Cloning and Expression of a Fibrinolytic Enzyme Gene, aprECJ1, from Bacillus velezensis CJ1 Isolated from Myeolchi Jeotgal
2
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
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.
Abstract
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.
참고문헌 (23)
- Kho CW, "Confirmation of Vpr as a fbrinolytic enzyme present in extracellular proteins of Bacillus subtilis", Protein Expr. Purif, 39, pp.1~7 (2005)
- Chen H, "Nattokinase : a promising alternative in prevention and treatment of cardiovascular diseases", Biomark. Insights, 13, pp.1177271918~ (2018)
- Uesugi Y, "Highly potent fibrinolytic serine protease from Streptomyces", Enzyme Microb. Technol, 48, pp.7~12 (2011)
- Danilova L, "The practical potential of bacilli and their enzymes for industrial production", Front. Microbiol, 11, pp.1782~ (2020)
- Weng Y, "Nattokinase : an oral antithrombotic agent for the prevention of cardiovascular disease", Int. J. Mol. Sci, 18, pp.523~ (2017)
- Kurosawa Y, "A single-dose of oral nattokinase potentiates thrombolysis and anti-coagulation profiles", Sci. Rep, 5, pp.11601~ (2015)
- Mine Y, "Fibrinolytic enzymes in Asian traditional fermented foods", Food Res. Int, 38, pp.243~250 (2005)
- 정선주, "Cloning of Fibrinolytic Enzyme Gene from Bacillus subtilis Isolated from Cheonggukjang and Its Expression in Protease-deficient Bacillus subtilis Strains", Journal of Microbiology and Biotechnology, 17(6), pp.1018~1023 (2007), 한국미생물·생명공학회
- Kim SH, "Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. strain DJ-4 screened from Doen-Jang", Biosci. Biotechnol. Biochem, 64, pp.1722~1725 (2000)
- Fujita M, "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, pp.1340~1347 (1993)
- Peng Y, "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, pp.45~52 (2003)
- Rabbee MF, "Bacillus velezensis: a valuable member of bioactive molecules within plant microbiomes", Molecules, 24, pp.1046~ (2019)
- 구옥경, "Korean traditional fermented fish products: jeotgal", Journal of Ethnic Foods, 3(2), pp.107~116 (2016), 한국식품연구원
- Astrup T, "The fibrin plate method for estimating fibrinolytic activity", Arch. Biochem. Biophys, 40, pp.346~351 (1952)
- Kwon GH, "Development of a RAPD-PCR method for identification of Bacillus species isolated from cheonggukjang", Int. J. Food Microbiol, 129, pp.282~287 (2009)
- 김경민, "Characterization of a 27 kDa Fibrinolytic Enzyme from Bacillus amyloliquefaciens CH51 Isolated from Cheonggukjang", Journal of Microbiology and Biotechnology, 19(9), pp.997~1004 (2009), 한국미생물·생명공학회
- Sambrook J, "Molecular cloning a laboratory manual, chapter 5", pp.1~90 (2001), Cold Spring Harbor laboratory Press
- Nack-Shick Choi, "Expression and Identification of a Minor Extracellular Fibrinolytic Enzyme (Vpr) from Bacillus subtilis KCTC 3014", Biotechnology and Bioprocess Engineering, 15(3), pp.446~452 (2010), 한국생물공학회
- CHOI, NACK-SHICK, "Identification of Three Extracellular Proteases from Bacillus subtilis KCTC 3014", Journal of Microbiology and Biotechnology, 16(3), pp.457~464 (2006), 한국미생물·생명공학회
- Zhuang Yao, "Cloning of a Novel vpr Gene Encoding a Minor Fibrinolytic Enzyme from Bacillus subtilis SJ4 and the Properties of Vpr", Journal of Microbiology and Biotechnology, 30(11), pp.1720~1728 (2020), 한국미생물·생명공학회[DOI]
- Wu XC, "Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases", J. Bacteriol, 173, pp.4952~4958 (1991)
- Dabbagh F, "Nattokinase : production and application", Appl. Microbiol. Biotechnol, 98, pp.9199~9206 (2014)
- Peng Y, "Microbial fibrinolytic enzymes : an overview of source, production, properties, and thrombolytic activity in vivo", Appl. Microbiol. Biotechnol, 69, pp.126~132 (2005)