PRODUCTION OPTIMIZATION, PARTIAL PURIFICATION, AND THROMBOLYTIC ACTIVITY EVALUATION OF PROTEASE OF Bacillus cereus HSFI-10
Downloads
Cardiovascular disease is the primary cause of mortality in the world due to the formation of blood clots or thrombi in blood vessels. Bacterial proteases commonly function as thrombus dissolver agents in the pharmaceutical industry. Bacterial isolate HSFI-10 (Holothuria scabra Fermented Intestine-10) previously isolated from Rusip fermented sea cucumber had demonstrated thrombolytic activity. This study aimed to produce crude protease of HSFI-10 strain at an optimized incubation time and determine the thrombolytic activity of crude and dialysate proteases on A, B, AB, and O blood types. Isolate HSFI-10 was first molecularly identified and found to be Bacillus cereus with a homology level of 99.80% with Bacillus cereus strain ST06. The optimum crude enzyme was obtained after 48-h incubation with an activity of 222.52 U/mL, which increased to 438.84 U/mL after ammonium sulfate precipitation and dialysis. Clot lysis activity of crude enzymes was measured based on the gravimetry method on blood in the ABO system, showing results that ranged from 68.99% to 69.76%, while the dialysate ranged from 81.16% to 82.52%. In conclusion, partial purification of bacterial protease could increase both its specific and thrombolytic activities on human blood in the ABO system, with only 1% activity variability between A, B, AB, and O blood types.
Downloads
Abbas, N., Siddique, H.N., Masood, F., Shehzadi, A., Abbas, Z. and Ali, S., 2020. Production of protease enzyme from Bacillus subtilis using skimmed milk. Science International (Lahore), 32(2), pp.211-214. https://inis.iaea.org/search/search.aspx?orig_q=RN:51088719
Ahmadpour F, Yakhchali B. Development of an Asporogenic Bacillus cereus Strain to Improve keratinase production in exponential phase by switching sigmaH on and sigmaF off, 2017. FEMS Microbiology Letters, Volume 364, Issue 24, fnx216, https://doi.org/10.1093/femsle/fnx216
Artha, O.A., Pramono, H. and Sari, L.A., 2019, February. Identification of extracellular enzyme-producing bacteria (proteolytic, cellulolytic, and amylolytic) in the sediment of extensive ponds in Tanggulrejo, Gresik. In IOP Conference Series: Earth and Environmental Science (Vol. 236, No. 1, p. 012003). IOP Publishing. https://iopscience.iop.org/article/10.1088/1755-1315/236/1/012003
Assaf DD, Kenan E, Sionov R, Steinberg D, Shemesh M. Proteolytic Activity of Bacillus subtilis upon κ-Casein Undermines Its “Caries-Safe” Effect. Microorganisms. 2020; 8(2):221. https://doi.org/10.3390/microorganisms8020221
Battinelli, E.M., Leopold, J.A. and Loscalzo, J., 2019. Pathobiology of Thrombosis. Vascular Medicine: A Companion to Braunwald's Heart Disease E-Book, p.120.
Bergmeyer, H.U., GraB, M. and Walter, H.E., 1983. Alcohol oxidase. In Methods of enzymatic analysis, vol 2 (pp. 143-144). Weinheim: Verlag Chemie. https://www.sciencedirect.com/book/9780120913022/methods-of-enzymatic-analysis
Bottone EJ. Bacillus cereus a volatile human pathogen. Clinical microbiology reviews. 2010 Apr; 23(2): 382-98. https://doi.org/10.1128/cmr.00073-09
Chen H, Chen J, Zhang F, Li Y, Wang R, Zheng Q, Zhang X, Zeng J, Xu F, Lin Y. Effective management of atherosclerosis progress and hyperlipidemia with nattokinase: A clinical study with 1,062 participants. Front Cardiovasc Med. 2022 Aug 22;9:964977. Erratum in: Front Cardiovasc Med. 2022 Dec 05;9:1076420. PMID: 36072877; PMCID: PMC9441630. https://doi.org/10.3389/fcvm.2022.964977
Darmawati, S., 2015. Identification of Staphylococcus sp. strains isolated from positive widal blood based on 16s rRNA gene sequences. International Journal of Science and Engineering, 9(2), pp.97-100. https://doi.org/10.12777/ijse.9.2.97-100
Dewi OY, Ethica SN, Sukeksi A, Rakhmawatie MD, Darmawati S. Prospective In Vivo Assays on the Antithrombotic Potential of Protease Extracted from Bacillus sp. HSFI-12. In 7th International Conference on Biological Science (ICBS 2021) 2022 May 2 (pp. 394-403). Atlantis Press. https://doi.org/10.2991/absr.k.220406.056
Durachim A dan Astuti D. Hemostasis. Kemenkes RI, Jakarta. 2018. http://bppsdmk.kemkes.go.id/ pusdiksdmk/wp-content/uploads/2018/09/ Hemostasis_SC.pdf
Ethica, S.N., Oedjijono, O., Semiarti, E., Widada, J. and Raharjo, T.J., 2018. Genotypic and phenotypic characterization of Alcaligenes javaensis JG3 potential as an effective biodegrader. BIOTROPIA-The Southeast Asian Journal of Tropical Biology, 25(1), pp.1-10. https://doi.org/10.11598/btb.2018.25.1.583
Farooq, S., Nazir, R., Ganai, S.A. and Ganai, B.A., 2021. Isolation and characterization of a new cold-active protease from psychrotrophic bacteria of Western Himalayan glacial soil. Scientific reports, 11(1), pp.1-14. https://doi.org/10.1007/s11756-021-00915-6
Fuad, H., Hidayati, N., Darmawati, S., Munandar, H., Sulistyaningtyas, A.R., Nurrahman, N., Ernanto, A.R., Zilda, D.S., Widjanarka, W. and Ethica, S.N., 2020. Prospects of fibrinolytic proteases of bacteria from sea cucumber fermentation products as antithrombotic agent. In BIO Web of Conferences (Vol. 28, p. 02006). EDP Sciences. https://doi.org/10.1051/bioconf/20202802006
Gallelli G, Di Mizio G, Palleria C, Siniscalchi A, Rubino P, Muraca L, Cione E, Salerno M, De Sarro G, Gallelli L. Data Recorded in Real Life Support the Safety of Nattokinase in Patients with Vascular Diseases. Nutrients. 2021 Jun 13;13(6):2031. PMID: 34199189; PMCID: PMC8231931. https://doi.org/10.3390/nu13062031
Geneser, F., 1994. Buku teks histologi. Jilid I. Binapura Aksara. Jakarta. https://lib.ui.ac.id/ detail.jsp?id=140493
Green, M.R. and Sambrook, J., 2021. Cloning polymerase chain reaction (PCR) products: Blunt-end cloning. Cold Spring Harbor Protocols, 2021(4), pp.pdb-prot101287. https://doi.org/10.1101/ pdb.prot101287
Gupta N. DNA extraction and polymerase chain reaction. Journal of cytology. 2019 Apr 1; 36(2).
Hall, T., 2004. BioEdit version 7.0. 0. Distributed by the author, website: www.mbio.ncsu.edu/BioEdit/ bioedit.html
Hidayati, N., Fuad, H., Munandar, H., Zilda, D.S., Nurrahman, N., Fattah, M., Oedjijono, O., Samiasih, A. and Ethica, S.N., 2021, April. Proteolytic and Clot Lysis Activity Screening of Crude Proteases Extracted from Tissues and Bacterial Isolates of Holothuria scabra. In IOP Conference Series: Earth and Environmental Science (Vol. 755, No. 1, p. 012016). IOP Publishing. http://dx.doi.org/10.1088/1755-1315/755/1/012016
Holle LA, Monroe DM, Wolberg AS. VWF (von Willebrand Factor) is not required for red blood cell retention in clots in mice. Arteriosclerosis, Thrombosis, and Vascular Biology. 2020 Aug;40(8):1952-4. https://doi.org/10.1161/ ATVBAHA. 120.314575
Krishnamurthy, A., Belur, P.D. and Subramanya, S.B., 2018. Methods available to assess therapeutic potential of fibrinolytic enzymes of microbial origin: a review. Journal of Analytical Science and Technology, 9(1), pp.1-11. https://doi.org/ 10.5958/0974-360X.2020.00730.1
Lu Z, Guo W, Liu C. Isolation, identification and characterization of novel Bacillus subtilis. J Vet Med Sci. 2018 Mar 24;80(3):427-433. Epub 2018 Jan 23. PMID: 29367516; PMCID: PMC5880821. https://doi:10.1292/jvms.16-0572
Martina, S.J., Ramar, L.A., Silaban, M.R., Luthfi, M. and Govindan, P.A., 2019. Anti-platelet effectivity between aspirin with honey on cardiovascular disease based on bleeding time taken on mice. Open Access Macedonian Journal of Medical Sciences, 7(20), p.3416. https://doi.org/10.3889/ oamjms.2019.431
Mehic, D., Hofer, S., Jungbauer, C., Kaider, A., Haslacher, H., Eigenbauer, E., Rejtő, J., Schwartz, D., Jilma, B., Ay, C. and Pabinger, I., 2020. Association of ABO blood group with bleeding severity in patients with bleeding of unknown cause. Blood advances, 4(20), pp.5157-5164. https://dx.doi.org/ 10.1182%2Fbloodadvances.2020002452
Mogrovejo, D.C., Perini, L., Gostinčar, C., Sepčić, K., Turk, M., Ambrožič-Avguštin, J., Brill, F.H. and Gunde-Cimerman, N., 2020. Prevalence of antimicrobial resistance and hemolytic phenotypes in culturable arctic bacteria. Frontiers in microbiology, 11, p.570. https://doi.org/10.3389/ fmicb.2020.00570
Mohamed ABO, Al-Ama N, Al Kreathy H, et al. Oral Contraceptive Types in Relation to ABO Blood Groups Among Saudi Women of Different Reproductive Age Groups and Impact on Venous Thromboembolism. Clinical and Applied Thrombosis/Hemostasis. January 2020. https://doi:10.1177/1076029620966051
Mukherjee, S., 2019. Isolation and purification of industrial enzymes: advances in enzyme technology. In Advances in Enzyme Technology (pp. 41-70). Elsevier. https://doi.org/10.1016/ b978-0-444-64114-4.00002-9
Natsir, H., 2015. Production of protease enzyme from bacteria in hot spring of South Sulawesi, Bacillus licheniformis Hsa3-1a. Marina Chimica Acta, 16(1). https://doi.org/10.20956/mca.v16i1.949
Pagarra H, Hartati H, Ali A, Salempa P, Muharram M. Isolation and Optimization of Endophytic Bacteria from Roots of Karst Area Ecosystems Producing Protease Enzymes. The Journal of Research on the Lepidoptera. 2020;51(2):431-9. https://doi.org/ 10.36872/LEPI/V51I2/301110
Pagarra H, Hartati H, Ali A, Salempa P, Muharram M. Isolation and Optimization of Endophytic Bacteria from Roots of Karst Area Ecosystems Producing Protease Enzymes. The Journal of Research on the Lepidoptera. 2020;51(2):431-9. https://doi.org/ 10.36872/LEPI/V51I2/301110
Pendu JL, Breiman A, Rocher J, Dion M, Ruvoën-Clouet N. ABO blood types and COVID-19: spurious, anecdotal, or truly important relationships? A reasoned review of available data. Viruses 2021, Feb;13(2):160. https://doi.org/10.3390/ v13020160
Prasad, S., R.S. Kashyap, J.Y. Deopujari, H.J. Purohit, G.M. Taori, and H.F. Daginawala. 2006. ‗Development of an in vitro model to study clot lysis activity of thrombolytic drugs', BioMed Central, 4, pp. 9–12. https://doi.org/10.1186/ 1477-9560-4-14
Razzaq, A., Shamsi, S., Ali, A., Ali, Q., Sajjad, M., Malik, A. and Ashraf, M., 2019. Microbial proteases applications. Frontiers in bioengineering and biotechnology, 7, p.110. https://doi.org/10.3389/fbioe.2019.00110
Sambrook, J., 1989. Molecular cloning: a laboratory manual. Synthetic oligonucleotides. https://www.cabdirect.org/cabdirect/abstract/19901616061
Saxena, V., Gacchina Johnson, C., Negussie, A.H., Sharma, K.V., Dreher, M.R. and Wood, B.J., 2015. Temperature-sensitive liposome-mediated delivery of thrombolytic agents. International Journal of Hyperthermia, 31(1), pp.67-73. https://doi.org/10.5853/jos.2020.03349
Scheres, L.J., Lijfering, W.M. and Cannegieter, S.C., 2018. Current and future burden of venous thrombosis: not simply predictable. Research and practice in thrombosis and haemostasis, 2(2), pp.199-208. https://dx.doi.org/10.1002%2Frth2.12101
Separham, A., Dinparvar, S., Savadi-Oskouei, S., Pourafkari, L., Baghbani-Oskouei, A. and Nader, N.D., 2020. Association of ABO blood types with ST resolution following thrombolysis in acute ST elevation myocardial infarction. Journal of Cardiovascular and Thoracic Research, 12(2), p.106. https://dx.doi.org/10.34172%2Fjcvtr.2020.18
Shahi, S., Vahed, S.Z., Fathi, N. and Sharifi, S., 2018. Polymerase chain reaction (PCR)-based methods: promising molecular tools in dentistry. International journal of biological macromolecules, 117, pp.983-992. https://doi.org/10.1016/ j.ijbiomac.2018.05.085
Sharma, C., Salem, G.E.M., Sharma, N., Gautam, P. and Singh, R., 2020. Thrombolytic potential of novel thiol-dependent fibrinolytic protease from Bacillus cereus RSA1. Biomolecules, 10(1), p.3. https://doi.org/10.3390/biom10010003
Si, J.B., Jang, E.J., Charalampopoulos, D. and Wee, Y.J., 2018. Purification and characterization of microbial protease produced extracellularly from Bacillus subtilis FBL-1. Biotechnology and Bioprocess Engineering, 23(2), pp.176-182. https://doi.org/10.1007/s12257-017-0495-3
Suleiman AD, Abdul Rahman N, Mohd Yusof H, Mohd Shariff F, Yasid NA. Effect of Cultural Conditions on Protease Production by a Thermophilic Geobacillus thermoglucosidasius SKF4 Isolated from Sungai Klah Hot Spring Park, Malaysia. Molecules. 2020 Jun 4;25(11):2609. PMID: 32512695; PMCID: PMC7321352. https://doi.org/10.3390/ molecules25112609
Westphal, A.H. and van Berkel, W.J., 2021. Techniques for Enzyme Purification. Biocatalysis for Practitioners: Techniques, Reactions and Applications, pp.1-31. https://application.wiley-vch.de/books/sample/352734683X_c01.pdf
World Health Organization (WHO). Cardiovascular diseases (CVDs): 2017. Accessed from https://www.who.int/en/news-room/factsheets/ detail/cardiovascular diseases(cvds)
Copyright (c) 2023 Ainutajriani Ainutajriani, Sri Darmawati, Muhammad Ardi Afriansyah, Ragil Saptaningtyas, Stalis Norma Ethica, Dewi Seswita Zilda
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Authors who publish with this journal agree with the following terms:
- Authors retain copyright and grant the journal right of first publication, with the work 1 year after publication simultaneously licensed under a Creative Commons attribution-noncommerical-noderivates 4.0 International License that allows others to share, copy and redistribute the work in any medium or format, but only where the use is for non-commercial purposes and an acknowledgement of the work's authorship and initial publication in this journal is mentioned.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
