Abstract
Intravascular thrombosis is one of the major causes of variety of cardiovascular disorders leading to high mortality worldwide. Fibrinolytic enzymes from microbial sources possess ability to dissolve these clots and help to circumvent these problems in more efficient and safer way. In the present study, fibrinolytic protease with higher fibrinolytic activity than plasmin was obtained from Serratia sp. KG-2-1 isolated from garbage dump soil. Response surface methodology was used to study the interactive effect of concentration of maltose, yeast extract + peptone (1:1), incubation time, and pH on enzyme production and biomass. Maximum enzyme production was achieved at 33 °C after 24 h at neutral pH in media containing 1.5% Maltose, 4.0% yeast extract + peptone and other trace elements resulting in 1.82 folds increased production. The enzyme was purified from crude extract using ammonium sulfate precipitation and DEAE-Sephadex chromatography resulting in 12.9 fold purification with 14.9% yield. The purified enzyme belongs to metalloprotease class and had optimal activity in conditions similar to physiological environment with temperature optima of 40 °C and pH optima of 8. The enzyme was found to be stable in various solvents and its activity was enhanced in presence of Na+, K+, Ba2+, Cu2+, Mn2+, Hg2+ but inhibited by Ca2+ and Fe3+. Hence, the obtained enzyme may be used as potential therapeutic agent in combating various thrombolytic disorders.
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Agrebi R, Haddar A, Hajji M, Frikha F, Manni L, Jellouli K, Nasri M (2009) Fibrinolytic enzymes from a newly isolated marine bacterium Bacillus subtilis A26 characterization and statistical media optimization. Can J Microbiol 55:1049–1061
Ahn MJ, Ku HJ, Lee SH, Lee JH (2015) Characterization of novel fibrinolytic enzyme, BsfA, from Bacillus subtilis ZA400 in Kimchi reveals its pertinence to thrombosis treatment. J Microbiol Biotechnol 25:2090–2099
Astrup T, Mullertz S (1952) The fibrin plate method for estimating fibrinolytic activity. Arch Biochem 40:346–351
Bajaj BK, Sharma P (2011) An alkali-thermotolerant extracellular protease from a newly isolated Streptomyces sp. DP2. New Biotechnol 28:725–732
Bajaj BK, Singh S, Khullar M, Singh K, Bhardwaj S (2014) Optimization of fibrinolytic protease production from Bacillus subtilis I-2 using agro-residues. Braz Archives Bio Technol 57:653–662
Basu B, Apte SK (2008) A novel serralysin metalloprotease from Deinococcus radiodurans. Biochem Biophys Acta 1784:1256–1264
Bhargavi PL, Prakasham RS (2012) Proteolytic enzyme production by isolated Serratia sp RSPB11: role of environmental parameters. Curr Trends Biotechnol Pharm 6:55–65
Bhargavi PL, Prakasham RS (2013) A fibrinolytic, alkaline and thermostable metalloprotease from the newly isolated Serratia sp RSPB11. Int J Biol Macromol 61:479–486
Bizuye A, Sago A, Admasu G, Getachew H, Kassa P, Amsaya M (2013) Isolation, optimization and characterization of protease producing bacteria from soil and water in Gondar town, North West Ethiopia. Int J Bacteriol Virol Immunol 3:20–24
Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254
Choi NS, Song JJ, Chung DM, Kim YJ, Maeng PJ, Kim SH (2009) Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet. J Ind Microbiol Biotechnol 36:417–426
Chung DM, Choi NS, Chun HK, Maeng PJ, Park SB, Kim SH (2010) A new fibrinolytic enzyme (55 kDa) from Allium tuberosum: purification, characterization, and comparison. J Med Food 13:1532–1536
Gupta A, Khare SK (2009) Enzymes from solvent-tolerant microbes: useful biocatalysts for non-aqueous enzymology. Crit Rev Biotechnol 29:44–54
Hassanein WA, Kotb E, Awny NM, El-Zawahry YA (2011) Fibrinolysis and anticoagulant potential of a metallo protease produced by Bacillus subtilis K42. J Biosci 36:773–779
Huang MX, Ye Y, Chen YX, Han YL (2012) Partial purification and characterization of fibrinolytic enzymes from yellow mealworm. Int J Pept Res Ther 18:153–161
Ju X, Cao X, Sun Y, Wang Z, Cao C, Liu J, Jiang J (2012) Purification and characterization of a fibrinolytic enzyme from Streptomyces sp. XZNUM 00004. World J Microbiol Biotechnol 28:2479–2486
Kim SH, Choi NS (2000) Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp strain DJ-4 screened from Doen-Jang. Biosc Biotechnol Biochem 64:1722–1725
Kim SB, Lee DW, Cheigh CI, Choe EA, Lee SJ, Hong YH, Choi HJ, Pyun YR (2006) Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, Tempeh. J Ind Microbiol Biotechnol 33:436–444
Kim HC, Choi BS, Sapkota K, Kim S, Lee HJ, Yoo JC, Kim SJ (2011) Purification and characterization of a novel, highly potent fibrinolytic enzyme from Paecilomyces tenuipes. Process Biochem 46:1545–1553
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lee AR, Kim GM, Park JY, Jo HD, Cha J, Song YS, Chun J, Kim JH (2010) Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH86-1 isolated from Cheonggukjang. J Korean Soc Appl Biol Chem 53:56–61
Liu XL, Du LX, Lu FP, Zheng XQ, Xiao J (2005) Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12. Appl Microbiol Biotechnol 67:209–214
Mahajan PM, Nayak S, Lele SS (2012) Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: media optimization, purification and characterization. J Biosci Bioeng 113:307–314
Niyonzima FN, More SS (2015) Purification and characterization of detergent-compatible protease from Aspergillus terreus gr. 3. Biotech 5:61–70
Peng Y, Yang X, Zhang Y (2005) Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo. Appl Microbiol Biotechnol 69:126–132
Ponnuswamy V, Arumugaperumal A, Samue GPV, Mariadhas VA, Naif AA (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
Rashad MM, Mahmoud AE, Al-Kashef AS, Nooman MU (2012) Purification and characterization of a novel fibrinolytic enzyme by Candida guilliermondii grown on sunflower oil cake. J Appl Sci Res 8:635–645
Romero CM, Baigori MD, Baron AM, Krieger N, Pera LM (2014) Activity and stability of lipase preparations from Penicillium corylophilum: potential use in biocatalysis. Chem Eng Technol 37:1987–1992
Sellek GA, Chaudhuri JB (1999) Biocatalysis in organic media using enzymes from extremophiles. Enzyme Microb Technol 25:471–482
Simkhada JR, Mander P, Cho SS, Yoo JC (2010) A novel fibrinolytic protease from Streptomyces sp. CS684. Process Biochem 45:88–93
Singh S, Bajaj BK (2015) Medium optimization for enhanced production of protease with industrially desirable attributes from Bacillus subtilis K-1. Chem Eng Commun 202:1051–1060
Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H (1987) A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia 43:1110–1111
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729
Tariq AL, Reyaz AL (2012) Characterization of UTI associated protease from Serratia marcescens strain TW1 and its immunogenic properties. Int Res J Biotechnol 3:88–95
Valls C, Rojas C, Pujadas G, Garcia-Vallve S, Mulero M (2012) Characterization of the activity and stability of amylase from saliva and detergent. Biochem Mol Bio Edu 40:254–265
Venkataraman D, Ilangovan S, Sampathkumar MV, Victoria MJ, Pasha SPBS, Pandian SBRK, Gurunathan S (2010) Medium optimization and immobilization of purified fibrinolytic URAK from Bacillus cereus NK1 on PHB nanoparticles. Enzy Microbial Technol 47:297–304
Vijayaraghavan P, Vincent SGP, Arasu MV, Al-Dhabi NA (2016) Bioconversion of agro-industrial wastes for the production of fibrinolytic enzyme from Bacillus halodurans IND18: purification and biochemical characterization. Electro J Biotechnol 20:1–8
Wang S, Chen H, Liang T, Lin Y (2009) A novel nattokinase produced by Pseudomonas sp. TKU015 using shrimp shells as substrate. Process Biochem 44:70–76
Wu B, Wu L, Chen D, Yang Z, Luo M (2009) Purification and characterization of a novel fibrinolytic protease from Fusarium sp. CPCC 480097. J Ind Microbiol Biotechnol 36:451–459
Wu D, Ran T, Wang W, Xu D (2016) Structure of a thermostable serralysin from Serratia sp. FS14 at 1.1A resolution. Acta Cryst 72:10–15
Acknowledgements
The author Kapila Taneja gratefully acknowledges Department of Science and Technology, Govt. of India for providing financial support in the form of DST-INSPIRE Fellowship. Authors also thank Microbial Culture Collection, NCCS, DBT, Govt. of India for phenotypic characterization of samples.
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Taneja, K., Bajaj, B.K., Kumar, S. et al. Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media. 3 Biotech 7, 184 (2017). https://doi.org/10.1007/s13205-017-0808-4
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DOI: https://doi.org/10.1007/s13205-017-0808-4