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Fibrinolytic and ACE Inhibitory Activity of Nattokinase Extracted from Bacillus subtilis VITMS 2: A Strain Isolated from Fermented Milk of Vigna unguiculata

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Abstract

The current research work aims at optimization, production, purification and evaluation of fibrinolytic extracellular protease from Bacillus subtilis VITMS2 isolated from fermented milk of Vigna unguiculata. The optimal production was achieved at 4.0% inoculum, pH7.0, 30 °C with (1% w/v) sucrose, (2% w/v) soya bean meal and (2% w/v) malt extract and 10 mM of CaCl2, MgSO4, Na2HPO4 and K2HPO4. The clear cell-free supernatant was purified using conventional ammonium sulphate salt fractionation (75%), ultrafiltration, ion-exchange (DEAE Sepharose FF) and gel filtration (Sephadex G-50). The molecular mass was determined to be 29 kDa using SDS-PAGE analysis. The purified enzyme showed strong fibrinolytic activity with a specific activity of 2418.85 U/mg and has a yield of 12.01%. The enzyme was highly stable up to 60 °C and a pH range of 10.0 until 72 h of incubation. The purified enzyme showed 97.4% in vitro thrombolytic activity. The Km and Vmax values of the enzyme was determined to be 0.0114 mM and 147.8 µmol min−1 using the chromogenic substrate S-7388. IC50 of ace inhibition was assessed to be 0.06 mg/mL suggesting anti-hypertensive property of the fibrinolytic enzyme. The above-obtained ace-inhibition results was supported by in silico molecular docking studies which revealed better binding affinity of nattokinase with a HADDOCK score of − 22.0 ± 8.5 confirms affinity towards angiotensin converting enzyme.

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References

  1. Ouriel K (2002) Current status of thrombolysis for peripheral arterial occlusive disease. Ann Vasc Surg 16:797–804

    Article  PubMed  Google Scholar 

  2. Collen D, Lijnen HR (1991) Basic and clinical aspects of fibrinolysis and thrombolysis. Blood 78:3114–3124

    Article  PubMed  CAS  Google Scholar 

  3. Collen D, Lijnen HR (1994) Staphylokinase, a fibrin-specific plasminogen activator with therapeutic potential? Blood 84:680–686

    Article  PubMed  CAS  Google Scholar 

  4. Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H (1987) A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia 43:1110–1111

    Article  PubMed  CAS  Google Scholar 

  5. Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, Oh H, Kwon I, Lee S (1996) Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11–4 screened from Chungkook-Jang. Appl Environ Microbiol 62:2482–2488

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Zhuang Yao XL, Shim JM, Lee KW, Kim HJ, Kim JH (2017) Properties of a fibrinolytic enzyme secreted by Bacillus amyloliquefaciens RSB34, isolated from Doenjang. J Microbiol Biotechnol 27:9–18

    Article  PubMed  Google Scholar 

  7. Peng Y, Zhang YZ (2002) Optimization of fermentation conditions of douchi fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. Chin Food Ferment Ind 28:19–23

    CAS  Google Scholar 

  8. Kim JH, Kim YSA (1999) fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea. Biosci Biotechnol Biochem 63:2130–2136

    Article  PubMed  CAS  Google Scholar 

  9. Yao Z, Kim JA, Kim JH (2018) Properties of a fibrinolytic enzyme secreted by Bacillus subtilis JS2 isolated from saeu (small shrimp) jeotgal. Food Sci Biotechnol 27:765–772

    Article  PubMed  CAS  Google Scholar 

  10. Wong AH, Mine Y (2004) Novel fibrinolytic enzyme in fermented shrimp paste, a traditional Asian fermented seasoning. J Agric Food Chem 52:980–986

    Article  PubMed  CAS  Google Scholar 

  11. Kim HK, Kim GT, Kim DK, Choi WA, Park SH, Jeon YK, Kong IS (1997) Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. KA38 originated from fermented fish. J Ferment Bioeng 84:307–312

    Article  CAS  Google Scholar 

  12. Noh KA, Kim DH, Choi NS, Kim SH (1999) Isolation of fibrinolytic enzyme producing strains from kimchi. Korean J Food Sci Technol 31:219–223

    Google Scholar 

  13. 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 21:1166–1173

    Article  PubMed  CAS  Google Scholar 

  14. Afifah DN, Rustanti N, Anjani G, Syah D, Suhartono MT (2017) Proteomics study of extracellular fibrinolytic proteases from Bacillus licheniformis RO3 and Bacillus pumilus 2. g isolated from Indonesian fermented food. IOP conference series: earth and environmental science, vol 55. IOP Publishing, Bristol, p 012025

    Google Scholar 

  15. Oh SM, Seo JH, Lee SP (2005) Production of fibrinolytic enzyme and peptides from alkaline fermentation of soybean curd residue by Bacillus firmus NA-1. J Korean Soc Food Sci Nutr 34:904–909

    Article  CAS  Google Scholar 

  16. Lucy J, Raharjo PF, Elvina E, Florencia L, Susanti AI, Pinontoan R (2019) Clot lysis activity of Bacillus subtilis G8 isolated from Japanese fermented natto soybeans. Appl Food Biotechnol 6:101–109

    CAS  Google Scholar 

  17. Prihanto AA, Firdaus M (2019) Proteolytic and fibrinolytic activities of halophilic lactic acid bacteria from two Indonesian fermented foods. J Microbiol Biotechnol Food Sci 2021:2291–2293

    Google Scholar 

  18. Alanazi OA, El-Fetoh NM, Mohammed NA, Alanizy TM, Alanazi YW, Alanazi MS, Alrwaili AA, Alruwaili AH, Alanazi AH, Alanazi AS (2017) Deep venous thrombosis among hypertensive patients in King Abdulaziz University (KAU) Hospital, Jeddah, Kingdom of Saudi Arabia. Electron Physician 9:5472

    Article  PubMed  PubMed Central  Google Scholar 

  19. Masuda O, Nakamura Y, Takano T (1996) Antihypertensive peptides are present in aorta after oral administration of sour milk containing these peptides to spontaneously hypertensive rats. J Nutr 126:3063–3068

    Article  PubMed  CAS  Google Scholar 

  20. McCarron DA, Morris CD, Henry HJ, Stanton JL (1984) Blood pressure and nutrient intake in the United States. Science 224:1392–1398

    Article  PubMed  CAS  Google Scholar 

  21. Meisel H (1998) Overview on milk protein-derived peptides. Int Dairy J 8:363–373

    Article  CAS  Google Scholar 

  22. Teschemacher H, Koch G, Brantl V (1997) Milk protein-derived opioid receptor ligands. Pept Sci 43:99–117

    Article  CAS  Google Scholar 

  23. Yang NC, Chou CW, Chen CY, Hwang KL, Yang YC (2009) Combined nattokinase with red yeast rice but not nattokinase alone has potent effects on blood lipids in human subjects with hyperlipidemia. Asia Pac J Clin Nutr 18:310–317

    PubMed  CAS  Google Scholar 

  24. Fujita M, Ohnishi K, Takaoka S, Ogasawara K, Fukuyama R, Nakamuta H (2011) Antihypertensive effects of continuous oral administration of nattokinase and its fragments in spontaneously hypertensive rats. Biol Pharm Bull 34:1696–1701

    Article  PubMed  CAS  Google Scholar 

  25. Fadl NN, Ahmed HH, Booles HF, Sayed AH (2013) Serrapeptase and nattokinase intervention for relieving Alzheimer’s disease pathophysiology in rat model. Hum Exp Toxicol 32:721–735

    Article  PubMed  CAS  Google Scholar 

  26. Takabayashi T, Imoto Y, Sakashita M, Kato Y, Tokunaga T, Yoshida K, Narita N, Ishizuka T, Fujieda S (2017) Nattokinase, profibrinolytic enzyme, effectively shrinks the nasal polyp tissue and decreases viscosity of mucus. Allergol Int 66:594–602

    Article  PubMed  CAS  Google Scholar 

  27. Lordan R, Tsoupras A, Mitra B, Zabetakis I (2018) Dairy fats and cardiovascular disease: do we really need to be concerned? Foods 7:29

    Article  PubMed Central  Google Scholar 

  28. Lee BH, Lai YS, Wu SC (2015) Antioxidation, angiotensin converting enzyme inhibition activity, nattokinase, and antihypertension of Bacillus subtilis (natto)-fermented pigeon pea. J Food Drug Anal 23:750–757

    Article  PubMed  CAS  Google Scholar 

  29. Matsuoka H, Sasago K, Sekiguchi M (1968) Manufacturing of a cheese-like product from soybean milk part I. Nippon Shokuhin Kogyo Gakkaishi 15:103–108

    Article  CAS  Google Scholar 

  30. Maniatis T (1989) Molecular cloning. Decontamination of Dilute Solutions of Ethidium Bromide

  31. Box JF (1980) RA Fisher and the design of experiments, 1922–1926. Am Stat 34:1–79

    Google Scholar 

  32. Green AA, Hughes WL (1955) Protein fractionation on the basis of solubility in aqueous solutions of salts and organic solvents. Elsevier, Amsterdam

    Book  Google Scholar 

  33. Phillips AT, Signs MW (2004) Desalting, concentration, and buffer exchange by dialysis and ultrafiltration. Curr Protoc Protein Sci 38:4–4

    Article  Google Scholar 

  34. Walker JM (1996) The protein protocols handbook. Springer, Berlin

    Book  Google Scholar 

  35. Andrews P (1965) The gel-filtration behavior of proteins related to their molecular weights over a wide range. Biochem J 96:595–606

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Anson ML (1938) The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J Gen Physiol 22:79

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Tomarelli RM (1949) The use of azoalbumin as a substrate in the colorimetric determination of peptic and tryptic activity. J Lab Clin Med 34:428–433

    PubMed  CAS  Google Scholar 

  38. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

    Article  PubMed  CAS  Google Scholar 

  39. Holmström B (1968) Production of streptokinase in continuous culture. Appl Microbiol 16:73–77

    Article  PubMed  PubMed Central  Google Scholar 

  40. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  41. Uscanga A, Moyano FJ, Alvarez CA (2010) Assessment of enzymatic efficiency on protein digestion in the tilapia Oreochromis niloticus. Fish Physiol Biochem 36:1079–1085

    Article  PubMed  CAS  Google Scholar 

  42. Cushman DW, Cheung HS (1971) Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem Pharmacol 20:1637–1648

    Article  PubMed  CAS  Google Scholar 

  43. Van Zundert GC, Rodrigues JP, Trellet M, Schmitz C, Kastritis PL, Karaca E, Melquiond AS, van Dijk M, De Vries SJ, Bonvin AM (2016) The HADDOCK2. 2 web server: user-friendly integrative modeling of biomolecular complexes. J Mol Biol 428:720–725

    Article  PubMed  Google Scholar 

  44. Wassenaar TA, Van Dijk M, Loureiro-Ferreira N, Van Der Schot G, De Vries SJ, Schmitz C, Van Der Zwan J, Boelens R, Giachetti A, Ferella L, Rosato A (2012) WeNMR: structural biology on the grid. J Grid Comput 10:743–767

    Article  Google Scholar 

  45. National Federation of Cooperatives on Natto in: A Historical Record of Natto 19. Ed. (1977) Food Pionia, Natto Research Center, Tokyo 20: 1637–1648.

  46. Bode C, Runge MS, Smalling RW (1996) The future of thrombolysis in the treatment of acute myocardial infarction. Eur Heart J 17:55–60

    Article  PubMed  Google Scholar 

  47. 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

    Article  PubMed  CAS  Google Scholar 

  48. Chang CT, Fan MH, Kuo FC, Sung HY (2000) Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1. J Agric Food Chem 48:3210–3216

    Article  PubMed  CAS  Google Scholar 

  49. Wang CT, Ji BP, Li B, Nout R, Li PL, Ji H, Chen LF (2006) Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. J Ind Microbiol Biotechnol 33:750–758

    Article  PubMed  CAS  Google Scholar 

  50. Seo JH, Lee SP (2004) Production of fibrinolytic enzyme from soybean grits fermented by Bacillus firmus NA-1. J Med Food 7:442–449

    Article  PubMed  CAS  Google Scholar 

  51. Wu R, Chen G, Pan S, Zeng J, Liang Z (2019) Cost-effective fibrinolytic enzyme production by Bacillus subtilis WR350 using medium supplemented with corn steep powder and sucrose. Sci Rep 9:1

    Google Scholar 

  52. Jeong SJ, Kwon GH, Chun J, Kim JS, Park CS, Kwon DY (2007) Cloning of fibrinolytic enzyme gene from Bacillus subtilis isolated from Cheonggukjung and its expression in protease-deficient Bacillus subtilis. J Microbiol Biotechnol 17:1018–1023

    PubMed  CAS  Google Scholar 

  53. Yin LJ, Lin HH, Jiang ST (2010) Bioproperties of potent nattokinase from Bacillus subtilis YJ1. J Agric Food Chem 58:5737–5742

    Article  PubMed  CAS  Google Scholar 

  54. Kim SB, Lee DW, Cheigh CI, Choe EA, Lee SJ, Hong YH, Pyun YR (2006) Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, Tempeh. J Ind Microbiol Biotechnol 33:436–444

    Article  PubMed  CAS  Google Scholar 

  55. Yoo CK, Seo WS, Lee CS, Kang SM (1998) Purification and characterization of fibrinolytic enzyme excreted by Bacillus subtilis K-54 isolated from Chung Guk Jang. San’oeb Misaengmul Haghoeji 26:507–514

    CAS  Google Scholar 

  56. Wang C, Du M, Zheng D, Kong F, Zu G, Feng Y (2009) Purification and characterization of nattokinase from Bacillus subtilis natto B-12. J Agric Food Chem 57(20):9722–9729

    Article  PubMed  CAS  Google Scholar 

  57. Murakami K, Yamanaka N, Ohnishi K, Fukayama M, Yoshino M (2012) Inhibition of angiotensin I converting enzyme by subtilisin NAT (nattokinase) in natto, a Japanese traditional fermented food. Food Funct 3:674–678

    Article  PubMed  CAS  Google Scholar 

  58. Wu XC, Lee WI, Tran LO, Wong SL (1991) Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J Bacteriol 173:4952–4958

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Dominguez C, Boelens R, Bonvin AM (2003) HADDOCK: a protein− protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125:1731–1737

    Article  PubMed  CAS  Google Scholar 

  60. Fernández-Recio J, Totrov M, Abagyan R (2004) Identification of protein–protein interaction sites from docking energy landscapes. J Mol Biol 335:843–865

    Article  PubMed  Google Scholar 

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Acknowledgements

The authors thank VIT University for providing ‘VIT SEED GRANT’ for carrying out this research work. The authors are thankful to Vellore Institute of Technology for the constant encouragement, help and support for extending necessary facilities.

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  1. Department of Biotechnology, School of Biosciences and Technology, Vellore Institute of Technology, Vellore, 632014, Tamil Nadu, India

    S. Merlyn Keziah & C. Subathra Devi

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Correspondence to C. Subathra Devi.

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Keziah, S.M., Devi, C.S. Fibrinolytic and ACE Inhibitory Activity of Nattokinase Extracted from Bacillus subtilis VITMS 2: A Strain Isolated from Fermented Milk of Vigna unguiculata. Protein J 40, 876–890 (2021). https://doi.org/10.1007/s10930-021-10023-8

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