Molecular Characterization of Bacterial Fibrinolytic Proteins from Indonesian Traditional Fermented Foods

Abstract

Previously, the crude extracts of recombinant Nattokinase (NK) variants i.e. NatTK and NatOC and one wild type Douchi Fibrinolytic Enzyme (DFE) from Indonesian traditional fermented foods has been shown to demonstrate fibrinolytic activity. Both NKs contain substitutions of D41N, V192A and 252-RLQHTLEALSTM-263 but NatOC has additional V4F. In the present study, the effects of amino acid substitutions in NK variants and G169A in DFE on their enzyme characteristics were evaluated. Pure proteins were obtained using two sequential steps chromatography using ion exchange and a gel filtration columns. Their activities were determined with fibrin plate, fibrin zymography, fibrinogen hydrolysis, and chromogenic assays. The fibrinogen degradation profile of the wild type NK (NatWT) was different to the NK variants but similar to DFEs. Optimum activity of all the NKs and DFEs was achieved at 50 °C while the optimum pH for NatWT/DFEs and NK variants were 8 and 7, respectively. DFEG169A exhibited higher fibrinogen degradation rate and fibrin specific activity than DFE. PMSF inhibited all the NKs and DFEs while SDS and EDTA caused lower activity. The NK variants were more resistant towards Na+ and Ca2+ but more sensitive to K+. The amino acid substitutions in NK variants alter their fibrinogen degradation profile, optimum working pH, working pH range, and resistance to some ions. Substitutions in NK variants likely promote structural changes, particularly with the binding mode of the calcium ion cofactor. The results provide a beneficial basis for future development of fibrino(gen)olytic proteins with improved properties for cardiovascular diseases therapy.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Abbreviations

NatTK:

Recombinant nattokinase from bacteria in dried tauco (with amino acid variation D41N and V192A)

NatOC:

Recombinant nattokinase from bacteria in yellow oncom (with amino acid variation V4F, D41N and V192A)

NatWT:

Recombinant nattokinase

NatBS:

Nattokinase from Bacillus subtilis, AAO65246 (NCBI)

DFE:

Douchi fibrinolytic enzyme, ACA34903 (NCBI)

DFEG169A:

DFE with glycine substitution in position 169 with alanine

SDS:

Sodium dodecyl sulphate

EDTA:

Ethylenediaminetetraacetic acid

PMSF:

Phenylmethylsulfonyl fluoride

CN:

Coordination number

References

  1. 1.

    Wander GS, Chhabra ST (2013) Critical analysis of various drugs used for thrombolytic therapy in acute myocardial infarction. Med Updat 23:109–116

    Google Scholar 

  2. 2.

    Lin HV, Wu G, Hsieh M, Chang S, Tsai G (2015) Purification and characterization of nattokinase from cultural filtrate of red alga Porphyra dentata fermented by Bacillus subtilis N1. J Mar Sci Technol 23(2):240–248

    Google Scholar 

  3. 3.

    Singh TA, Devi KR, Ahmed G, Jeyaram K (2014) Microbial and endogenous origin of fibrinolytic activity in traditional fermented foods of Northeast India. Food Res Int 55:356–362

    Google Scholar 

  4. 4.

    Wei X, Luo M, Xu L, Zhang Y, Lin X, Kong P (2011) Production of fibrinolytic enzyme from Bacillus amyloliquefaciens by fermentation of chickpeas, with the evaluation of the anticoagulant and antioxidant properties of chickpeas. J Agric Food Chem 59(8):3957–3963

    Article  CAS  PubMed  Google Scholar 

  5. 5.

    Kumar A, Pulicherla KK, Seetha RK, Rao SRKS (2010) Evolutionary trend of thrombolytics. Int J Biosci Biotechnol 2(4):51–68

    CAS  Google Scholar 

  6. 6.

    Jayalakshmi T, Krishnamoorthy P, Ramesh Babu P, Vidhya B (2012) Production, purification and biochemical characterization of alkaline fibrinolytic enzyme from Bacillus subtilis strain-GBRC1. J Chem Pharm Res 4(12):5027–5031

    CAS  Google Scholar 

  7. 7.

    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(3):523

    Article  CAS  PubMed Central  Google Scholar 

  8. 8.

    Zhang RH, Xiao L, Peng Y, Wang HY, Bai F, Zhang Y (2015) Gene expression and characteristics of a novel fibrinolytic enzyme (subtilisin DFE) in Escherichia coli. Microbiology 41(2):190–195

    Google Scholar 

  9. 9.

    Motaal AA, Fahmy I, El-Halawany A, Ibrahim N (2015) Comparative fibrinolytic activities of nattokinases from Bacillus subtilis var. natto. J Pharm Sci Res 7(2):63–66

    Google Scholar 

  10. 10.

    Ghasemi Y, Dabbagh F, Ghasemina A (2012) Cloning of a fibrinolytic enzyme (subtilisin) gene from Bacillus subtilis in Escherichia coli. Mol Biotechnol 52:1–7

    Article  CAS  Google Scholar 

  11. 11.

    Ni H, Guo PC, Jiang WL, Fan XM, Luo XY, Li HH (2016) Expression of nattokinase in Escherichia coli and renaturation of its inclusion body. J Biotechnol 231:65–71

    Article  CAS  PubMed  Google Scholar 

  12. 12.

    Dabbagh F, Negahdaripour M, Berenjian A, Behfar A, Mohammadi F, Zamani M, Irajie C, Ghasemi Y (2014) Nattokinase : production and application. Appl Microbiol Biotechnol 98(22):9199–9206

    Article  CAS  Google Scholar 

  13. 13.

    Nascimento TP, Sales AE, Porto CS, Marcos R, Brandão RMP, Takaki GMC, Teixeira JAC, Porto TS, Porto ALF (2015) Production and characterization of new fibrinolytic protease from mucor subtillissimus UCP 1262 in solid-state fermentation. Adv Enzyme Res 3(3):81–91

    CAS  Google Scholar 

  14. 14.

    Kim J, Kim J, Choi K, Kim JH, Song Y, Cha J (2011) Enhancement of the catalytic activity of a 27 kDa subtilisin-like enzyme from Bacillus amyloliquefaciens CH51 by in vitro mutagenesis. J Agric Food Chem 59(16):8675–8682

    Article  CAS  PubMed  Google Scholar 

  15. 15.

    Purwaeni E, Darojatin I, Riani C, Retnoningrum DS (2018) Bacterial fibrinolytic enzyme coding sequences from indonesian traditional fermented foods isolated using metagenomic approach and their expression in Escherichia coli. Food Biotechnol 32(1):47–59

    Article  CAS  Google Scholar 

  16. 16.

    Ginther CL (1979) Sporulation and the production of serine protease and cephamycin c by Streptomyces lactamdurans. Antimicrob Agents Chemother 15(4):522–526

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Kim J, Choi K, Kim JH, Song Y, Cha J (2013) Enhancement of the thermostability of a fibrinolytic enzyme from Bacillus amyloliquefaciens CH51. J Life Sci 23(1):15–23

    Article  Google Scholar 

  18. 18.

    Heo K, Cho KM, Lee CK, Kim GM, Shin JH, Kim JS, Kim JH (2013) Characterization of a fibrinolytic enzyme secreted by Bacillus amyloliquefaciens CB1 and its gene cloning. J Microbiol Biotechnol 23(7):974–983

    Article  CAS  PubMed  Google Scholar 

  19. 19.

    Hu Y, Yu D, Zhaoting W, Hou J, Tyagi R, Liang Y (2019) 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 9:9235

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Wang C, Ji B, Li B, Ji H (2006) Enzymatic properties and identification of a fibrinolytic serine protease purified from Bacillus subtilis DC33. World J Microbiol Biotechnol 22(12):1365–1371

    Article  CAS  Google Scholar 

  21. 21.

    Choi NS, Song JJ, Chung DM, Kim YJ, Maeng PJ, Kim SH (2009) Purification and characterization of a novel thermoacid-stable W brinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet. J Ind Microbiol Biotechnol 36(3):417–426

    Article  CAS  PubMed  Google Scholar 

  22. 22.

    Matsubara K, Hori K, Matsuura Y, Miyazawa K (2000) Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum. Comp Biochem Physiol B Biochem Mol Biol 125(1):137–143

    Article  CAS  PubMed  Google Scholar 

  23. 23.

    Jo HD, Lee HA, Jeong SJ, Kim JH (2011) Purification and characterization of a major fibrinolytic enzyme from Bacillus amyloliquefaciens MJ5-41 Isolated from Meju. J Microbiol Biotechnol 21(11):1166–1173

    Article  CAS  PubMed  Google Scholar 

  24. 24.

    Kim DW, Choi JH, Park SE, Kim S, Sapkota K, Kim SJ (2015) Purification and characterization of a fibrinolytic enzyme from Petasites japonicus. Int J Biol Macromol 72:1159–1167

    Article  CAS  PubMed  Google Scholar 

  25. 25.

    Jeong SJ, Heo K, Park JY, Lee KW, Park JY, Joo SH, Kim JH (2015) Characterization of AprE176, a fibrinolytic enzyme from Bacillus subtilis HK176. J Microbiol Biotechnol 25(1):89–97

    Article  CAS  PubMed  Google Scholar 

  26. 26.

    Choi N, Chang K, Jae Maeng P, Kim S (2004) Cloning, expression, and fibrin(ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. DJ-4. FEMS Microbiol Lett 236(2):325–331

    Article  CAS  PubMed  Google Scholar 

  27. 27.

    Borah D, Sangra A, Shahin L (2012) Production, purification and characterization of nattokinase from Bacillus subtilis, isolated from tea garden soil samples of Dibrugarh, Assam. Asian J Pharm Clin Res 5(3):124–125

    CAS  Google Scholar 

  28. 28.

    Peng Y, Huang Q, Zhang R, Zhang Y (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 B Biochem Mol Biol 134(1):45–52

    Article  PubMed  Google Scholar 

  29. 29.

    Wang S, Deng Z, Li Q, Ge X, Bo Q, Liu J, Cui J, Jiang X, Liu J, Zhang L, Hong M (2011)  A novel alkaline serine protease with fi brinolytic activity from the polychaete, Neanthes japonica. Comp Biochem Physiol B Biochem Mol Biol 159(1):18–25

    Article  CAS  PubMed  Google Scholar 

  30. 30.

    Cheng G, He L, Sun Z, Cui Z, Du Y, Kong Y (2015) Purification and biochemical characterization of a novel fibrinolytic enzyme from Streptomyces sp. P3. J Microbiol Biotechnol 25(9):1449–1459

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Wangsa Tirta Ismaya for productive discussion of structural analysis and Rahmat Muliadi for technical assistance. This research was funded by Science and Technology Research Grant (STRG) from the Indonesian Toray Science Foundation (ITSF) and the RISBIN IPTEKDOK (Riset Pembinaan Ilmu Pengetahuan dan Teknologi Kedokteran) grant from the Health Research and Development Agency in the Ministry of Health, Republic Indonesia.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Debbie Soefie Retnoningrum.

Ethics declarations

Conflict of interest

All authors declare that they no conflict of interest.

Research Involving Human Participants and/or Animals

This article does not contain any studies with animals and human participants performed by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 2.80 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Purwaeni, E., Riani, C. & Retnoningrum, D.S. Molecular Characterization of Bacterial Fibrinolytic Proteins from Indonesian Traditional Fermented Foods. Protein J 39, 258–267 (2020). https://doi.org/10.1007/s10930-020-09897-x

Download citation

Keywords

  • DFE G169A
  • Fibrin specificity
  • Fibrinogen degradation
  • Ion resistance
  • NK variants