Biosynthesis of fibrinolytic agent urokinase by Enterococcus gallinarum isolated from sardine

  • A. Nawaz
  • S.Q. Gillani
  • S.F. Tahir
  • K.A. Shah
  • S. Ashraf
  • H. Mukhtar
  • I.U. Haq
Keywords: Fibrinogen, Thrombin, Fibrin clots, In vitro, Thrombosis

Abstract

Fibrinogen degradation by thrombin leads to the formation of fibrin clots in the blood which results in cardiovascular diseases due to which certain fibrinolytic agents are used to treat cardiovascular complications. Urokinase is being favored as a fibrinolytic agent due to its high fibrin specificity and fewer side effects. Therefore, the main objective of this research was the isolation of potent urokinase producing bacteria from soil, seawater, and fermented food samples. Thirty-five isolates that were capable of producing urokinase were isolated using sixty samples and subjected to secondary screening for urokinase estimation. Bacterial isolate QGA-20 that showed maximum enzyme activity 52.6±0.03 FU/ml/min was identified by 18s RNA sequencing as Enterococcus gallinarum which has never been reported before for urokinase production. Different physical and cultural parameters such as fermentation medium, incubation time, temperature, pH, carbon source, nitrogen source, inoculum size, and trace elements were optimized for submerged fermentation to synthesize urokinase. Maximum production of urokinase 81.3±0.02 FU/ml/min was obtained with M-5 fermentation media at 96 hrs of incubation, 37oC temperature, 7 pH, sucrose as carbon, and soya flour as a nitrogen source, 3% inoculum size and CaCl2 as a trace element. In in vitro studies, urokinase enzyme was also applied for the disintegration of clot successfully. This study revealed a potent urokinase producer, reported first time, with appreciable clot lysis ability.   

References

Singh, T.A., Devi, K.R., Ahmed, G. and Jeyaram, K. (2014). Microbial and endogenous origin of fibrinolytic activity in traditional fermented foods of Northeast India. Food research international 55, 356-362. https://doi.org/10.1016/j.foodres.2013.11.028
Bajaj, B.K., Singh, S., Khullar, M., Singh, K. and Bhardwaj, S. (2014). Optimization of fibrinolytic protease production from Bacillus subtilis I-2 using agro-residues. Brazilian Archives of Biology and Technology 57, 653-662. https://doi.org/10.1590/S1516-8913201402132
Anh, D.B.Q., Mi, N.T.T. and Van Hung, P. (2015). Isolation and optimization of growth condition of Bacillus sp. from fermented shrimp paste for high fibrinolytic enzyme production. Arabian Journal for Science and Engineering 40, 23-28. https://doi.org/10.1007/s13369-014-1506-8
Chen, B., Huo, J., He, Z., He, Q., Hao, Y. and Chen, Z. (2013). Isolation and identification of an effective fibrinolytic strain Bacillus subtilis FR-33 from the Chinese doufuru and primary analysis of its fibrinolytic enzyme. African Journal of Microbiology Research 7, 2001-2009. https://DOI:10.5897/AJMR12.282
Chen, H., Wei-sei, X. and Qun, XI. (2011). Optimization of cultural conditions for the production of fibrinolytic enzyme with Cordyceps militaris. Science and Technology of Food Industry 1, 18-34. https://doi.org/10.1021/jf1049535
Dong, P., Wang, L., Sun, M., Yan, P., Zhang, X. and Yang, X. (2016). Comparison of antithrombotic effect between tirofiban and urokinase in emergency percutaneous coronary intervention. Biomedical Journal of Research 27, 1275-1279.
Dubey, R., Kumar, J., Agrawala, D., Char, T. and Pusp, P. (2011). Isolation, production, purification, assay and characterization of fibrinolytic enzymes (Nattokinase, Streptokinase and Urokinase) from bacterial sources. African Journal of Biotechnology 10, 1408-1420. https://DOI:10.5897/AJB10.1268
Ebben, H., Johanna, H. and Jeroen, S. (2015). Therapeutic application of contrast-enhanced ultrasound and low-dose urokinase for thrombolysis in a porcine model of acute peripheral arterial occlusion. Journal of Vascular surgery 1, 477-501. https://doi.org/10.1016/j.jvs.2014.02.057
Gupta, R., Beg, Q. and Lorenz, P. (2002). Bacterial alkaline proteases: molecular approaches and industrial applications. Applied microbiology and biotechnology 59, 15-32. https://doi.org/10.1007/s00253-002-0975-y
Huy, D.N.A., Hao, P. A. and Hung, P.V. (2016). Screening and identification of Bacillus sp. isolated from traditional Vietnamese soybean-fermented products for high fibrinolytic enzyme production. International Food Research Journal, 23, 326-331.
Ju, X., Cao, X., Sun, Y., Wang, Z., Cao, C., Liu, J. and Jiang, J. (2012). Purification and characterization of a fibrinolytic enzyme from Streptomyces sp. XZNUM 00004. World Journal of Microbiology and Biotechnology 28, 2479-2486. https://doi.org/10.1007/s11274-012-1055-9
Kotb, E., Helal, G.E.D.A. and Edries, F.M. (2015). Screening for fibrinolytic filamentous fungi and enzymatic properties of the most potent producer, Aspergillus brasiliensis AUMC 9735. Biologia 70, 1565-1574. https://doi.org/10.1515/biolog-2015-0192
Kumaran, S., Palani, P., Chellaram, C., Anand, T.P. and Kaviyarasan, V. (2011). Screening of fibrinolytic protease from South Indian isolates of Ganoderma lucidum. International Journal of Pharma and Bio Sciences 2, 419-431. http://www.ijpbs.net/volume2/issue1/pharma/_41.pdf
Mahajan, P.M., Nayak, S. and Lele, S.S. (2012). Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: Media optimization, purification and characterization. Journal of bioscience and bioengineering 113, 307-314. https://doi.org/10.1016/j.jbiosc.2011.10.023
Moharam, M.E., El-Bendary, M.A., El-Beih, F., Easa, S.M.H., Elsoud, M.M.A., Azzam, M.I. and Elgamal, N.N. (2019). Optimization of fibrinolytic enzyme production by newly isolated Bacillus subtilis Egy using central composite design. Biocatalysis and agricultural biotechnology 17, 43-50. https://doi.org/10.1016/j.bcab.2018.11.003
Moukhametova, L.I., Aisina, R.B., Lomakina, G.Y. and Varfolomeev, S.D. (2002). Properties of the urokinase-type plasminogen activator modified with phenylglyoxal. Russian Journal of Bioorganic Chemistry 28, 278-283. https://doi.org/10.1023/A:1019535606678
Pan, S., Chen, G., Wu, R., Cao, X., Zeng, W. and Liang, Z. (2019). Non-sterile submerged fermentation of fibrinolytic enzyme by marine Bacillus subtilis harboring antibacterial activity with starvation strategy. Frontiers in microbiology 10, 1025. https://doi.org/10.3389/fmicb.2019.01025
Reddy, L.V.A., Wee, Y.J., Yun, J.S. and Ryu, H.W. (2008). Optimization of alkaline protease production by batch culture of Bacillus sp. RKY3 through Plackett–Burman and response surface methodological approaches. Bioresource technology 99, 2242-2249. https://doi.org/10.1016/j.biortech.2007.05.006
Rovati, J.I., Delgado, O.D., Figueroa, L.I. and Fariña, J.I. (2010). A novel source of fibrinolytic activity: Bionectria sp., an unconventional enzyme-producing fungus isolated from Las Yungas rainforest (Tucumán, Argentina). World Journal of Microbiology and Biotechnology, 26(1), 55. https://doi.org/10.1007/s11274-009-0142-z
Sharma, A., Sharma, A., & Shivlata, L. (2015). Optimization of Medium Components for Enhanced Production of Extracellular Fibrinolytic Protease from Citrobacter braakii. International Journal of Current Microbiology and Applied Sciences 4, 248-259.
Smitha, K.V. and Pradeep, B. V. (2018). Optimization of Physical and Cultural Conditions of Fibrinolytic Enzyme from Bacillus altitudinis S-CSR 0020. Journal of Pure and Applied Microbiology 12, 343-354. http://dx.doi.org/10.22207/JPAM.12.1.40
Velumani, S. (2016). Isolation, Screening, Characterization and Production of Fibrinolytic enzyme from marine microorganism. International Journal of Advanced Research 2, 2395-4396.
Vijayaraghavan, P., Vincent, P. and Gnana, S. (2014). Medium optimization for the production of fibrinolytic enzyme by Paenibacillus sp. IND8 using response surface methodology. The Scientific World Journal 9. https://doi.org/10.1155/2014/276942
Wei, X., Luo, M., Xu, L., Zhang, Y., Lin, X., Kong, P. and Liu, H. (2011). Production of fibrinolytic enzyme from Bacillus amyloliquefaciens by fermentation of chickpeas, with the evaluation of the anticoagulant and antioxidant properties of chickpeas. Journal of agricultural and food chemistry 59, 3957-3963. https://doi.org/10.1021/jf1049535
Wang, C.T., Ji, B. P., Li, B., Nout, R., Li, P.L., Ji, H. and Chen, L.F. (2006). Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. Journal of Industrial Microbiology and Biotechnology 33, 750-758. https://doi.org/10.1007/s10295-006-0111-6
Wen, A.Y., Wu, B.T., Xu, X.B. and Liu, S.Q. (2018). Clinical study on the early application and ideal dosage of urokinase after surgery for hypertensive intracerebral hemorrhage. European Review for Medical and Pharmacological Sciences 22, 4663-4668.
Wu, R., Chen, G., Pan, S., Zeng, J. and Liang, Z. (2019). Cost-effective fibrinolytic enzyme production by Bacillus subtilis WR350 using medium supplemented with corn steep powder and sucrose. Scientific reports 9, 1-10. https://doi.org/10.1038/s41598-019-43371-8
Yoon, S.J., Yu, M.A., Sim, G.S., Kwon, S.T., Hwang, J.K., Shin, J.K, and Pyun, Y.R. (2002). Screening and characterization of microorganisms with fibrinolytic activity from fermented foods. Journal of microbiology and biotechnology 12, 649-656.
Published
2020-07-01
How to Cite
Nawaz, A., Gillani, S., Tahir, S., Shah, K., Ashraf, S., Mukhtar, H., & Haq, I. (2020). Biosynthesis of fibrinolytic agent urokinase by Enterococcus gallinarum isolated from sardine. Revista Mexicana De Ingeniería Química, 19(Sup. 1), 213-225. https://doi.org/10.24275/rmiq/Bio1654
Section
Biotechnology

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