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Purification, biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262

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Abstract

Fibrinolytic proteases are enzymes that degrade fibrin. They provide a promising alternative to existing drugs for thrombolytic therapy. A protease isolated from the filamentous fungus Mucor subtilissimus UCP 1262 was purified in three steps by ammonium sulfate fractionation, ion exchange, and molecular exclusion chromatographies, and characterized biochemically and structurally. The purified protease exhibited a molecular mass of 20 kDa, an apparent isoelectric point of 4.94 and a secondary structure composed mainly of α-helices. Selectivity for N-succinyl-Ala–Ala–Pro–Phe-p-nitroanilide as substrate suggests that this enzyme is a chymotrypsin-like serine protease, whose activity was enhanced by the addition of Cu2+, Mg2+, and Fe2+. The enzyme showed a fibrinolytic activity of 22.53 U/mL at 40 °C and its contact with polyethylene glycol did not lead to any significant alteration of its secondary structure. This protein represents an important example of a novel fibrinolytic enzyme with potential use in the treatment of thromboembolic disorders such as strokes, pulmonary emboli, and deep vein thrombosis.

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References

  1. Mendis S, Puska P, Norrving B; World Health Organization; World Heart Federation; World Stroke Organization (2011) Global atlas on cardiovascular disease prevention and control. World Health Organization in collaboration with the World Heart Federation and the World Stroke Organization, Geneva

  2. Fu T, Yang F, Zhu H, Zhu H, Guo L (2016) Rapid extraction and purification of lumbrokinase from Lumbricus rubellus using a hollow fiber membrane and size exclusion chromatography. Biotechnol Lett 38:251–258

    Article  CAS  Google Scholar 

  3. Sun Z, Liu P, Cheng G, Zhang B, Dong W, Su X, Huang Y, Cui Z, Kong Y (2016) A fibrinolytic protease AfeE from Streptomyces sp. CC5, with potent thrombolytic activity in a mouse model. Int J Biol Macromol 85:346–354

    Article  CAS  Google Scholar 

  4. Devi CS, Mohanasrinivasan V, Sharma P, Das D, Vaishnavi B, Naine SJ (2016) Production, purification and stability studies on nattokinase: a therapeutic protein extracted from mutant Pseudomonas aeruginosa CMSS isolated from bovine milk. Int J Pept Res Ther 22:263–269

    Article  CAS  Google Scholar 

  5. Shirasaka N, Naitou M, Okamura K, Kusuda M, Fukuta Y, Terashita T (2012) Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3. Mycoscience 53:354–364

    Article  CAS  Google Scholar 

  6. Uesugi Y, Kawata H, Jo J, Saito Y, Tabata Y (2010) An ultrasound-responsive nano delivery system of tissue-type plasminogen activator for thrombolytic therapy. J Control Release 147:269–277

    Article  CAS  Google Scholar 

  7. Bagchi SN, Sondhia S, Agrawal MK, Banerjee S (2016) An angiotensin-converting enzyme-inhibitory metabolite with partial structure of microginin in a cyanobacterium Anabaena fertilissima CCC597, producing fibrinolytic protease. J Appl Phycol 28:177–180

    Article  CAS  Google Scholar 

  8. Mohanasrinivasan V, Subathra CD, Biswas R, Mitra FP, Selvarajan E, Suganthi V (2013) Enhanced production of nattokinase from UV mutated Bacillus sp. Bangladesh J Pharmacol 8:110–115

    Article  Google Scholar 

  9. Kim HC, Choi B-S, Sapkota K, Kim S, Lee HJ, Yoo JC, Kim S-J (2011) Purification and characterization of a novel, highly potent fibrinolytic enzyme from Paecilomyces tenuipes. Process Biochem 46:1545–1553

    Article  CAS  Google Scholar 

  10. Nascimento TP, Sales AE, Porto CS, Brandão RMP, Takaki GMC, Teixeira JAC, Porto TS, Porto ALF (2015) Production and characterization of new fibrinolytic protease from Mucor subtilissimus UCP 1262 in solid-state fermentation. Adv Enzyme Res 8:81–91

    Article  Google Scholar 

  11. Zhang X, Yun LJ, Peng LB, Lu Y, Ma KP, Tang F (2013) Optimization of Douchi fibrinolytic enzyme production by statistical experimental methods. J Huazhong Univ Sci Technol Med Sci 33:153–158

    Article  Google Scholar 

  12. Yegin S, Fernandez-Lahore M, Jose Gama Salgado A, Guvenc U, Goksungur Y, Tari C (2011) Aspartic proteinases from Mucor spp. in cheese manufacturing. Appl Microbiol Biotechnol 89:949–960

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Astrup T, Mullertz S (1952) The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 40:346–351

    Article  CAS  Google Scholar 

  15. Wang SL, Wu YY, Liang TW (2011) Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007. New Biotechnol 28:196–202

    Article  CAS  Google Scholar 

  16. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85

    Article  CAS  Google Scholar 

  17. 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

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Kim SH, Choi NS, Lee WY (1998) Fibrin zymography: a direct analysis of fibrinolytic enzymes on gels. Anal Biochem 263:115–116

    Article  CAS  Google Scholar 

  20. Moon SM, Kim JS, Kim HJ, Choi MS, Park BR, Kim SG, Ahn H, Chun HS, Shin YK, Kim JJ, Kim DK, Lee SY, Seo YW, Kim YH, Kim CS (2014) Purification and characterization of a novel fibrinolytic alpha chymotrypsin like serine metalloprotease from the edible mushroom, Lyophyllum shimeji. J Biosci Bioeng 117:544–550

    Article  CAS  Google Scholar 

  21. Koh YS, Chung KH, Kim DS (2001) Biochemical characterization of a thrombin-like enzyme and a fibrinolytic serine protease from snake (Agkistrodon saxatilis) venom. Toxicon 39:555–560

    Article  CAS  Google Scholar 

  22. Montriwong A, Kaewphuak S, Rodtong S, Roytrakul S, Yongsawatdigul J (2012) Novel fibrinolytic enzymes from Virgibacillus halodenitrificans SK1-3-7 isolated from fish sauce fermentation. Process Biochem 47:2379–2387

    Article  CAS  Google Scholar 

  23. Yeo WS, Seo MJ, Kim MJ, Lee HH, Kang BW, Park JU, Choi YH, Jeong YK (2011) Biochemical analysis of a fibrinolytic enzyme purified from Bacillus subtilis strain A1. J Microbiol 49:376–380

    Article  CAS  Google Scholar 

  24. Bi Q, Han B, Feng Y, Jiang Z, Yang Y, Liu W (2013) Antithrombotic effects of a newly purified fibrinolytic protease from Urechis unicinctus. Thromb Res 132:e135–144

    Article  CAS  Google Scholar 

  25. Ju X, Cao X, Sun Y, Wang Z, Cao C, Liu J, Jiang J (2012) Purification and characterization of a fibrinolytic enzyme from Streptomyces sp. XZNUM 00004. World J Microbiol Biotechnol 28:2479–2486

    Article  CAS  Google Scholar 

  26. Huang S, Pan S, Chen G, Huang S, Zhang Z, Li Y, Liang Z (2013) Biochemical characteristics of a fibrinolytic enzyme purified from a marine bacterium, Bacillus subtilis HQS-3. Int J Biol Macromol 62:124–130

    Article  CAS  Google Scholar 

  27. Tharwat NA (2006) Purification and biochemical characterization of fibrinolytic enzyme produced by thermophilic fungus Oidiodendron flavum. Biotechnology 5:160–165

    Article  CAS  Google Scholar 

  28. Lu CL, Chen S, Chen SN (2010) Purification and characterization of a novel fibrinolytic protease from Schizophyllum commune. J Food Drug Anal 18:69–76

    CAS  Google Scholar 

  29. Mander P, Cho SS, Simkhada JR, Choi YH, Yoo JC (2011) A low molecular weight chymotrypsin-like novel fibrinolytic enzyme from Streptomyces sp. CS624. Process Biochem 46:1449–1455

    Article  CAS  Google Scholar 

  30. Wang X, Rosell A, Lo EH (2008) Targeting extracellular matrix proteolysis for hemorrhagic complications of tPA stroke therapy. CNS Neurol Disord Drug Targets 7:235–242

    Article  CAS  Google Scholar 

  31. Nakajima N, Mihara H, Sumi H (1993) Characterization of potent fibrinolytic enzymes in earthworm, Lumbricus rubellus. Biosci Biotechnol Biochem 57:1726–1730

    Article  CAS  Google Scholar 

  32. Deng Z, Wang S, Li Q, Ji X, Zhang L, Hong M (2010) Purification and characterization of a novel fibrinolytic enzyme from the polychaete, Neanthes japonica (Iznka). Bioresour Technol 101:1954–1960

    Article  CAS  Google Scholar 

  33. Wang F, Wang C, Li M, Gui L, Zhang J, Chang W (2003) Purification, characterization and crystallization of a group of earthworm fibrinolytic enzymes from Eisenia fetida. Biotechnol Lett 25:1105–1109

    Article  CAS  Google Scholar 

  34. You WK, Sohn YD, Kim KY, Park DH, Jang Y, Chung KH (2004) Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans. Insect Biochem Mol Biol 34:239–250

    Article  CAS  Google Scholar 

  35. Wang SL, Yeh PY (2006) Production of a surfactant-and solvent stable alkaliphilic protease by bioconversion of shrimp shell wastes fermented by Bacillus subtilis TKU007. Process Biochem 41:1545–1552

    Article  CAS  Google Scholar 

  36. Lu F, Lu Z, Bie X, Yao Z, Wang Y, Lu Y, Guo Y (2010) Purification and characterization of a novel anticoagulant and fibrinolytic enzyme produced by endophytic bacterium Paenibacillus polymyxa EJS-3. Thromb Res 126:e349–355

    Article  CAS  Google Scholar 

  37. Cha WS, Park SS, Kim SJ, Choi D (2010) Biochemical and enzymatic properties of a fibrinolytic enzyme from Pleurotus eryngii cultivated under solid-state conditions using corn cob. Bioresour Technol 101:6475–6481

    Article  CAS  Google Scholar 

  38. Bajaj BK, Sharma N, Singh S (2013) Enhanced production of fibrinolytic protease from Bacillus cereus NS-2 using cotton cake as nitrogen source. Biocatal Agric Biotechnol 2:204–209

    Google Scholar 

  39. Majumdar S, Sarmah B, Gogoi D, Banerjee S, Ghosh SS, Banerjee S, Chattopadhyay P, Mukherjee AK (2014) Characterization, mechanism of anticoagulant action, and assessment of therapeutic potential of a fibrinolytic serine protease (Brevithrombolase) purified from Brevibacillus brevis strain FF02B. Biochimie 103:50–60

    Article  CAS  Google Scholar 

  40. Li H, Sun Y, Jiao X, Wang H, Zhu H (2016) Purification and characterization of thermostable serine proteases encoded by the genes ttha0099 and ttha01320 from Thermus thermophilus HB8. Extremophiles 20:493–502

    Article  CAS  Google Scholar 

  41. Thaz CJ, Jayaraman G (2014) Stability and detergent compatibility of a predominantly beta-sheet serine protease from halotolerant B. aquimaris VITP4 strain. Appl Biochem Biotechnol 172:687–700

    Article  CAS  Google Scholar 

  42. Chittoor JT, Balaji L, Jayaraman G (2016) Optimization of parameters that affect the activity of the alkaline protease from halotolerant bacterium, Bacillus acquimaris VITP4, by the application of response surface methodology and evaluation of the storage stability of the enzyme. Iran J Biotech 14:e1269

    Article  Google Scholar 

  43. Burstein EA, Vedenkina NS, Ivkova MN (1973) Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol 18:263–279

    Article  CAS  Google Scholar 

  44. Nascimento TP, Sales AE, Porto CS, Brandão RM, de Campos-Takaki GM, Teixeira JA, Porto TS, Porto AL, Converti A (2016) Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate). J Chromatogr B Anal Technol Biomed Life Sci 1025:16–24

    Article  CAS  Google Scholar 

  45. Chapanian R, Kwan DH, Constantinescu I, Shaikh FA, Rossi NA, Withers SG, Kizhakkedathu JN (2014) Enhancement of biological reactions on cell surfaces via macromolecular crowding. Nat Commun 5:e4683

    Article  Google Scholar 

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Acknowledgements

The authors would like to thank the Projects RENNORFUN/CNPq process No. 563382/2010-4 and SISBIOTA/FACEPE process No. APQ-0086-2.12/11 for financial support of the present work. The authors also appreciate FACEPE and CNPq for the scholarship of the first author and the Laboratory of Bioactive Technology (LABTECBIO/UFRPE) that provided the necessary structure for the research.

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Correspondence to Attilio Converti.

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Nascimento, T.P., Sales, A.E., Porto, T.S. et al. Purification, biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262. Bioprocess Biosyst Eng 40, 1209–1219 (2017). https://doi.org/10.1007/s00449-017-1781-3

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