| Peer-Reviewed

New Phylogenetic Molecular Markers in Bacteria of the Genus Bacillus: Fibrinolytic Proteases

Received: 7 February 2021    Accepted: 16 March 2021    Published: 18 August 2021
Views:       Downloads:
Abstract

The fibrinolytic proteases developed by bacteria have become substances of medical interest, as they have been recognized as antithrombotic substances in the blood. It is in this context that a study has been carried out with the aim of studying the fibrinolytic proteases produced by bacteria isolated from food in the Congo. Four strains of bacteria of the genus Bacillus isolated from crushed and cooked squash seeds, namely Bacillus sp strain ASM7, Bacillus sp strain CRK, Bacillus pumilus strain ASM5, Bacillus subtilis strain ASM1, identified by their rDNA16S, were tested positive for the production of fibrinolytic proteases. The fibrin box technique was used. The diameters on the fibrin boxes prove a significant production of fibrinolytic proteases. The genes coding for these fibrinolytic proteases were amplified by PCR and 1% Agarose gel electrophoresis shows that the size of the amplicons for the four strains is between1200-1450bp. The sequences of these coding genes have been for the four strains studied submitted to GenBank and the assigned accession numbers are respectively: Bacillus sp strain ASM7 MT743004, Bacillus sp strain CRK MT743005, Bacillus pumilus strain ASM5 MT743006, Bacillus subtilis strain ASM1 MT743007. These genes show a high degree of similarity of almost 99.50% with their counterparts in the databases, are all coding and show some observable differences. The translation of these genes in coherent reading frames confirms the amino acids already known in the active sites relating to their fibrinolysis role. The fibrinolytic protease CFE1 (Id=QNJ60181) is for Bacillus sp strain ASM7, the fibrinolytic protease CFE2 (Id=QNJ60182) is for Bacillus sp strain CRK, the fibrinolytic protease CFE3 (Id=QNJ60183) is for Bacillus pumilus strain ASM5, the fibrinolytic protease CFE4 (Id=QNJ60184) is for Bacillus subtilis strain ASM1. All these fibrinolytic proteases show a strong similarity (99.51-99.76%) with the Bacillus AprX serine protease, reference sequence. The phylogenetic inference test based on these fibrinolytic proteases shows that these proteases form a highly conserved characteristic group in bacteria of the genus Bacillus. This allowed us to retain these fibrinolytic proteases as an important phylogenetic molecular marker.

Published in International Journal of Microbiology and Biotechnology (Volume 6, Issue 3)
DOI 10.11648/j.ijmb.20210603.14
Page(s) 86-94
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

Fibrinolytic Proteases, Sequencing, Phylogenetic Molecular Markers, Bacillus

References
[1] Gupta, R., Beg, Q., Lorenz, P. (2002). Bacterial alkaline proteases: molecular approaches and industrial applications. Applied Microbiology and Biotechnology, 59: 15-32.
[2] Chang, C, T., Wang, P, M., Hung, Y, F., Chung, Y, C. (2012): Purification and biochemical properties of a fibrinolytic enzyme from Bacillus subtilis-fermented red bean. Food Chemistry, Volume 133, Issue 4, 15 August 2012, Pages 1611-1617.
[3] Jeong, Y, K. Park, J, U., Baek, H., Park, S, H., Kong, I, S., Kim, D, W., Joo, W, H. (2001) Purification and biochemical characterization of a fibrinolytic enzyme from Bacillus subtilis BK-17. World J Microbiol Biotechnol 17: 89–92.
[4] Kim, W., Choi, K., Kim, Y., Park, H., Choi, J., Lee, Y., Lee, S. (1996). Purification and Characterization of a Fibrinolytic Enzyme Produced from Bacillus sp. strain CK 11-4 Screened from Chungkook-Jang, Applied and Environmental Microbiology, 62 (7), 2482–2488. DOI: 10.1128/AEM.62.7.2482-2488.1996.
[5] Bijender, K, B., Satbir, S., Mowkshi, K., Kaurab, S., Shikha, B. (2014): Optimization of Fibrinolytic Protease Production from Bacillus subtilis I-2 Using Agro-Residues. Braz. Arch. Biol. Technol. v. 57 n. 5: pp. 653-662.
[6] Chang, C., Wang, P., Hung, Y., Chung, Y. (2012). Purification and biochemical properties of a fibrinolytic enzyme from Bacillus subtilis -fermented red bean. Food Chemistry, 133 (4), 1611–1617.
[7] Kim, H., Kim G., Kim D. (1997). Purification et caractérisation d'une nouvelle enzyme fibrinolytique du Bacillus sp. KA38 originaire de poisson fermenté. J. Fermenter. Bioeng, 84. 307-312.
[8] Sherry. S (1987). Appraisal of various thrombolytic agents in the treatment of acute yocardial infarction The American Journal of Medicine Volume 83, Issue 2, Supplement 1, Pages https://doi.org/10.1016/0002-9343(87)90885-0.
[9] Wang, C. T., Ji, B. P., Li, B., Nout, R., Li, P. L., Ji, H., Chen, L, F. (2006). Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. The Journal of Industrial Microbiology and Biotechnology, 33 (9), 750–758. Sumi, H., Hamada, H., Tsushima, H. (1987). A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto: A typical and popular soybean food in the Japanese diet, Experientia 43 (1987) 1110–1111.
[10] Sumi, H., Nakajima, N., C. Yatagai, A. (1995). Unique strong fibrinolytic enzyme (Katsuwokinase) in skipjack “shiokara”, a Japanese traditional fermented food, Biochem. Mol. Biol. 112 543–547.
[11] Kim, S, H., Choi, N, S. (2000). Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp strain DJ-4 screened from Doen-Jang. Biosci Biotechnol Biochem 64: 1722–1725.
[12] Wong, A, H, K., Mine Y. (2004). Novel fibrinolytic enzyme in fermented shrimp paste, a traditional Asian fermented seasoning. J Agr Food Chem. 52, 980–986, https://doi.org/10.1021/jf034535y.
[13] Kim, S, B., Lee D, W., Cheigh, C, I., Choe, E, A., Lee, S, J., Hong, Y, H., Choi, H, J., Pyun, Y, R. (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: 436–44.
[14] Nguimbi, E., Ahombo, G., Moyen, R., Ampa, R., Vouidibio, A., Nina, E.,… Louembe, D. (2014). Optimization of Growth, Fibrinolytic Enzyme Production and PCR Amplification of Encoding Fibrinolytic Enzyme Gene in Bacillus amyloliquefaciens Isolated from Ntoba mbodi at Brazzaville, International Journal of Science and Research (IJSR), 3 (11), 2799–2803.
[15] Hu, Y., Yu, D., Zhaoting, W., Hou, J., Tyagi, R., Liang, Y. (2019). Purification and characterization of a novel, highly potent fibrinolytic enzyme from Bacillus subtilis DC27 screened from Douchi, a traditional Chinese fermented soybean food, Scientific Reports 2019, N° 1, (May), 3–12.
[16] Soloka, M, F, A., Nguimbi, E., Kayath, A, C., Ahombo, G. (2020) Molecular characterization of Bacilllus-genus Bacteria with Fibrinolytique isolated from squashes <<NTETE>> in the Brazzaville in the republic Of Congo. American journal of Microbiological Research, Vol 8. No. 1, 7-18.
[17] Lagzouli, M., Charouf, R., Yachioui, El, M., Ouhssine, M,. Berny, El, H., Jadal, M. (2007). Optimisation de la croissance glucoamylase extracellulaire, Bull. Soc. Pharm. Bordeaux 146, 251-270.
[18] Soloka, M, F, A., Moyen, R., Nguimbi, E., Gabriel Ahombo., Raoul Ampa., Kayath, A, C., Vouidibio, A., Morabandza, C, J., Kobawila S, C. (2017) Production, Partial Purification and Based SDS-PAGE profiles of Caseinolytic Enzyme in two Bacillus strains isolated from fermented cassava leaves "ntoba mbodi" in congo Brazzaville. Journal of pure and applied microbiology, vol. 11 (1), p. 77-86.
[19] Wang, S, H., Zhang, C., Yang, Y, L, Diao, M., Bai, M, F. (2007). Projection de haute enzyme fibrinolytique production souche et la caractérisation de l'enzyme fibrinolytique produite à partir de Bacillus subtilis LD 8547. J. mondiale Microbiol. Biotechnol, 7. Epub.
[20] Kotb, E. (2015). Purification and partial characterization of serine fibrinolytic enzyme from Bacillus megaterium KSK-07 isolated from Kishk, a traditional egyptian fermented food. Appl Biochem Micro. 51, 34–43, https://doi.org/10.1134/s000368381501007x.
[21] Soloka, M, F, A., Nguimbi, E., Ampa, R.,; Kayath, A, C., Vouidibio, A., Ahombo, G. (2018). Production, Encoding Genes Variability and N- Terminal Sequences of Fibrinolytic Enzymes Produced by Bacillus Strains Isolated from Fermented Cassava Leaves “ Ntoba Mbodi ” at Brazzaville, Republic of Congo’ International Journal of Science and Research (IJSR) 7, 241–48.
[22] Mir M, S., H., Rabbani M., Naghitorabi, M. (2009): Cloning of alkaline protease gene from Bacillus subtilis 168 Research in Pharmaceutical Sciences, 4 (1): 43-46.
[23] Altschu, F, S., Gish, W., Miller, W., Myers, W. E., Lipman, J, D (1990): Basic Local Alignement search Tool. J. Mol. Biol. 215 403-410.
[24] Rouhou, C. (2006). Evaluation des classifications phylogénétiques des bacillaceae basées sur les gènes de l’opéron rrn et de gènes de ménage. Memoire pour l’obtention de la maîtrise en biologie.
[25] Kumar, S., Stecher G., Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.
[26] Barboza-Corona, J, E., De la Fuente-Salcido, N., Alva-Murillo N., Ochoa-Zarzosa A., LopezMeza, J, E. (2009). Activity of bacteriocins synthesized by Bacillus thuringiensis against Staphylococcus aureus isolates associated to bovine mastitis. Veterinary Microbiology 138, n° 1: 179-183.
[27] Pacheco Cano, R, d., Norm, M., De la Fuente-Salcido., Salcedo-Hermandez, R., José E. Barboza, C., Hernandez-Guzman G., Leon-Galvan, M, F., Bideshi, D, K. (2014). Characterization, N-terminal sequencing and classification of Tolworthcin 524: a bacteriocin produced by Bacillus thuringiensis subsp. tolworthi. Microbiological research 169: 948-953.
[28] Mokemiabeka, N, S., Kayath, C, A., Nguimbi, E., Augustin, A., Guy, A., Eboungabeka, M., Botteaux, A. (2016). Microbiological and biochemical assessment of crushed red pepper from Capsicum frutescens preserved in jars and manufactured in local markets in Republic of Congo, International Journal of Biotechnology Research 4 (October), 1–10.
[29] Heo, K., Cho, K. M., Lee, C. K., Kim, G. M., Shin, J., Kim, J. S., & Kim, J. H. (2013). Characterization of a Fibrinolytic Enzyme Secreted by Bacillus amyloliquefaciens CB1 and Its Gene Cloning. J. Microbiol. Biotechnol. (2013), 23 (7), 974–983 23 (7), 974–983.
[30] Kodama, T., Endo, K., Sawada, K., Ara, K., Ozaki, K., Kakeshita, H., Sekiguchi, J. (2007). Bacillus subtilis AprX Involved in Degradation of a Heterologous Protein During the Late Stationary Growth Phase, Journal of bi osci ence and bi oengi ne ering 104 (2), 135–143.
[31] Peng, Y., Yang, X., Xiao, L., & Zhang, Y. (2004). Cloning and expression of a fibrinolytic enzyme (subtilisin DFE) gene from Bacillus amyloliquefaciens DC-4 in Bacillus subtilis, Research in Microbiology 155 155, 167–173.
[32] Agrebi, R., Haddar, A., Hmidet, N., Jellouli, K., Manni, L., Nasri, M. (2009). BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification, biochemical and molecular characterization, Process Biochemistry journal 44: 1252–1259.
Cite This Article
  • APA Style

    Nguimbi Etienne, Soloka Mabika Faly Armel, Dibangou Valentin, Nzaou Stech Anomène Eckzechel, Kayath Aimé Christian, et al. (2021). New Phylogenetic Molecular Markers in Bacteria of the Genus Bacillus: Fibrinolytic Proteases. International Journal of Microbiology and Biotechnology, 6(3), 86-94. https://doi.org/10.11648/j.ijmb.20210603.14

    Copy | Download

    ACS Style

    Nguimbi Etienne; Soloka Mabika Faly Armel; Dibangou Valentin; Nzaou Stech Anomène Eckzechel; Kayath Aimé Christian, et al. New Phylogenetic Molecular Markers in Bacteria of the Genus Bacillus: Fibrinolytic Proteases. Int. J. Microbiol. Biotechnol. 2021, 6(3), 86-94. doi: 10.11648/j.ijmb.20210603.14

    Copy | Download

    AMA Style

    Nguimbi Etienne, Soloka Mabika Faly Armel, Dibangou Valentin, Nzaou Stech Anomène Eckzechel, Kayath Aimé Christian, et al. New Phylogenetic Molecular Markers in Bacteria of the Genus Bacillus: Fibrinolytic Proteases. Int J Microbiol Biotechnol. 2021;6(3):86-94. doi: 10.11648/j.ijmb.20210603.14

    Copy | Download

  • @article{10.11648/j.ijmb.20210603.14,
      author = {Nguimbi Etienne and Soloka Mabika Faly Armel and Dibangou Valentin and Nzaou Stech Anomène Eckzechel and Kayath Aimé Christian and Moyen Rachel},
      title = {New Phylogenetic Molecular Markers in Bacteria of the Genus Bacillus: Fibrinolytic Proteases},
      journal = {International Journal of Microbiology and Biotechnology},
      volume = {6},
      number = {3},
      pages = {86-94},
      doi = {10.11648/j.ijmb.20210603.14},
      url = {https://doi.org/10.11648/j.ijmb.20210603.14},
      eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijmb.20210603.14},
      abstract = {The fibrinolytic proteases developed by bacteria have become substances of medical interest, as they have been recognized as antithrombotic substances in the blood. It is in this context that a study has been carried out with the aim of studying the fibrinolytic proteases produced by bacteria isolated from food in the Congo. Four strains of bacteria of the genus Bacillus isolated from crushed and cooked squash seeds, namely Bacillus sp strain ASM7, Bacillus sp strain CRK, Bacillus pumilus strain ASM5, Bacillus subtilis strain ASM1, identified by their rDNA16S, were tested positive for the production of fibrinolytic proteases. The fibrin box technique was used. The diameters on the fibrin boxes prove a significant production of fibrinolytic proteases. The genes coding for these fibrinolytic proteases were amplified by PCR and 1% Agarose gel electrophoresis shows that the size of the amplicons for the four strains is between1200-1450bp. The sequences of these coding genes have been for the four strains studied submitted to GenBank and the assigned accession numbers are respectively: Bacillus sp strain ASM7 MT743004, Bacillus sp strain CRK MT743005, Bacillus pumilus strain ASM5 MT743006, Bacillus subtilis strain ASM1 MT743007. These genes show a high degree of similarity of almost 99.50% with their counterparts in the databases, are all coding and show some observable differences. The translation of these genes in coherent reading frames confirms the amino acids already known in the active sites relating to their fibrinolysis role. The fibrinolytic protease CFE1 (Id=QNJ60181) is for Bacillus sp strain ASM7, the fibrinolytic protease CFE2 (Id=QNJ60182) is for Bacillus sp strain CRK, the fibrinolytic protease CFE3 (Id=QNJ60183) is for Bacillus pumilus strain ASM5, the fibrinolytic protease CFE4 (Id=QNJ60184) is for Bacillus subtilis strain ASM1. All these fibrinolytic proteases show a strong similarity (99.51-99.76%) with the Bacillus AprX serine protease, reference sequence. The phylogenetic inference test based on these fibrinolytic proteases shows that these proteases form a highly conserved characteristic group in bacteria of the genus Bacillus. This allowed us to retain these fibrinolytic proteases as an important phylogenetic molecular marker.},
     year = {2021}
    }
    

    Copy | Download

  • TY  - JOUR
    T1  - New Phylogenetic Molecular Markers in Bacteria of the Genus Bacillus: Fibrinolytic Proteases
    AU  - Nguimbi Etienne
    AU  - Soloka Mabika Faly Armel
    AU  - Dibangou Valentin
    AU  - Nzaou Stech Anomène Eckzechel
    AU  - Kayath Aimé Christian
    AU  - Moyen Rachel
    Y1  - 2021/08/18
    PY  - 2021
    N1  - https://doi.org/10.11648/j.ijmb.20210603.14
    DO  - 10.11648/j.ijmb.20210603.14
    T2  - International Journal of Microbiology and Biotechnology
    JF  - International Journal of Microbiology and Biotechnology
    JO  - International Journal of Microbiology and Biotechnology
    SP  - 86
    EP  - 94
    PB  - Science Publishing Group
    SN  - 2578-9686
    UR  - https://doi.org/10.11648/j.ijmb.20210603.14
    AB  - The fibrinolytic proteases developed by bacteria have become substances of medical interest, as they have been recognized as antithrombotic substances in the blood. It is in this context that a study has been carried out with the aim of studying the fibrinolytic proteases produced by bacteria isolated from food in the Congo. Four strains of bacteria of the genus Bacillus isolated from crushed and cooked squash seeds, namely Bacillus sp strain ASM7, Bacillus sp strain CRK, Bacillus pumilus strain ASM5, Bacillus subtilis strain ASM1, identified by their rDNA16S, were tested positive for the production of fibrinolytic proteases. The fibrin box technique was used. The diameters on the fibrin boxes prove a significant production of fibrinolytic proteases. The genes coding for these fibrinolytic proteases were amplified by PCR and 1% Agarose gel electrophoresis shows that the size of the amplicons for the four strains is between1200-1450bp. The sequences of these coding genes have been for the four strains studied submitted to GenBank and the assigned accession numbers are respectively: Bacillus sp strain ASM7 MT743004, Bacillus sp strain CRK MT743005, Bacillus pumilus strain ASM5 MT743006, Bacillus subtilis strain ASM1 MT743007. These genes show a high degree of similarity of almost 99.50% with their counterparts in the databases, are all coding and show some observable differences. The translation of these genes in coherent reading frames confirms the amino acids already known in the active sites relating to their fibrinolysis role. The fibrinolytic protease CFE1 (Id=QNJ60181) is for Bacillus sp strain ASM7, the fibrinolytic protease CFE2 (Id=QNJ60182) is for Bacillus sp strain CRK, the fibrinolytic protease CFE3 (Id=QNJ60183) is for Bacillus pumilus strain ASM5, the fibrinolytic protease CFE4 (Id=QNJ60184) is for Bacillus subtilis strain ASM1. All these fibrinolytic proteases show a strong similarity (99.51-99.76%) with the Bacillus AprX serine protease, reference sequence. The phylogenetic inference test based on these fibrinolytic proteases shows that these proteases form a highly conserved characteristic group in bacteria of the genus Bacillus. This allowed us to retain these fibrinolytic proteases as an important phylogenetic molecular marker.
    VL  - 6
    IS  - 3
    ER  - 

    Copy | Download

Author Information
  • Laboratory of Cell and Molecular Biology, Faculty of Sciences and Techniques, Marien Ngouabi University, Brazzaville, Republic of Congo

  • Laboratory of Cell and Molecular Biology, Faculty of Sciences and Techniques, Marien Ngouabi University, Brazzaville, Republic of Congo

  • Laboratory of Cell and Molecular Biology, Faculty of Sciences and Techniques, Marien Ngouabi University, Brazzaville, Republic of Congo

  • Laboratory of Cell and Molecular Biology, Faculty of Sciences and Techniques, Marien Ngouabi University, Brazzaville, Republic of Congo

  • Laboratory of Cell and Molecular Biology, Faculty of Sciences and Techniques, Marien Ngouabi University, Brazzaville, Republic of Congo

  • Laboratory of Cell and Molecular Biology, Faculty of Sciences and Techniques, Marien Ngouabi University, Brazzaville, Republic of Congo

  • Sections