PRODUCTION AND PURIFICATION OF PHARMACEUTICALLY IMPORTANT FIBRINOLYTIC ENZYME FROM BACILLUS SPECIES
Abstract
The medicinal and pharmaceutical importance of currently available thrombolytic agents like urokinase, t-PA, streptokinase, staphylokinase and others, demonstrated repeatedly since 1970s, however sometimes they cause undesirable side effects like bleeding and allergic responds. The present findings reports isolation, screening and identification of soil bacterium for production of fibrinolytic enzyme. Samples for the study were collected from different locations were first screened for proteolytic activity using skimmed milk agar plate and lastly fibrin plate method was used to evaluate fibrinolytic activity. The strain capable of producing fibrinolytic protein was identified as Bacillus Spp. Using both Bergery’s manual of systemic bacteriology and biochemical characterization simultaneously. Selected strain was than subjected to the process of fermentation using basal media for 5 days, 37°C and at 180rpm. Protein content and fibrinolytic activity were measured by Biuret method using bovine serum albumin as standard and fibrinolytic assay respectively. Three stage purification was done, that includes salting out with ammonium sulphate, followed by gel filtration chromatography and finally separated by RP-HPLC, proteins were eluted in peaks with a retention time of 2.092, 3.188, 5.178, 7.295, and 11.32 minutes. The fraction with retention time 7.295 minutes shows a maximum activity. The enzyme found to be having an optimum pH between 7.0 and 7.5. Enzyme is also stable at the optimum pH and found to lose its activity on higher side of acidity or alkalinity. It is more active at 40°C and is stable at 37°C to 43°C with slight modification in activity.
Keywords:
Bacillus Spp, Thrombus, clot, fibrin, fibrinolytic, fibrinogenDOI
https://doi.org/10.25004/IJPSDR.2015.070610References
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11. Hajji M, Kanoun S, Nasri M, Gharsallah N. Purification and characterization of an alkaline serine-protease produced by a new isolated Aspergillus clavatus ES1. Process Biochemistry. 2007; 42(5):791-7.
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14. Vranova V, Rejsek K, Formanek P. Proteolytic activity in soil: A review. Applied Soil Ecology. 2013; 70:23-32.
15. Chang CT, Wang PM, Hung YF, Chung YC. Purification and biochemical properties of a fibrinolytic enzyme from Bacillus subtilis-fermented red bean. Food Chemistry. 2012; 133(4):1611-7.
16. Balachandran C, Duraipandiyan V, Ignacimuthu S. Purification and characterization of protease enzyme from actinomycetes and its cytotoxic effect on cancer cell line (A549). Asian Pacific Journal of Tropical Biomedicine. 2012; 2(1):S392-S400.
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19. Mahajan PM, Nayak S, Lele SS. Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: media optimization, purification and characterization. J Biosci Bioeng. 2012; 113(3):307-14.
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22. Manivannan S. Madhavi P, Bhuvaneswari S. Production and Optimization of α-Amylase from Aspergillus flavus under Solid State Fermentation. International Journal of Pharmaceutical Sciences and Drug Research. 2015; 7(3):298-303.
23. Wu B, Wu L, Chen D, Yang Z, Luo M. Purification and characterization of a novel fibrinolytic protease from Fusarium sp. CPCC 480097. J Ind Microbiol Biotechnol. 2009; 36(3):451-9.
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25. George R, Witt W, Kreutzfeldt C. Determination of protein by Coomassie dye-binding in agarose gels. J Biochem Biophys Methods. 1986; 13(4–5):221-9.
26. Wang SL, Wu YY, Liang TW. Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007. N Biotechnol. 2011; 28(2):196-202.
27. Uesugi Y, Usuki H, Iwabuchi M, Hatanaka T. Highly potent fibrinolytic serine protease from Streptomyces. Enzyme Microb Technol. 2011; 48(1):7-12.
28. Dubey R, Kumar J, Agrawala D, Pusp P. Isolation, production, purification, assay and characterization of fibrinolytic enzymes (Nattokinase, Streptokinase and Urokinase) from bacterial sources. African Journal of Biotechnology. 2011; 10(8):1408-20.
29. Qiu Y, Choo YM, Yoon HJ, Jia J, Cui Z, Wang D, et al. Fibrinogenolytic activity of bumblebee venom serine protease. Toxicol Appl Pharmacol. 2011; 255(2):207-13.
30. Rahman JDMM. Antioxidant and thrombolytic activity of chloroform extract of Bacopa monniera (l.). Bulletin of Pharmaceutical Research. 2014; 4(3):133-9.
31. Simkhada JR, Cho SS, Mander P, Choi YH, Yoo JC. Purification, biochemical properties and antithrombotic effect of a novel Streptomyces enzyme on carrageenan-induced mice tail thrombosis model. Thromb Res. 2012; 129(2):176-82.
32. Sun MZ, Liu S, Greenaway FT. Characterization of a fibrinolytic enzyme (ussurenase) from Agkistrodon blomhoffii ussurensis snake venom: insights into the effects of Ca2+ on function and structure. Biochim Biophys Acta. 2006; 1764(8):1340-8.
33. Axelrod DA, Wakefield TW. Future directions in antithrombotic therapy: emphasis on venous thromboembolism. J Am Coll Surg. 2001; 192(5):641-51.
34. Verstraete M. Third-generation thrombolytic drugs. The American Journal of Medicine. 2000; 109(1):52-8.
35. Leadley Jr RJ, Chi L, Rebello SS, Gagnon A. Contribution of in vivo models of thrombosis to the discovery and development of novel antithrombotic agents. J Pharmacol Toxicol Methods. 2000; 43(2):101-16.
36. Toombs CF. New directions in thrombolytic therapy. Curr Opin Pharmacol. 2001; 1(2):164-8.
37. Sinnaeve P, Van De Werf F. Thrombolytic Therapy: State of the Art. Thromb Res. 2001; 103, Supplement 1(0):S71-S9.
38. Gulba DC, Bode C, Runge MS, Huber K. Thrombolytic agents-an updated overview. Fibrinolysis and Proteolysis. 1998; 12, Supplement 2(0):39-58.
39. Vanderschueren S, Van De Werf F, Collen D. Recombinant staphylokinase for thrombolytic therapy. Fibrinolysis and Proteolysis. 1997; 11, Supplement 2(0):39-44.
40. Schussheim AE, Fuster V. Thrombosis, antithrombotic agents, and the antithrombotic approach in cardiac disease. Prog Cardiovasc Dis. 1997; 40(3):205-38.
2. Cavello IA, Hours RA, Rojas NL, Cavalitto SF. Purification and characterization of a keratinolytic serine protease from Purpureocillium lilacinum LPS # 876. Process Biochemistry. 2013; 48(5-6):972-8.
3. Bilheiro RP, Braga AD, Filho ML, Carvalho-Tavares J, Agero U, Carvalho M, et al. The thrombolytic action of a proteolytic fraction (P1G10) from Carica candamarcensis. Thromb Res. 2013; 131(4):e175-82.
4. Wolberg AS, Campbell RA. Thrombin generation, fibrin clot formation and hemostasis. Transfus Apher Sci. 2008; 38(1):15-23.
5. Wakefield TW, Caprini J, Comerota AJ. Thromboembolic diseases. Curr Probl Surg. 2008; 45(12):844-99.
6. De Laforcade A. Diseases associated with thrombosis. Top Companion Anim Med. 2012; 27(2):59-64.
7. Atici AG, Findik S, Light RW, Ozkaya S, Erkan L, Akan H. Vena caval thromboses. J Crit Care. 2010; 25(2):336-42.
8. Marder VJ. Thrombolytic therapy for deep vein thrombosis: potential application of plasmin. Thromb Res. 2009; 123, Supplement 4(0):S56-S61.
9. López JA, Chen J. Pathophysiology of venous thrombosis. Thromb Res. 2009; 123, Supplement 4(0):S30-S4.
10. Longstaff C, Thelwell C. Understanding the enzymology of fibrinolysis and improving thrombolytic therapy. FEBS Lett. 2005; 579(15):3303-9.
11. Hajji M, Kanoun S, Nasri M, Gharsallah N. Purification and characterization of an alkaline serine-protease produced by a new isolated Aspergillus clavatus ES1. Process Biochemistry. 2007; 42(5):791-7.
12. Doddapaneni KK, Tatineni R, Vellanki RN, Gandu B, Panyala NR, Chakali B, et al. Purification and characterization of two novel extra cellular proteases from Serratia rubidaea. Process Biochemistry. 2007; 42(8):1229-36.
13. Joo HS, Chang CS. Production of protease from a new alkalophilic Bacillus sp. I-312 grown on soybean meal: optimization and some properties. Process Biochemistry. 2005; 40(3-4):1263-70.
14. Vranova V, Rejsek K, Formanek P. Proteolytic activity in soil: A review. Applied Soil Ecology. 2013; 70:23-32.
15. Chang CT, Wang PM, Hung YF, Chung YC. Purification and biochemical properties of a fibrinolytic enzyme from Bacillus subtilis-fermented red bean. Food Chemistry. 2012; 133(4):1611-7.
16. Balachandran C, Duraipandiyan V, Ignacimuthu S. Purification and characterization of protease enzyme from actinomycetes and its cytotoxic effect on cancer cell line (A549). Asian Pacific Journal of Tropical Biomedicine. 2012; 2(1):S392-S400.
17. Zanphorlin LM, Cabral H, Arantes E, Assis D, Juliano L, Juliano MA, et al. Purification and characterization of a new alkaline serine protease from the thermophilic fungus Myceliophthora sp. Process Biochemistry. 2011; 46(11):2137-43.
18. Choi D, Cha WS, Park N, Kim HW, Lee JH, Park JS, et al. Purification and characterization of a novel fibrinolytic enzyme from fruiting bodies of Korean Cordyceps militaris. Bioresour Technol. 2011; 102(3):3279-85.
19. Mahajan PM, Nayak S, Lele SS. Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: media optimization, purification and characterization. J Biosci Bioeng. 2012; 113(3):307-14.
20. Kumar S, Sharma NS, Saharan MR, Singh R. Extracellular acid protease from Rhizopus oryzae: purification and characterization. Process Biochemistry. 2005; 40(5):1701-5.
21. Mukherjee AK, Rai SK, Thakur R, Chattopadhyay P, Kar SK. Bafibrinase: A non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency. Biochimie. 2012; 94(6):1300-8.
22. Manivannan S. Madhavi P, Bhuvaneswari S. Production and Optimization of α-Amylase from Aspergillus flavus under Solid State Fermentation. International Journal of Pharmaceutical Sciences and Drug Research. 2015; 7(3):298-303.
23. Wu B, Wu L, Chen D, Yang Z, Luo M. Purification and characterization of a novel fibrinolytic protease from Fusarium sp. CPCC 480097. J Ind Microbiol Biotechnol. 2009; 36(3):451-9.
24. Ledoux M, Lamy F. Determination of proteins and sulfobetaine with the folin-phenol reagent. Anal Biochem. 1986; 157(1):28-31.
25. George R, Witt W, Kreutzfeldt C. Determination of protein by Coomassie dye-binding in agarose gels. J Biochem Biophys Methods. 1986; 13(4–5):221-9.
26. Wang SL, Wu YY, Liang TW. Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007. N Biotechnol. 2011; 28(2):196-202.
27. Uesugi Y, Usuki H, Iwabuchi M, Hatanaka T. Highly potent fibrinolytic serine protease from Streptomyces. Enzyme Microb Technol. 2011; 48(1):7-12.
28. Dubey R, Kumar J, Agrawala D, Pusp P. Isolation, production, purification, assay and characterization of fibrinolytic enzymes (Nattokinase, Streptokinase and Urokinase) from bacterial sources. African Journal of Biotechnology. 2011; 10(8):1408-20.
29. Qiu Y, Choo YM, Yoon HJ, Jia J, Cui Z, Wang D, et al. Fibrinogenolytic activity of bumblebee venom serine protease. Toxicol Appl Pharmacol. 2011; 255(2):207-13.
30. Rahman JDMM. Antioxidant and thrombolytic activity of chloroform extract of Bacopa monniera (l.). Bulletin of Pharmaceutical Research. 2014; 4(3):133-9.
31. Simkhada JR, Cho SS, Mander P, Choi YH, Yoo JC. Purification, biochemical properties and antithrombotic effect of a novel Streptomyces enzyme on carrageenan-induced mice tail thrombosis model. Thromb Res. 2012; 129(2):176-82.
32. Sun MZ, Liu S, Greenaway FT. Characterization of a fibrinolytic enzyme (ussurenase) from Agkistrodon blomhoffii ussurensis snake venom: insights into the effects of Ca2+ on function and structure. Biochim Biophys Acta. 2006; 1764(8):1340-8.
33. Axelrod DA, Wakefield TW. Future directions in antithrombotic therapy: emphasis on venous thromboembolism. J Am Coll Surg. 2001; 192(5):641-51.
34. Verstraete M. Third-generation thrombolytic drugs. The American Journal of Medicine. 2000; 109(1):52-8.
35. Leadley Jr RJ, Chi L, Rebello SS, Gagnon A. Contribution of in vivo models of thrombosis to the discovery and development of novel antithrombotic agents. J Pharmacol Toxicol Methods. 2000; 43(2):101-16.
36. Toombs CF. New directions in thrombolytic therapy. Curr Opin Pharmacol. 2001; 1(2):164-8.
37. Sinnaeve P, Van De Werf F. Thrombolytic Therapy: State of the Art. Thromb Res. 2001; 103, Supplement 1(0):S71-S9.
38. Gulba DC, Bode C, Runge MS, Huber K. Thrombolytic agents-an updated overview. Fibrinolysis and Proteolysis. 1998; 12, Supplement 2(0):39-58.
39. Vanderschueren S, Van De Werf F, Collen D. Recombinant staphylokinase for thrombolytic therapy. Fibrinolysis and Proteolysis. 1997; 11, Supplement 2(0):39-44.
40. Schussheim AE, Fuster V. Thrombosis, antithrombotic agents, and the antithrombotic approach in cardiac disease. Prog Cardiovasc Dis. 1997; 40(3):205-38.
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01-11-2015
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“PRODUCTION AND PURIFICATION OF PHARMACEUTICALLY IMPORTANT FIBRINOLYTIC ENZYME FROM BACILLUS SPECIES”. International Journal of Pharmaceutical Sciences and Drug Research, vol. 7, no. 6, Nov. 2015, pp. 493-7, https://doi.org/10.25004/IJPSDR.2015.070610.
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How to Cite
“PRODUCTION AND PURIFICATION OF PHARMACEUTICALLY IMPORTANT FIBRINOLYTIC ENZYME FROM BACILLUS SPECIES”. International Journal of Pharmaceutical Sciences and Drug Research, vol. 7, no. 6, Nov. 2015, pp. 493-7, https://doi.org/10.25004/IJPSDR.2015.070610.
