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Screening and Isolation of Fibrinolytic Enzymes from Bacteria using Agro-waste for Thrombolytic Treatment


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1 Department of Microbiology, Ethiraj College for Women, Chennai, India
     

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Bacterial fibrinolytic enzymes find great applications to treat and prevent cardiovascular diseases (CVD). The novel food-grade microorganisms are useful for thrombolytic therapy. Agro-residues were used for the production of fibrinolytic enzyme in solid-state fermentation. In this study, four different food samples were used and two Bacillus sp were isolated. The bacterial isolates were cultured and screened for its fibrinolytic activity. Under SSF, the production of fibrinolytic enzyme was enhanced by using cow dung substrate. The enzyme was then further purified by ammonium sulphate precipitation and dialysis method. After the successive purification steps, the molecular weight was estimated. The efficiency of the fibrinolytic enzyme produced was determined by its clot lytic activity on fibrin clot with the known concentration of the standard ‘Streptokinase’.

Keywords

Corn, Pearl Millet (Kambu), Finger Millet (Ragi), Wheat, Bacillus subtilis, Solid-Substrate Fermentation, Cow Dung, Fibrinolytic Enzyme, SDS-PAGE, Thrombolytic Treatment.
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  • Sumi, H. (1989) Fibrinolytic enzyme: property of Nattokinase and its application to oral fibrinolytic therapy. In "Functional" Food Materiah': Research and Development of Physiologically Active Substances Derived from Food, pp. 88-%. Industry and Technology Society, Tokyo.

  • Kotb, E. (2012). Fibrinolytic Bacterial Enzymes with Thrombolytic Activity.ISBN 978-3-642-24980-8 DOI: 10.1007/978-3-642-24980-8 series ISBN 1 2191-5385 softcover ISBN 978-3-642-24979-2.

  • Vijayaraghavan, P and Vincent S. G. P. (2014). Medium Optimization for the Production of Fibrinolytic Enzyme by Paenibacillus sp. IND8 Using Response Surface Methodology, Hindawi Publishing Corporation, The Scientific World Journal Volume 2014, Article ID 276942, doi: 10.1155/2014/276942

  • Collen. D and H. R. Lijnen. (2004). Tissue-type plasminogen activator: A historical perspective and personal account, J. Thromb. Haemost, 2: 541 - 546.

  • Duffy, M. J. (2002). Urokinase plasminogen activator and its inhibitor, PAI-1, as prognostic markers in breast cancer: From pilot to level 1 studies. Clin. Chem . 48: 1194 - 1197.

  • Sumi, H. (1989) Fibrinolytic enzyme: property of Nattokinase and its application to oral fibrinolytic therapy. In "Functional" Food Materiah': Research and Development of Physiologically Active Substances Derived from Food, pp. 88-%. Industry and Technology Society, Tokyo.

  • Bode. C et al. (1996). The future of thrombolysis in the treatment of acute myocardial infarction. Eur Heart J 17:55–60

  • Mukherjee, A. K et al. (2008). Production of alkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using Imperata cylindrica grass and potato peel as low-cost medium: characterization and application of enzyme in detergent formulation. Biochem. Eng. J. 39, 353–361. doi: 10.1016/j.bej.2007. 09.017

  • Ambrus, J. L et al. (1979) Studies on platelet aggregation with pentoxifylline: Effect in neoplastic disorders and other new indications. J. Med. 10, 339-345

  • Hwang, C.M et al. (2002). In vivo evaluation of lumbrokinase, a fibrinolytic enzyme extracted from Lumbricus rubellus, in a prosthetic vascular graft. J. Cardiovasc. Surg., 43: 891-894.

  • Kim, W et al. (1996). Purification and Characterization of a Fibrinolytic Enzyme Produced from Bacillus sp. strain CK 11-4 Screened from Chungkook-Jang, Applied and Environmental Microbiology, July 1996, p. 2482–2488 Vol. 62, No. 7 0099-2240/96/$04.0010

  • Sumi, H et al. (1987). A Novel Fibrinolytic Enzyme (Nattokinase) in the Vegetable Cheese Natto; a Typical and Popular Soybean Food in The Japanese Diet. Experientia. 43(10): 1110–1111.

  • Matseliukh, O.V et al. (1999). Purification and physicochemical properties of Bacillus thuringiensis IMBB-7324 peptidase with elastolytic and fibrinolytic activity. Ukr.Biokhim.Zh.84, 25–36.

  • Nidialkova, N. A et al (2012). Isolation of Bacillus thuringiensis IMVB-7324 fibrinolytic peptidase. Microbiol.Z.74,9–15.

  • Chitte, R. R et al. (2011). Production, purification, and biochemical characterization of a fibrinolytic enzyme from thermophilic Streptomyces sp. MCMB-379. Appl.Biochem.165,1406–1413. doi: 10.1007/s12010-011-9356-2

  • Lapsongphon, N et al. (2013). Spent brewery yeast sludge as a single nitrogen source for fibrinolytic enzyme production of Virgibacillus sp.SK37. FoodSci.Biotechnol.22, 71–78. doi: 10.1007/s10068-0130010-3

  • Nidialkova, N. A et al. (2013). Physicochemical properties of Bacillus thuringiensis IMVB-7324 fibrinolytic peptidase. Mikrobiol.Z.75,3–7.

  • Kumar, D. J. M et al. (2014). Production, optimization and characterization of fibrinolytic enzyme by Bacillus subtilis RJAS19. Pak. J. Biol. Sci. 17,529–534. doi:10.3923/pjbs.2014.529.534

  • Jhample, S. B et al. (2015). Statistical media optimization for enhanced production of fibrinolytic enzyme from newly isolated Proteus penneri SP-20. Biocatal. Agric. Biotechnol. 4, 370–379. doi: 10.1016/j.bcab.2015.05.006

  • Pandey, A et al. (2000). Biotechnological potential of coffee pulp and coffee husk for bioprocesses. Biochem. Eng. J. 6, 153–162. doi: 10.1016/S1369-703X(00) 00084-X

  • Johnvesly, B et al. (2002). Pigeon pea waste as a novel, inexpensive, substrate for productionof a thermostable alkaline protease from thermoalkalophilic Bacillus sp. JB-99. Bioresour. Technol. 82, 61–64. doi: 10.1016/S0960-8524(01)00147-X

  • Prakasham, R. S et al. (2006). Green gram husk an inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation. Bioresour. Technol. 97, 1449–1454. doi: 10.1016/j.biortech.2005.07.015

  • Mukherjee, A. K et al. (2008). Production of alkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using Imperata cylindrica grass and potato peel as low-cost medium: characterization and application of enzyme in detergent formulation. Biochem. Eng. J. 39, 353–361. doi: 10.1016/j.bej.2007. 09.017

  • Mahanta, N et al. (2008). Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using Jatropha curcas seed cake as substrate. Bioresour.Technol.99,1729–1735. doi: 10.1016/j.biortech.2007.03.046

  • Rajendran, A., and Thangavelu, V. (2013). Utilizing agricultural wastes as substrates for lipase production by Candida rugosa NCIM 3462 in solid-state fermentation: response surface optimization of fermentation parameters. Waste Biomass Valori 4,347–357. doi: 10.1007/s12649-012-9140-8

  • Salihu, A et al. (2014). Utilization of groundnut husk as a solid substrate for cellulase production by Aspergillus niger using response surface methodology. Waste Biomass Valorization 5,585–593. doi: 10.1007/s12649-013-9268-1

  • Fulhage, C. D. (2000). Reduce Environmental Problems with Proper Land Application of Animal Manure. Columbia, MO: University of Missouri Extension

  • Adegunloye, D. V et al. (2007). Microbial analysis of compost using cowdung as booster. Pak. J. Nut. 6, 506-510. doi: 10.3923/pjn.2007.506.510

  • Vijayaraghavan, P et al. (2016). Cow Dung Is a Novel Feedstock for Fibrinolytic Enzyme Production from Newly Isolated Bacillus sp. IND7 and Its Application in In Vitro Clot Lysis. Front. Microbiol. 7:361. doi: 10.3389/fmicb.2016.00361

  • Vijayaraghavan, P., and Vincent, S.G.P. (2012). Cow dung as a novel, inexpensive substrate for the production of a halo-tolerant alkaline protease by Halomonas sp. PV1 for eco-friendly applications. Biochem. Eng. J. 69, 57–60. doi: 10.1016/j.bej.2012.08.014

  • Vijayaraghavan, P et al. (2012). Cow dung: a potential biomass substrate for the production of detergent-stable dehairing protease by alkaliphilic Bacillus subtilis strain VV. Springer Plus1,76. doi: 10.1186/2193-1801-1-76

  • Vijayaraghavan, P et al. (2016). Bioconversion of agro-industrial wastes for the production of fibrinolytic enzyme from Bacillus halodurans IND18: Purification and biochemical characterization, Production and hosting by Elsevier, Pontificia Universidad CatólicadeValparaíso 0717-3458/© 2016 doi: 10.1016/j.ejbt.2016.01.002

  • Vijayaraghavan, P., and Vincent, S. G. P. (2015). A low cost fermentation medium for potential fibrinolytic enzyme production by a newly isolated marine bacterium, Shewanella sp. IND20. Biotechnol. Rep. 7, 135–142. doi: 10.1016/j.btre.2015.06.005

  • Hmood, S. A, Aziz, G. M. (2016). Optimum conditions for fibrinolytic enzyme (Nattokinase) production by Bacillus sp. B24 using solid state fermentation, Iraqi Journal of Science, 2016, Vol. 57, No.2C, pp:1391-1401

  • Sanusia, N. A, Jamaluddina, H. (2012). Purification of a Fibrinolytic Enzyme from Bacillus Sp. Isolated from Budu, 59 (2012) 63–68 |www.jurnalteknologi.utm.my | eISSN 2180–3722 | ISSN 0127–9696

  • Yuan, J et al. (2012). Thrombolytic effects of Douchi Fibrinolytic enzyme from Bacillus subtilis LD-8547 in vitro and in vivo, BMC Biotechnology 2012, | http://www.biomedcentral.com/1472-6750/12/36.

  • Huy et al. (2016). Screening and identification of Bacillus sp. isolated from traditional Vietnamese soybean-fermented products for high fibrinolytic enzyme production, International Food Research Journal 23(1): 326-331 (2016) Journal homepage | http://www.ifrj.upm.edu.my|

  • Chang, C. T et al. (2012). Purification and biochemical properties of a fibrinolytic enzyme from Bacillus subtilis-fermented red bean. Food Chem. 133, 1611–1617. doi: 10.1016/j.foodchem.2012.02.061

  • Vijayaraghavan, P et al. (2016). Bioconversion of agro-industrial wastes for the production of fibrinolytic enzyme from Bacillus halodurans IND18: Purification and biochemical characterization, Production and hosting by Elsevier, Pontificia Universidad CatólicadeValparaíso 0717-3458/© 2016 doi: 10.1016/j.ejbt.2016.01.002

  • Biji, G. D et al. (2016). Bio-prospecting of cuttle fish waste and cow dung for the production of fibrinolytic enzyme from Bacillus cereus IND5 in solid state fermentation, 3 Biotech (2016) 6:231 DOI 10.1007/s13205-016-0553-0

  • Balaraman, K. and Prakabaran, G. (2007). Production and Purification of a Fibrinolytic Enzyme (Thrombinase) from Bacillus Sphaericus. Indian J Med Res. 126: 459–464

  • Jeong, Y. K et al. (2001). Purification and Biochemical Characterization of a Fibrinolytic Enzyme from Bacillus subtilis BK-17. World Journal of Microbiology and Biotechnology. 17: 89–92

  • Kim, W et al. (1996). Purification and Characterization of a Fibrinolytic Enzyme Produced from Bacillus sp. strain CK 11-4 Screened from Chungkook-Jang, Applied and Environmental Microbiology, July 1996, p. 2482–2488 Vol. 62, No. 7 0099-2240/96/$04.0010

  • Hwang et al. (2007). Purification and Characterization of a New Fibrinolytic Enzyme of Bacillus licheniformis KJ-31, Isolated from Korean Traditional Jeot-gal. Journal of Microbiol Biotechnol. Vol 17(9): 1469-1476.

  • Jo et al. (2011). Purification and Characterization of a Major Fibrinolytic Enzyme from Bacillus amyloliquefaciens MJ5-41 Isolated from Meju. J. Microbiol. Biotechnol. 21(11): 1166–1173

  • Peng, Y et al. (2003). Purification and Characterization of a Fibrinolytic Enzyme Produced by Bacillus Amyloliquefaciens DC-4 Screened from Douchi, A Traditional Chinese Soybean Food. Comparative Biochemistry and Physiology, Part B. 134: 45–52.

  • Sumi, H et al. (1987). A Novel Fibrinolytic Enzyme (Nattokinase) in the Vegetable Cheese Natto; a Typical and Popular Soybean Food in The Japanese Diet. Experientia. 43(10): 1110–1111.

  • Deepak, V et al. (2008). Optimization of media composition for nattokinase production by Bacillus subtilis using response surface methodology. Bioresour.Technol. 99, 81708174. doi: 10.1016/j.biortech.2008.03.018

  • Mahajan, P. M et al. (2012). Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: media optimization, purification and characterization. J. Biosci. Bioeng. 113, 307–314. doi: 10.1016/j.jbiosc.2011.10.023

  • Yuan, J et al. (2012). Thrombolytic effects of Douchi Fibrinolytic enzyme from Bacillus subtilis LD-8547 in vitro and in vivo, BMC Biotechnology 2012, | http://www.biomedcentral.com/1472-6750/12/36.

  • Vijayaraghavan, P and Vincent S. G. P. (2014). Statistical Optimization of Fibrinolytic Enzyme Production Using Agroresidues by Bacillus cereus IND1 and Its Thrombolytic Activity In Vitro, Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 725064, 11 pages doi: 10.1155/2014/725064

  • Vijayaraghavan, P et al. (2016). Cow Dung Is a Novel Feedstock for Fibrinolytic Enzyme Production from Newly Isolated Bacillus sp. IND7 and Its Application in In Vitro Clot Lysis. Front. Microbiol. 7:361. doi: 10.3389/fmicb.2016.00361


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  • Screening and Isolation of Fibrinolytic Enzymes from Bacteria using Agro-waste for Thrombolytic Treatment

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Authors

S. Lakshmi Priya
Department of Microbiology, Ethiraj College for Women, Chennai, India
K. Krishna Prema
Department of Microbiology, Ethiraj College for Women, Chennai, India

Abstract


Bacterial fibrinolytic enzymes find great applications to treat and prevent cardiovascular diseases (CVD). The novel food-grade microorganisms are useful for thrombolytic therapy. Agro-residues were used for the production of fibrinolytic enzyme in solid-state fermentation. In this study, four different food samples were used and two Bacillus sp were isolated. The bacterial isolates were cultured and screened for its fibrinolytic activity. Under SSF, the production of fibrinolytic enzyme was enhanced by using cow dung substrate. The enzyme was then further purified by ammonium sulphate precipitation and dialysis method. After the successive purification steps, the molecular weight was estimated. The efficiency of the fibrinolytic enzyme produced was determined by its clot lytic activity on fibrin clot with the known concentration of the standard ‘Streptokinase’.

Keywords


Corn, Pearl Millet (Kambu), Finger Millet (Ragi), Wheat, Bacillus subtilis, Solid-Substrate Fermentation, Cow Dung, Fibrinolytic Enzyme, SDS-PAGE, Thrombolytic Treatment.

References