Y. 2005. Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo. Appl. Microbiol. Biotechnol. 69: 126-132.

Starts of Metrics

Share this article on :

  • mail

Related articles in MBL

Most KeyWord ?

What is Most Keyword?

  • It is most registrated keyword in articles at this journal during for 2 years.