Abstract
Thrombosis is a hematological disorder characterized by the formation of intravascular thrombi, which contributes to the development of cardiovascular diseases. Fibrinolytic enzymes are proteases that promote the hydrolysis of fibrin, promoting the dissolution of thrombi, contributing to the maintenance of adequate blood flow. The characterization of new effective, safe and low-cost fibrinolytic agents is an important strategy for the prevention and treatment of thrombosis. However, the development of new fibrinolytics requires the use of complex methodologies for purification, physicochemical characterization and evaluation of the action potential and toxicity of these enzymes. In this context, microbial enzymes produced by bacteria of the Bacillus genus are promising and widely researched sources to produce new fibrinolytics, with high thrombolytic potential and reduced toxicity. Thus, this review aims to provide a current and comprehensive understanding of the different Bacillus species used for the production of fibrinolytic proteases, highlighting the purification techniques, biochemical characteristics, enzymatic activity and toxicological evaluations used.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ahn MJ, Ku HJ, Lee SH et al (2015) Characterization of a novel fibrinolytic enzyme, BsfA, from Bacillus subtilis ZA400 in kimchi reveals its pertinence to thrombosis treatment. J Microbiol Biotechnol 25:2090–2099. https://doi.org/10.4014/jmb.1509.09048
Ali AMM, Bavisetty SCB (2020) Purification, physicochemical properties, and statistical optimization of fibrinolytic enzymes especially from fermented foods: a comprehensive review. Int J Biol Macromol 163:1498–1517. https://doi.org/10.1016/j.ijbiomac.2020.07.303
Amartely H, Some D, Tsadok A et al (2019) Ion exchange chromatography (IEX) coupled to multi-angle light scattering (MALS) for protein separation and characterization. J vis Exp. https://doi.org/10.3791/59408
Asgari M, Javaran MJ, Moieni A et al (2014) Production of human tissue plasminogen activator (tPA) in Cucumis sativus. Prep Biochem Biotechnol 44:82–192. https://doi.org/10.1080/10826068.2013.803480
Astrup T, Müllertz S (1952) The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 40:346–351. https://doi.org/10.1016/0003-9861(52)90121-5
Bajaj BK, Sharma N, Singh S (2013) Enhanced production of fibrinolytic protease from Bacillus cereus NS-2 using cotton seed cake as nitrogen source. Biocatal Agric Biotechnol 2:204–209. https://doi.org/10.1016/j.bcab.2013.04.003
Bajaj BK, Singh S, Khullar M et al (2014) Optimization of fibrinolytic protease production from Bacillus subtilis I-2 using agro-residues. Braz Arch Biol Technol 57:653–662. https://doi.org/10.1590/S1516-8913201402132
Begum R, Sharma M, Pillai KK et al (2014) Inhibitory effect of Careya arborea on inflammatory biomarkers in carrageenan-induced inflammation. Pharm Biol 53:437–445. https://doi.org/10.3109/13880209.2014.923005
Benjamin EJ, Virani SS, Callaway CW et al (2018) Heart disease and stroke statistics-2018 update: a report from the American Heart Association. Circulation 137:467–492. https://doi.org/10.1161/cir.0000000000000558
Biçer A, Taslimi P, Yakali G et al (2019) Synthesis, characterization, crystal structure of novel bis-thiomethylcyclohexanone derivatives and their inhibitory properties against some metabolic enzymes. Bioorg Chem 82:393–404. https://doi.org/10.1016/j.bioorg.2018.11.001
Biji GD, Arun A, Muthulakshmi E et al (2016) Bio-prospecting of cuttle fish waste and cow dung for the production of fibrinolytic enzyme from Bacillus cereus IND5 in solid state fermentation. 3 Biotech. https://doi.org/10.1007/s13205-016-0553-0
Bonnard T, Law LS, Tennant Z et al (2017) Development and validation of a high throughput whole blood thrombolysis plate assay. Sci Rep 7:2346. https://doi.org/10.1038/s41598-017-02498-2
Butler M, Acosta AM (2012) Recent advances in technology supporting biopharmaceutical production from mammalian cells. Appl Microbiol Biotechnol 96:885–894. https://doi.org/10.1007/s00253-012-4451-z
Cate TH, Hackeng TM, Frutos GP (2017) Coagulation factor and protease pathways in thrombosis and cardiovascular disease. Thromb Haemost 117:1265–1271. https://doi.org/10.1160/th17-02-0079
Cheng Q, Xu F, Hu N et al (2015) A novel Ca2+-dependent alkaline serine-protease (Bvsp) from Bacillus sp. with high fibrinolytic activity. J Mol Catal B Enzym 117:69–74. https://doi.org/10.1016/2Fj.molcatb.2015.04.006
Choi JH, Kim JE, Kim S et al (2017) Purification and partial characterization of a low molecular fibrinolytic serine metalloprotease C142 from the culture supernatant of Bacillus subtilis C142. Int J Bio Macromol 104:724–731. https://doi.org/10.1016/j.ijbiomac.2017.06.025
Dabbagh F, Negahdaripour M, Berenjian A et al (2014) Nattokinase: production and application. Appl Microbiol Biotechnol 98:9199–9206. https://doi.org/10.1007/s00253-014-6135-3
Devaraj Y, Rajender SK, Halami PM (2018) Purification and characterization of fibrinolytic protease from Bacillus amyloliquefaciens MCC2606 and analysis of fibrin degradation product by MS/MS. Prep Biochem Biotechnol 48:172–180. https://doi.org/10.1080/10826068.2017.1421964
Drejer EB, Hakvag S, Irla M et al (2018) Genetic tools and techniques for recombinant expression in thermophilic bacillaceae. Microorganisms 6:42. https://doi.org/10.3390/microorganisms6020042
Farraj DAAL, Kumar TSJ, Vijayaraghavan P et al (2020) Enhanced production, purification and biochemical characterization of therapeutic potential fibrinolytic enzyme from a new Bacillus flexus from marine environment. J King Saud Uni Sci 32:3174–3180. https://doi.org/10.1016/j.jksus.2020.09.004
Fekete S, Beck A, Veuthey JL et al (2015) Ion-exchange chromatography for the characterization of biopharmaceuticals. J Pharm Biomed Anal 113:43–55. https://doi.org/10.1016/j.jpba.2015.02.037
Hao C, Sun M, Wang H et al (2019) Low molecular weight heparins and their clinical applications. Prog Mol Biol Transl Sci 163:21–39. https://doi.org/10.1016/bs.pmbts.2019.02.003
Heo K, Cho KM, Lee CK et al (2013) Characterization of a fibrinolytic enzyme secreted by Bacillus amyloliquefaciens CB1 and its gene cloning. J Microbiol Biotechnol 23:974–983. https://doi.org/10.4014/jmb.1302.02065
Hogas S, Bilha SC, Branisteanu D et al (2017) Potential novel biomarkers of cardiovascular dysfunction and disease: Cardiotrophin-1, adipokines and galectin-3. Arch Med Sci 13:897–913. https://doi.org/10.5114/aoms.2016.58664
Hu Y, Yu D, Wang Z et al (2019) Purification and characterization of a novel, highly potent fibrinolytic enzyme from Bacillus subtilis DC27 screened from Douchi, a traditional Chinese fermented soybean food. Sci Rep. https://www.nature.com/articles/s41598-019-45686-y
Huang S, Pan S, Chen G et al (2013) Biochemical characteristics of a fibrinolytic enzyme purified from amarine bacterium, Bacillus subtilis HQS-3. Int J Biol 62:124–130. https://doi.org/10.1016/j.ijbiomac.2013.08.048
Jacquemin M, Vanlinthout I, Horenbeeck IV et al (2017) I. The amplitude of coagulation curves from thrombin time tests allows dysfibrinogenemia caused by the common mutation FGG-Arg301 to be distinguished from hypofibrinogenemia. Int J Lab Hematol 39:301–307. https://doi.org/10.1111/ijlh.12625
Jansen KA, Zhmurov A, Vos BE et al (2020) Molecular packing structure of fibrin fibers resolved by X-ray scattering and molecular modeling. Soft Matter 16:8272–8283. https://doi.org/10.1039/d0sm00916d
Jeong SJ, Heo K, Park JY et al (2015) Characterization of AprE176, a fibrinolytic enzyme from Bacillus subtilis HK176. J Microbiol Biotechnol 25:89–97. https://doi.org/10.4014/jmb.1409.09087
Jo HD, Lee HA, Jeong SJ et al (2011) Purification and characterization of a major fibrinolytic enzyme from Bacillus amyloliquefaciens MJ5-41 isolated from meju. J Microbiol Biotechnol 21:1166–1173. https://doi.org/10.4014/jmb.1106.06008
Kalyanpur M (2002) Downstream processing in the biotechnology industry. Mol Biotechnol 22:87–98. https://doi.org/10.1385/mb:22:1:087
Kim GM, Lee AR, Lee KW et al (2009) Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from cheonggukjang. J Microbiol Biotechnol 19:997–1004. https://doi.org/10.4014/jmb.0811.600
Kim C, Ri K, Choe S (2020a) A novel fibrinolytic enzymes from the Korean traditional fermented food-Jeotgal: purification and characterization. J Food Biochem. https://doi.org/10.1111/jfbc.13255
Kim DH, Nguyen QT, Ko GS et al (2020b) Molecular and Enzymatic Features of Homoserine Dehydrogenase from Bacillus subtilis. J Microbiol Biotechnol 30:1905–1911. https://doi.org/10.4014/jmb.2004.04060
Klykov O, Zwaan CVD, Heck AJR et al (2020) Missing regions within the molecular architecture of human fibrin clots structurally resolved by XL-MS and integrative structural modeling. Proc Natl Acad Sci USA 117:976–1987. https://doi.org/10.1073/pnas.1911785117
Kotb E (2014a) Purification and partial characterization of a chymotrypsin-like serine fibrinolytic enzyme from Bacillus amyloliquefaciens FCF-11 using corn husk as a novel substrate. World J Microbiol Biotechnol 30:2071–2080. https://doi.org/10.1007/s11274-014-1632-1
Kotb E (2014b) The biotechnological potential of fibrinolytic enzymes in the dissolution of endogenous blood thrombi. Biotechnol Prog 30:656–672. https://doi.org/10.1002/btpr.1918
Lee AR, Kim GM, Kwon GH et al (2009) Cloning of aprE86-1 gene encoding a 27-kDa mature fibrinolytic enzyme from Bacillus amyloliquefaciens CH86-1. J Microbiol Biotechnol 20:370–374. https://doi.org/10.4014/jmb.0906.06029
Li A, Garcia DA, Lyman GH et al (2019) Direct oral anticoagulant (DOAC) versus low-molecular-weight heparin (LMWH) for treatment of cancer associated thrombosis (CAT): a systematic review and meta-analysis. Thromb Res 173:158–163. https://doi.org/10.1016/j.thromres.2018.02.144
Lima LA, Filho RFC, Santos JG et al (2014) Produção de protease colagenolítica por Bacillus stearothermophillus de solo amazônico. Acta Amaz 44:403–410. https://doi.org/10.1590/1809-4392201305074
Liu X, Kopparapu NK, Shi X et al (2015) Purification and biochemical characterization of a novel fibrinolytic enzyme from culture supernatant of Cordyceps militaris. J Agric Food Chem 63:2215–2224. https://doi.org/10.1021/jf505717e
Lu M, Gao Z, Xing S et al (2021) Purification, characterization, and chemical modification of Bacillus velezensis SN-14 fibrinolytic enzyme. Int J Biol Macromol 177:601–609. https://doi.org/10.1016/j.ijbiomac.2021.02.167
Ma N, Liu XW, Yang YJ et al (2015) Preventive effect of aspirin eugenol ester on thrombosis in κ-carrageenan-induced rat tail thrombosis model. PLoS ONE. https://doi.org/10.1371/journal.pone.0133125
Macrae FL, Duval C, Papareddy P et al (2018) A fibrin biofilm covers blood clots and protects from microbial invasion. J Clin Invest 128:3356–3368. https://doi.org/10.1172/jci98734
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. https://doi.org/10.1016/j.jbiosc.2011.10.023
Majumdar S, Dutta S, Das T et al (2015) Antiplatelet and antithrombotic activity of a fibrin(ogen)olytic protease from Bacillus cereus strain FF01. Int J Biol Macromol 79:477–489. https://doi.org/10.1016/j.ijbiomac.2015.04.075
Maqsood M, Mushtaq Z, Rasheed T et al (2021) Thrombolytic and cytotoxic activity of different bioactive extracts of E. coli. CSCEE. https://doi.org/10.1016/j.cscee.2021.100080
Meshram V, Saxena S, Paul K et al (2017) Production, purification and characterisation of a potential fibrinolytic protease from Endophytic Xylaria curta by solid substrate fermentation. Appl Biochem Biotechnol 181:1496–1512. https://doi.org/10.1007/s12010-016-2298-y
Moharam ME, He Q (2019) Optimization of fibrinolytic enzyme production by newly isolated Bacillus subtilis Egy using central composite design. Biocatal Agric Biotechnol 17:43–50. https://doi.org/10.1016/j.biortech.2007.09.029
Mukherjee AK, Rai SK, Thakur R et al (2012) Bafibrinase: a non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency. Biochimie 94:1300–1308. https://doi.org/10.1016/j.biochi.2012.02.027
Nagata C, Wada K, Tamura T et al (2017) Dietary soy and natto intake and cardiovascular disease mortality in Japanese adults: the Takayama study. Am J Clin Nutr 105:426–431. https://doi.org/10.3945/ajcn.116.137281
Narasimhan MK, Chandrasekaran M, Rajesh M (2015) Fibrinolytic enzyme production by newly isolated Bacillus cereus SRM-001 with enhanced in-vitro blood clot lysis potential. J Gen Appl Microbiol 61:157–164. https://doi.org/10.2323/jgam.61.157
Narasimhan MK, Ethiraj S, Krishnamurthi T et al (2018) Purification, biochemical and thermal properties of fibrinolytic enzyme secreted by Bacillus cereus SRM-001. Prep Biochem Biotechnol 48:34–42. https://doi.org/10.1080/10826068.2017.1387560
Paik HD, Lee SK, Heo S et al (2004) Purification and characterization of the fibrinolytic enzyme produced by Bacillus subtilis KCK-7 from Chungkookjang. J Microbiol BiotechnoL 14:829–835
Prasad S, Kashyap RS, Deopujari JY et al (2006) Development of an in vitro model to study clot lysis activity of thrombolytic drugs. Thromb J. https://doi.org/10.1186/1477-9560-4-14
Raafat AI, Araby E, Lotfy S (2012) Enhancement of fibrinolytic enzyme production from Bacillus subtilis via immobilization process onto radiation synthesized starch/dimethylaminoethyl methacrylate hydrogel. Carbohydr Polym 87:1369–1374. https://doi.org/10.1016/2Fj.carbpol.2011.09.029
Sales AE, Souza FASD, Teixeira JA et al (2013) Integrated process production and extraction of the fibrinolytic protease from Bacillus sp. UFPEDA 485. Appl Biochem Biotechnol 170:1676–1688. https://doi.org/10.1007/s12010-013-0306-z
Sharma C, Salem GEM, Sharma N et al (2019) Thrombolytic potential of novel thiol-dependent fibrinolytic protease from Bacillus cereus RSA1. Biomolecules. https://doi.org/10.3390/biom10010003
Silva PEC, Barros RC, Albuquerque WWC et al (2018) In vitro thrombolytic activity of a purified fibrinolytic enzyme from Chlorella vulgaris. J Chromatogr B Analyt Technol Biomed Life Sci 1092:524–529. https://doi.org/10.1016/j.jchromb.2018.04.040
Singh S, Bajaj BK (2017) Potential application spectrum of microbial proteases for clean and green industrial production. Energ Ecol Environ 2:370–386. https://doi.org/10.1007/S40974-017-0076-5
Toledo AL, Severo JB, Souza RR et al (2007) Purification by expanded bed adsorption and characterization of an alpha-amylases FORILASE NTL from A. niger. J Chromatogr B Analyt Technol Biomed Life Sci 846:51–56. https://doi.org/10.1016/j.jchromb.2006.08.011
Venkataraman D, Ilangovan S, Sampathkumar MV et al (2010) Medium optimization and immobilization of purifed fbrinolytic URAK from Bacillus cereus NK1 on PHB nanoparticles. Enzyme Microb Technol 47:297–304. https://doi.org/10.1016/j.enzmictec.2010.07.004
Vijayaraghavan P, Arasu MV, Rajan RA et al (2019) Enhanced production of fibrinolytic enzyme by a new Xanthomonas oryzae IND3 using low-cost culture medium by response surface methodology. Saudi J Biol Sci 26:217–224. https://doi.org/10.1016/j.sjbs.2018.08.029
Wang SL, Wu YY, Liang TW (2011) Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007. N Biotechnol 28:196–202. https://doi.org/10.1016/j.nbt.2010.09.003
Weisel JW, Litvinov RI (2017) Fibrin formation, structure and properties. Subcell Biochem 82:405–456. https://doi.org/10.1007/978-3-319-49674-0_13
Wilken LR, Nikolov ZL (2012) Recovery and purification of plant-made recombinant proteins. Biotechnol Adv 30:419–433. https://doi.org/10.1016/j.biotechadv.2011.07.020
World Health Organization (2020) World Health Statistics 2019: Monitoring Health for the SDGs: Sustainable Development Goals. World Health Organization, Geneva
Wu S, Lu S, Liu J et al (2021) Physicochemical properties and bioactivities of rice beans fermented by Bacillus amyloliquefaciens. Engineering 7:2019–2225. https://doi.org/10.1016/j.eng.2020.10.010
Xin X, Ambati RR, Cai Z et al (2018) Purification and characterization of fibrinolytic enzyme from a bacterium isolated from soil. 3 Biotech. https://doi.org/10.1007/s13205-018-1115-4
Yang H, Yang L, Li X et al (2019) Genome sequencing, purification, and biochemical characterization of a strongly fibrinolytic enzyme from Bacillus amyloliquefaciens Jxnuwx-1 isolated from Chinese traditional Douchi. J Gen Appl Microbiol 66:153–162. https://doi.org/10.2323/jgam.2019.04.005
Yang H, Liu Y, Ning Y et al (2020a) Characterization of an intracellular alkaline serine protease from Bacillus velezensis SW5 with Fibrinolytic activity. Curr Microbiol 77:1610–1621. https://doi.org/10.1007/s00284-020-01977-6
Yang H, Yang L, Li X et al (2020b) Genome sequencing, purification, and biochemical characterization of a strongly fibrinolytic enzyme from Bacillus amyloliquefaciens Jxnuwx-1 isolated from Chinese traditional Douchi. J Gen Appl Microbiol 66:153–162. https://doi.org/10.2323/jgam.2019.04.005
Yao Z, Liu X, Shim JM et al (2017) Properties of a fibrinolytic enzyme secreted by Bacillus amyloliquefaciens RSB34, isolated from Doenjang. J Microbiol Biotechnol 27:9–18. https://doi.org/10.4014/jmb.1608.08034
Yao Z, Kim JA, Kim JH (2018) Properties of a fibrinolytic enzyme secreted by Bacillus subtilis JS2 isolated from saeu (small shrimp) jeotgal. Food Sci Biotechnol 27:765–772. https://doi.org/10.1007/s10068-017-0299-4
Yao Z, Kim JA, Kim JH (2019) Characterization of a fibrinolytic enzyme secreted by Bacillus velezensis BS2 isolated from sea squirt Jeotgal. J Microbiol Biotechnol 29:347–356. https://doi.org/10.4014/jmb.1810.10053
Yogesh D, Halami PM (2015) A fibrin degrading serine metallo protease of Bacillus circulans with α-chain specificity. Food Biosci 11:72–78. https://doi.org/10.1016/2Fj.fbio.2015.04.007
Zhou SS, Jin JP, Wang JQ et al (2018) miRNAS in cardiovascular diseases: potential biomarkers, therapeutic targets and challenges. Acta Pharmacol Sin 39:1073–1084. https://doi.org/10.1038/aps.2018.30
Zhou Y, Chen H, Yu B et al (2022) Purification and characterization of a fibrinolytic enzyme from marine Bacillus velezensis Z01 and assessment of its therapeutic efficacy in vivo. Microorganisms 10:843. https://doi.org/10.3390/microorganisms10050843
Zhu L, Li M, Liu Y (2019) Intravenous administration of low-molecular-weight heparin. Am J Ther 26:426–428. https://doi.org/10.1097/mjt.0000000000000841
Acknowledgements
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES, PRINT, Finance Code 460 88887.568973/2020-00) and Fundação de Ciência e Tecnologia do Estado de Pernambuco, Brazil (FACEPE, Finance Code BFP-0213-5.05/20).
Funding
No funding was received to assist with the preparation of this manuscript.
Author information
Authors and Affiliations
Contributions
All authors contributed to the writing and development of the study. The idea of writing this review, selecting and analyzing the data obtained were carried out by AHPL, IHAS and TPN. AMST and ALFP reviewed the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Conflict of interest
The authors declared that they have no conflict of interest.
Additional information
Communicated by Erko Stackebrandt.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Leite, A.H.P., da Silva, Í.H.A., Pastrana, L. et al. Purification, biochemical characterization and fibrinolytic potential of proteases produced by bacteria of the genus Bacillus: a systematic literature review. Arch Microbiol 204, 503 (2022). https://doi.org/10.1007/s00203-022-03134-y
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00203-022-03134-y