ISOLATION OF THERMOTOLERANT BACTERIA PRODUCING FIBRINOLYTIC ENZYME
Keywords:Fibrinolytic Enzyme, Thermotolerant Bacteria, Bacillus licheniformis
AbstractFibrinolytic enzymes were widely used in the treatment of cardiovascular diseases. However, the efficiency of the commercial enzymes are still lack of perfection because there are many side effects as well as not tolerant to downstream processing such as heat sensitive during spray drying process. Therefore, this study presents newly isolated thermophiles bacteria producing fibrinolytic enzyme. Sample was collected from Hot Spring Selayang at Selayang Selangor. Spread plate agar containing skim milk powder growth at pH 7, 53 Â for 24 hours was utilized to isolate thermotolerant bacteria producing protease. Further isolation on bacteria producing fibrinolytic enzyme was carried out using fibrin plate. 16S rDNA gene sequence analysis was used to identify the genotype of the isolates. 27 colonies of thermotolerant bacteria were isolated, however, only 19 of them showing proteolytic activity. All of the 19 isolates are motile and cocci in shapes, with 4 types of arrangement, which are single, diplo (pair), strepto (chain) and staphylo (cluster). HSP04 and HSP11 are gram positive bacteria and others are gram negative. From 19 isolates only 6 were chosen for further analysis. HSP23 showed the highest fibrinolytic activity compared with others. HSP23 was identified as Bacillus licheniformis with 98 % similarity to Bacillus licheniformis DCM 13 and Bacillus licheniformis strain ATCC 14580.
WHO 2011. Cardiovascular Disease. [Online]. From: www.who.int/cardiovascular_diseases/en/. [Acessed on 1 June 2015].
Voet, D., & Voet, J. G. 1990. Biochemistry. 2nd ed. John Wiley and Sons, NY, USA. 1087-1095
Ghupta, R., Sharma, V., Sridhara, S., Singh, B. P., & Arora, N. 2004. Identification of Serine Protease as a Mojor Allergen of Curvularia lunata. Allergy. 59: 421-427
Collen, D. & Lijnenen, H. R. 1994. Staphylokinase, a Fibrin Specific Plasminogen Activator with Therapeutic Potential. Blood. 84(3): 680-686.
Raju, E. V. N., & Divakar, G. 2013. Screening and Isolation of Fibrinolytic Protease Producing Bacteria from Various Regions in Bangalore. Scholars Academic Journal of Pharmacy (SAJP). 2(1): 27-30.
Mukherjee, A. K., & Rai, S. K. 2011. A Statistical Approach for the Enhanced Production of Alkaline Protease Showing Fibrinolytic Activity from a Newly Isolated Gram-Negative Bacillus sp. strain AS-S20-1. New Biotechnology. 28(2).
Huang, S., Pan, S., Chen, G., Huang, S., Zhang, Z., Li. Y., & Liang, Z. 2013. Biochemical Characteristcs of a Fibrinolytic Enzyme Purified from a Marine Bacterium, Bacillus subtilis HQS-3. International Journal of Biological Macromolecules. 62: 124-130.
Mahajan, P. M., Nayak, S. & 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(3): 307-314.
Jeong, S., Heo, K., Park, J. Y., Lee, K.W., Park, J., Joo, S. H., & Kim, J. H. 2015. Characterization of AprE176, a Fibrinolytic Enzyme from Bacillus subtilis HK176. J. Microbiol. Biotechnol. 25(1): 89-97.
Afifah, D. N., Sulchan, M., Syah, D., Yanti, Suhartono, M. T., & Kim, J. H. 2014. Purification and Characterization of a Fibrinolytic Enzyme from Bacillus pumilus 2.g Isolated from Gembus, an Indonesian Fermented Food. Prev. Nutr. Food Sci. 19(3): 213-219.
Sanusi, N. A., & Jamaluddin, H. 2012. Purification of Fibrinolytic Enzyme from Bacillus Sp. Isolated from Budu. Jurnal Teknologi. 59: 63-68.
Vijayaraghavan, P. and Vincent, S. G. P. 2014. Statistical Optimization of Fibrinolytic Enzyme Production by Pseudoalteromonas sp. IND11 Using Cow Dung Substrate by Response Surface Methodology. SpringerPlus. 3(1): 1-10.
Prihanto, A. A., Darius, & Firdaus, M. 2013. Proteolytic and Fibrinolytic Activities of Halophilic Lactic Acid Bacteria from Two Indonesian Fermented Foods. Journal of Microbiology, Biotechnology and Food Sciences. 2(5): 2291-2293.
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 Biochemistry. 47: 2379-2387.
Raj, A., Nancy, K., Pujari, N., Bhattacharya, S., Das, A., & Rajan, S. S. 2012. Enhancement of Protease Production by Pseudomonas Aeruginosa Isolated from Dairy Effluent Sludge and Determination of Its Fibrinolytic Potential. Asian Pasific Journal of Tropical Biomedicine. 1845-1851.
Lee, J., Park, S., Choi, W. A., Lee, K. H., Jeong, Y. K., Kong, I. S., & Park, S. 1999. Production of Fibrinolytic Enzyme in Bioreactor Culture by Bacillus subtillis BK-17. J Microbiol Biotechnol. 9(4): 443-449.
Seo, J. H., & Lee, S. P. 2004. Production of Fibrinolytic Enzyme from Soybean Grits Feremeted by Bacillus firmus NA-I. J Med Food. 7(4): 442-449.
Al-Juamily, E. F & Al-Zaidy, B. H. 2012. Optimization Conditions of Production Fibrinolytic Enzyme from Bacillus lichniformis B4 Local Isolate. British Journal of Pharmacology and Toxicology. 3(6): 289-295.
Kumar, S., & Nussinov, R. 2011. How Do Thermophilic Proteins Deal with Heat? CMLS, Cell Mol. Life Sci. 58: 1216-1233
Everly, C., & Alberto, J. 2000. Stressors, Stress and Survival: Overview. Front. Bioscien. 5: 780-786.
Calaus, D., & Berkeley, C. W. 1986. The Genus Bacillus. In: Bergeyâ€™s Manual of Systematic Bacteriology. Vol 2. Sneath PHA edition. Williams, Wilkins, Baltimore. 34: 1105-1139.
Astrup, T., & Mullertz. S. 1952. The Fibrin Plate Method for Estimating Fibrinolytic Activity. Arch. Biochem. Biophy. 40: 346-351.
Kim, H. K., Kim, G. T., Kim, D. K., Choi, W. A., Park, S. H., Jeong, Y. K., & Kong, I. S. 1997. Purification and Characterization of a Novel Fibrinolytic Enzyme from Bacillus sp. KA38 Originated from Fermented Fish. J Ferment Bioeng. 84(4): 307-312.
Singh, T. A., Devi, K. H., Ahmed, G., & Jeyaram, K. 2014. Microbial and Endogenous Origin of Fibrinolytic Activity in Traditional Fermented Foods of Northeast India. Food Research International. 55: 356-362.
Studier, F. W. 2005. Protein Production by Auto-Induction in High-Density Shaking Cultures. Protein Expression and Purification. 41: 207-234.
Chitte, R. R., & Dey, S. 2000. Potent Fibrinolytic Enzyme from a Thermophilic Streptomyces Species. World J Microbiol Biotechnol. 18(4): 289-294.
Maria, G., Asma, A., Afsheen, A., Rashida R. Z., Nadir, N. S., & Shah, A. U. Q. 2013. Isolation and Characterization of Different Strains of Bacillus licheniformis for the production of Commercially Significant Enzymes. Pak J Pharm Sci 26(4): 691-697.
Frankena, J., Verseveld, H. W., & Stouthamer, A. H. 1985. A continuous Culture Study of the Bioenergetics Aspects of Growth and Production of Exocellular Protease in Bacillus licheniformis. Appl. Microbiol Biotechnol. 22: 169-176.
Schroeter, R., Voigt, B., Jurgen, B., Methling K., Pother, D. C., Schafer, H., Albrecht, D., Mostertz, J., Mader, U., Evers, S., Murer, K. H., Lalk, M., Mascher, T., Hecker, M., & Schweder, T. 2011. The Peroxide Stress Response of Bacillus Licheniformis. Proteomics.11: 2851-2866.
How to Cite
Copyright of articles that appear in Jurnal Teknologi belongs exclusively to Penerbit Universiti Teknologi Malaysia (Penerbit UTM Press). This copyright covers the rights to reproduce the article, including reprints, electronic reproductions, or any other reproductions of similar nature.