PRODUCTION AND PARTIAL CHARACTERIZATION OF FIBRINOLYTIC ENZYME FROM A SOIL ISOLATE ASPERGILLUS CARBONARIUS S-CSR-0007
Objective: This work was undertaken with the aim of isolating and screening fungal soil isolates with fibrinolytic activity.
Methods: Soil sample near slaughter house was collected and screened for fibrinolytic activity by using fibrin-agar. Enzyme production was optimized under various parameters like pH, temperature, substrate concentration and purified partially by ammonium sulphate precipitation. The stability of the partially purified enzyme was analyzed under the influence of a wide range of pH, temperature, and substrate concentrations.
Results: Among the seven isolates screened, Aspergillus carbonarius S-CSR-0007 exhibited largest clear zone and was selected for further studies. Among the various substrates tested casein was found to support the highest caseinolytic activity of 816 U/ml and fibrinolytic activity of 510 U/ml. The culture supernatant of A. carbonarius S-CSR-0007 was fractionated by ammonium sulfate precipitation followed by dialysis, and maximum activity was obtained in the fraction with 80% ammonium sulfate, with an enzyme activity of 1200 U/ml using tyrosine as standard. The partially purified fibrinolytic enzyme showed optimal activity at 45 Â°C and pH 7.0. The enzyme was stable up to a temperature of 50 Â°C and pH 8.0, and the optimum substrate concentration was 4%.
Conclusion: The crude enzyme showed high blood clot lysis activity, which may be a good candidate in the pharmaceutical industry. However, more studies need to be carried out to establish its clinical use.
2. Wang SL, Chen HJ, Liang TW, Lin YD. A novel nattokinase produced by Pseudomonas sp. TKU015 using shrimp shells as substrate. Process Biochem 2009;44:70-6.
3. Dubey R, Kumar J, Agrawala D, Char T, Pusp P. Isolation, production, purification, assay and characterization of the fibrinolytic enzyme (Nattokinase, Streptokinase and Urokinase) from bacterial sources. Afr J Biotechnol 2011;10:1408-20.
4. Peng Y, Yang X, Zhang Y. Microbial fibrinolytic enzymes: an overview of the source, production, properties, and thrombolytic activity in vivo. Appl Microbiol Biotechnol 2005;69:126-32.
5. Cesarman-Maus G, Hajjar KA. Molecular mechanisms of fibrinolysis. Br J Haematol 2005;129:307â€“21.
6. EI-Aassar SA, EI-Badry HM, Abdel-Fattah AF. The biosynthesis of proteases with fibrinolytic activity in immobilized cultures of Penicillium chrysogenum H9. Appl Microbiol Biotechnol 1990;33:26-30.
7. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75.
8. Al-Juamily EF, Al-Zaidy BH. Optimization conditions of production fibrinolytic enzyme from Bacillus lichniformis B4 local isolate. Br J Pharmacol Toxicol 2012;3:289-95.
9. Mohanakumar A. Production and characterization of serratiopeptidase enzyme from Serratia marcescens. Int J Biol 2011;3:39-51.
10. Borah D, Yadav RNS, Sangra A, Shahin L, Chaubey AK. Production, purification, and characterization of nattokinase from Bacillus subtilis isolated from tea garden soil samples of Dibrugarh, Assam. Asian J Pharm Clin Res 2012;5:124-5.
11. Yin LJ, Lin HH, Jiang ST. Bioproperties of potent nattokinase from Bacillus subtilis YJ1. J Agric Food Chemi 2010;58:5737-42.
12. Raju EVN, Divakar G. Non-recombinant mutagenesis of bacillus cereus for fibrinolytic protease production. World J Pharm Pharm Sci 2013;2:6189-201.
13. Sanusi NA, Jamaluddin H. Purification of a fibrinolytic enzyme from Bacillus Sp. isolated from Budu. J Teknol 2012;59:63â€“8.
14. Makino K, Koshikava T, Nishithara T, Ichikawa T, Kondo M. Studies on protease from marine bacteria. Isolation of marine Pseudomonas sp. 145-2 and purification of protease. Microbios 1981;31:103-12.
15. Lee BC, Bae JT, Pyo HB, Choe TB, Kim SW, Hwang HJ, et al. Submerged culture conditions for the production of mycelial biomass exopolysaccharides by the edible Basidiomycete Grifola frondosa. Enzyme Microb Technol 2004;35:369-76.
16. Kumaran S, Jagadish LK, Palani P, Chellaram C, Anand TP, Kaviyarasan V. Growth optimization conditions for the production of the fibrinolytic enzyme from Ganoderma lucidum. J Ecobiotechnol 2010;2:11-5.
17. Kim SA, Son HJ, Kim KK, Park HC, Lee SM, Cho BW, et al. Characterisation and production of thermostable and acid stable extracellular fibrinolytic enzyme from Cordyceps millitaris. Int J Indust Entamol 2011;22:83-93.
18. Whittaker JR. The principle of enzymology for the food science. Marcel Dekker INC., New York; 1972.
19. Segel IH. Biochemical calculations. 2nd edition. John and Sons. Inc. New York; 1976.
20. Xiao-Lan L, Lian-Xiang D, Fu-Ping L, Xi-Qun Z, Jing X. Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12. Appl Microbiol Biotechnol 2005;67:209-14.
21. Simkhada JR, Mander P, Cho SS, Yoo JC. A novel fibrinolytic protease from Streptomyces sp. CS684. Process Biochem 2010;45:88-93.
22. Abdel-Naby MA, EI-Diwani AI, Shaker HM, Ismail AMS. Production and properties of the fibrinolytic enzyme from Streptomyces sp. NRC 411. World J Microbiol Biotechnol 1992;8:267-9.
23. Kim JH, Kim YS. A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea. Biosci Biotechnol Biochem 1999;63:2130-6.
24. Kim J, Sapkota K, Park S, Choi B, Kim S, Hiep NT, et al. A fibrinolytic enzyme from the medicinal mushroom Cordyceps militaris. J Microbiol 2006;44:622-31.
25. Pandee P, Dissara Y. Production and properties of a fibrinolytic enzyme by Schizophyllum commune BL23. Songklanakarin J Sci Technol 2008;30:447-53.
26. Choi HS, Shin HH. Purification and characterization of a cysteine protease from Pleurotus ostreatus. Biosci Biotechnol Biochem 1998;62:1416-8.
27. Chang C, Fan M, Kuo F, Sung H. Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK. J Agric Food Chem 2000;48:3210-6.
28. Kim JH, Kim YS. Characterization of a metalloenzyme from a wild mushroom, Tricholoma saponaceum. Biosci Biotechnol Biochem 2001;65:356-62.
29. Lonsane BK, Saucedo-Castaneda G, Raimbault M, Roussos S, Viniegra-Gonzalez G, Ghildyal NP, et al. Scale-up strategies for solid state fermentation systems. Process Biochem 1992;27:259-73.