Login Register Cart 0
Generic placeholder image

Current Pharmaceutical Biotechnology

Editor-in-Chief

ISSN (Print): 1389-2010
ISSN (Online): 1873-4316

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Letter Article

Characterizations and Fibrinolytic Activity of Serine Protease from Bacillus subtilis C10

Author(s): Nguyen T.A. Thu, Nguyen T.M. Khue, Nguyen D. Huy, Nguyen Q.D. Tien and Nguyen H. Loc*

Volume 21, Issue 2, 2020

Page: [110 - 116] Pages: 7

DOI: 10.2174/1389201020666191002145415

Price: $65

Abstract

Background: Fibrinolytic enzymes, such as Nattokinases from Bacillus species are known to degrade the fibrin blood clots. They belong to serine protease group having commercial applications, such as therapeutic agents and functional food formulation.

Objective: The present study reports some characteristics and fibrinolytic activity of serine protease from B. subtilis C10 strain that was isolated from shrimp shell.

Methods: Extracellular enzyme from B. subtilis C10 culture was harvested and partially purified by ammonium sulphate precipitation. Fibrinolytic activity of the enzyme was determined by zymography and measured by spectrophotometry with fibrinogen and thrombin used as substrates. The optimal temperature and pH for fibrinolytic activity were studied in the range of 31-43ºC and 5-10, respectively. The thermal and pH stability of enzyme was studied by incubating enzyme for 30 min in the same range of temperature and pH as above. The effect of some metal ions and reagents on fibrinolytic activity of enzyme was evaluated by concentrations of 5 mM and 5%, respectively.

Results: Zymogram analysis indicated the presence of four fibrinolytic enzymes with molecular weights of approximately 69, 67, 39 and 36 kDa. The optimal temperature and pH for enzyme activity were 37°C and 9, respectively. The thermal and pH stability ranged from 35-39°C and 8-10, respectively. Fibrinolytic activity reached a maximum value of about 400 U/mg protein after 16 h of C10 strain culture. Enzyme has been drastically inhibited by PMSF and SDS, and partially inhibited by EDTA, while Triton X-100 has significantly increased enzyme activity. Effects of ions such as Mg2+, Ca2+ and Mn2+ on enzyme were negligible, except Cu2+ and Zn2+ have strongly decreased its activity.

Conclusion: Results from the present study suggested that enzyme obtained from B. subtilis C10 could be serine protease that has a high fibrinolytic activity up to about 400 U/mg protein at the most appropriate temperature and pH of 37ºC and 9. This activity can be improved up to 142% by incubating enzyme with 5% Triton X-100 for 30 min.

Keywords: Bacillus subtilis, fibrinolytic activity, nattokinase, serine protease, enzyme production, zymogram.

Next »
Graphical Abstract

[1]
Madala, P.K.; Tyndall, J.D.; Nall, T.; Fairlie, D.P. Update 1 of: Proteases universally recognize beta strands in their active sites. Chem. Rev., 2010, 110(6), PR1-PR31.
[http://dx.doi.org/10.1021/cr900368a] [PMID: 20377171]
[2]
Raju, E.V.N.; Divakar, G. An overview on microbial fibrinolytic proteases. Int. J. Pharm. Sci. Res., 2014, 5, 643-656.
[3]
Fujita, M.; Nomura, K.; Hong, K.; Ito, Y.; Asada, A.; Nishimuro, S. Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. Biochem. Biophys. Res. Commun., 1993, 197(3), 1340-1347.
[http://dx.doi.org/10.1006/bbrc.1993.2624] [PMID: 8280151]
[4]
Nagai, T.; Yamada, K.; Yoshimura, M.; Ishikawa, K.; Miyamoto, Y.; Hashimoto, K.; Noda, Y.; Nitta, A.; Nabeshima, T. The tissue plasminogen activator-plasmin system participates in the rewarding effect of morphine by regulating dopamine release. Proc. Natl. Acad. Sci. USA, 2004, 101(10), 3650-3655.
[http://dx.doi.org/10.1073/pnas.0306587101] [PMID: 14988509]
[5]
Di Cera, E. Serine proteases. IUBMB Life, 2009, 61(5), 510-515.
[http://dx.doi.org/10.1002/iub.186] [PMID: 19180666]
[6]
Rajagopalan, S.; Gonias, S.L.; Pizzo, S.V. A nonantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function. J. Clin. Invest., 1985, 75(2), 413-419.
[http://dx.doi.org/10.1172/JCI111715] [PMID: 3156148]
[7]
Ko, J.H.; Yan, J.P.; Zhu, L.; Qi, Y.P. Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02. Comp. Biochem. Physiol. C Toxicol. Pharmacol., 2004, 137(1), 65-74.
[http://dx.doi.org/10.1016/j.cca.2003.11.008] [PMID: 14984705]
[8]
Wang, C.; Ji, B.; Li, B.; Ji, H. Enzymatic properties and identification of a fibrinolytic serine protease purified from Bacillus subtilis DC33. World J. Microbiol. Biotechnol., 2006, 22, 1365-1371.
[http://dx.doi.org/10.1007/s11274-006-9184-7]
[9]
Kim, S.B.; Lee, D.W.; Cheigh, C.I.; Choe, E.A.; Lee, S.J.; Hong, Y.H.; Choi, H.J.; Pyun, Y.R. Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, Tempeh. J. Ind. Microbiol. Biotechnol., 2006, 33(6), 436-444.
[http://dx.doi.org/10.1007/s10295-006-0085-4] [PMID: 16470353]
[10]
Agrebi, R.; Hmidet, N.; Hajji, M.; Ktari, N.; Haddar, A.; Fakhfakh-Zouari, N.; Nasri, M. Fibrinolytic serine protease isolation from Bacillus amyloliquefaciens An6 grown on Mirabilis jalapa tuber powders. Appl. Biochem. Biotechnol., 2010, 162(1), 75-88.
[http://dx.doi.org/10.1007/s12010-009-8800-z] [PMID: 19842068]
[11]
Alekseev, A.A.; Koutsenogiy, P.K.; Miroshnikov, P.N.; Shilova, M.A. Isolation and properties of fibrinolytic subtilisin-like serine protease secreted by the Bacillus subtilis strain B-2805. Dokl. Biochem. Biophys., 2014, 455(1), 72-75.
[http://dx.doi.org/10.1134/S1607672914020082] [PMID: 24795104]
[12]
Yogesh, D.; Halami, P.M. Evidence that multiple proteases of Bacillus subtilis can degrade fibrin and fibrinogen. Int. Food Res. J., 2015, 22(4), 1662-1667.
[13]
Jeong, S.J.; Heo, K.; Park, J.Y.; Lee, K.W.; Park, J.Y.; Joo, S.H.; Kim, J.H. Characterization of AprE176, a fibrinolytic enzyme from Bacillus subtilis HK176. J. Microbiol. Biotechnol., 2015, 25(1), 89-97.
[http://dx.doi.org/10.4014/jmb.1409.09087] [PMID: 25315053]
[14]
Loc, N.H.; Song, N.V.; Quang, H.T.; Dung, L.D.; Loi, D.D.; Phuong, T.T.B.; Thuy, D.T.B. Cloning and expression of neutral protease gene from Bacillus subtilis. Vietnam J. Biotechnol., 2008, 6(4B), 963-970.
[15]
Roth, V. 2006.Doubling Time Computing, http://www.doubling-time.com/compute.php
[16]
Anson, M.L. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J. Gen. Physiol., 1938, 22(1), 79-89.
[http://dx.doi.org/10.1085/jgp.22.1.79] [PMID: 19873094]
[17]
Deepak, V.; Kalishwaralal, K.; Ramkumarpandian, S.; Babu, S.V.; Senthilkumar, S.R.; Sangiliyandi, G. Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology. Bioresour. Technol., 2008, 99(17), 8170-8174.
[http://dx.doi.org/10.1016/j.biortech.2008.03.018] [PMID: 18430568]
[18]
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72(1-2), 248-254.
[http://dx.doi.org/10.1016/0003-2697(76)90527-3] [PMID: 942051]
[19]
Choi, N.S.; Yoo, K.H.; Hahm, J.H.; Yoon, K.S.; Chang, K.T.; Hyun, B.H.; Maeng, P.J.; Kim, S.H. Purification and characterization of a new peptidase, bacillopeptidase DJ-2, having fibrinolytic activity: Produced by Bacillus sp. DJ-2 from Doen-Jang. J. Microbiol. Biotechnol., 2005, 15(1), 72-79.
[20]
Posada-Uribe, L.F.; Romero-Tabarez, M.; Villegas-Escobar, V. Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production. Bioprocess Biosyst. Eng., 2015, 38(10), 1879-1888.
[http://dx.doi.org/10.1007/s00449-015-1428-1] [PMID: 26135004]
[21]
Sahoo, K.K.; Arakha, M.; Sarkar, P.; Robin Davis, P.; Jha, S. Enhancement of properties of recycled coarse aggregate concrete using bacteria. Int. J. Smart Nano Mater., 2016.
[http://dx.doi.org/10.1080/19475411.2016.1152322]
[22]
Vijayakumar, G.; Tamilarasan, R.; Kumar, M.D. Removal of Cd2+ ions from aqueous solution using live and dead Bacillus subtilis. Chem. Engin. Res. Bullet., 2011, 15, 18-24.
[23]
Hakim, A.; Bhuiyan, F.R.; Iqbal, A.; Emon, T.H.; Ahmed, J.; Azad, A.K. Production and partial characterization of dehairing alkaline protease from Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 by using organic municipal solid wastes. Heliyon, 2018, 4(6)e00646
[http://dx.doi.org/10.1016/j.heliyon.2018.e00646] [PMID: 30009270]
[24]
Chantawannakula, P.; Oncharoena, A.; Klanbuta, K.; Chukeatiroteb, E.; Lumyonga, S. Characterization of proteases of Bacillus subtilis strain 38 isolated from traditionally fermented soybean in Northern Thailand. Sci. Asia, 2002, 28, 241-245.
[http://dx.doi.org/10.2306/scienceasia1513-1874.2002.28.241]
[25]
Pant, G.; Prakash, A.; Pavani, J.V.P.; Bera, S.; Deviram, G.V.N.S.; Kumar, A.; Panchpuri, M.; Prasuna, R.G. Production, optimization and partial purification of protease from Bacillus subtilis. J. Taibah Univ. Sci., 2015, 9, 50-55.
[http://dx.doi.org/10.1016/j.jtusci.2014.04.010]
[26]
Bajaj, B.K.; Singh, S.; Khullar, M.; Singh, K.; Bhardwaj, S. Optimization of fibrinolytic protease production from Bacillus subtilis I-2 using agro-residues. Braz. Arch. Biol. Technol., 2014, 57, 653-662.
[http://dx.doi.org/10.1590/S1516-8913201402132]
[27]
Ren, Y.; Pan, X.; Lyu, Q.; Liu, W. Biochemical characterization of a fibrinolytic enzyme composed of multiple fragments. Acta Biochim. Biophys. Sin. (Shanghai), 2018, 50(2), 227-229.
[http://dx.doi.org/10.1093/abbs/gmx125] [PMID: 29309704]
[28]
Bhatt, P.C.; Kapoor, R.; Panda, B.P. Purification and biochemical characterization of menaquione-free, pure nattokinase from Bacillus subtilis MTCC 2616. Indian J. Biochem. Biophys., 2015, 52, 248-253.
[29]
Kumar, D.J.M.; Rakshitha, R.; Vidhya, M.A.; Jennifer, P.S.; Prasad, S.; Kumar, M.R.; Kalaichelvan, P.T. Production, optimization and characterization of fibrinolytic enzyme by Bacillus subtilis RJAS19. Pak. J. Biol. Sci., 2014, 17(4), 529-534.
[http://dx.doi.org/10.3923/pjbs.2014.529.534] [PMID: 25911841]
[30]
Lin, H.T.V.; Wu, G.J.; Hsieh, M.C.; Chang, S.H.; Tsai, G.J. Purification and characterization of nattokinase from cultural filtrate of red alga Porphyra dentata fermented by Bacillus subtilis N1. J. Mar. Sci. Technol., 2015, 23, 240-248.
[31]
Matthews, D.J. Interfacial metal-binding site design. Curr. Opin. Biotechnol., 1995, 6(4), 419-424.
[http://dx.doi.org/10.1016/0958-1669(95)80071-9] [PMID: 7579652]
[32]
Nowak, P.; Zgirski, A. Effects of metal ions on activity of plasmin. Biol. Trace Elem. Res., 2003, 93(1-3), 87-94.
[http://dx.doi.org/10.1385/BTER:93:1-3:87] [PMID: 12835493]
[33]
Kim, G.M.; Lee, A.R.; Lee, K.W.; Park, J.Y.; Chun, J.; Cha, J.; Song, Y.S.; Kim, J.H. Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from cheonggukjang. J. Microbiol. Biotechnol., 2009, 19(9), 997-1004.
[http://dx.doi.org/10.4014/jmb.0811.600] [PMID: 19809258]
[34]
Amorim, F.G.; Menaldo, D.L.; Carone, S.E.I.; Silva, T.A.; Sartim, M.A.; De Pauw, E.; Quinton, L.; Sampaio, S.V. New insights on moojase, a thrombin-like serine protease from Bothrops moojeni snake venom. Toxins (Basel), 2018, 10(12), 500.
[http://dx.doi.org/10.3390/toxins10120500] [PMID: 30487389]

Rights & Permissions Print Cite
Article Metrics
25
2
© 2024 Bentham Science Publishers | Privacy Policy