Skip to main content
SpringerLink
Log in

Halobacillus blutaparonensis Strain M9 as a Source of Extracellular Serine Peptidases with Properties for Biotechnological Purposes

  • EXPERIMENTAL ARTICLES
  • Published:
Microbiology Aims and scope Submit manuscript

Abstract

In this work, we report on the halophilic bacterium Halobacillus blutaparonensis strain M9, which was isolated from a preserved Brazilian restinga, as a producer of alkaline serine peptidases with potential application as additives for biotechnological purposes. Gelatin-zymography assay revealed the presence of the major peptidase of 70 kDa and two minor ones (50 and 40 kDa) secreted by the living bacterial cells. When a specific chromogenic substrate for chymotrypsin-type serine peptidases was employed by means of an in-solution assay, the bacterial peptidases were completely inhibited by phenylmethylsulphonyl fluoride (PMSF) and slightly inhibited by benzamidine, N-tosyl-L-lysine chloromethyl ketone (TLCK) and 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), which are classical serine peptidase inhibitors. The proteolytic activity remained extremely stable within a broad pH range (from 5.0 to 9.0) and also displayed moderate thermostability. In addition, the effects of osmolytes/polyols, divalent cations, denaturing agents, and detergents on the enzymatic activity and stability of these serine peptidases were tested. The proteolytic activity increased in the presence of different osmolytes/polyols (e.g., glycerol, glutamate, and mannitol). The enzymatic activity increased to 110% when incubated in the presence of 10 mM Ca2+, while Mg2+ and Mn2+ (both at 10 mM) decreased the proteolytic activity to 95 and 85%, respectively. Interestingly, urea at 1% induced a significant increase (around 160%) in the proteolytic activity. The peptidases showed high stability when incubated with various detergent agents and commercial detergent powders. Collectively, all these properties indicate H. blutaparonensis as a producer of extracellular serine peptidases with possible industrial applications, such as in detergent formulations.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.

Similar content being viewed by others

REFERENCES

  1. Alvarez, V.M., Von der Weid, I., Seldin, L., and Santos, A.L.S., Influence of growth conditions on the production of extracellular proteolytic enzymes in Paenibacillus peoriae NRRL BD-62 and Paenibacillus polymyxa SCE2, Lett. Appl. Microbiol., 2006, vol. 43, pp. 625–630.

    Article  CAS  PubMed  Google Scholar 

  2. Anwar, A. and Saleemuddin, M., Alkaline protease from Spilosoma obliqua: potential applications in bio-formulations, Biotechnol. Appl. Biochem., 2000, vol. 31, pp. 85–89.

    Article  CAS  PubMed  Google Scholar 

  3. Badgujar, S.B. and Mahajan, R.T., Identification and characterization of Euphorbia nivulia latex proteins, Int. J. Biol. Macromol., 2014, vol. 64, pp. 193–201.

    Article  CAS  PubMed  Google Scholar 

  4. Benjamin, S., Smitha, R.B., Jisha, V.N., Pradeep, S., Sajith, S., Sreedevi, S., Priji, P., Unni, K., and Josh, M., A monograph on amylases from Bacillus spp, Adv. Biosci. Biotechnol., 2013, vol. 4, pp. 227–241.

    Article  Google Scholar 

  5. Betzel, C., Klupsch, S., Branner, S., and Wilson, K.S., Crystal structures of the alkaline proteases savinase and esperase from Bacillus lentus, Adv. Exp. Med. Biol., 1996, vol. 379, pp. 49–61.

    Article  CAS  PubMed  Google Scholar 

  6. Borgi, I. and Gargouri, A., Investigations on a hyper-proteolytic mutant of Beauveria bassiana: broad substrate specificity and high biotechnological potential of a serine protease, FEMS Microbiol. Lett., 2014, vol. 351, pp. 23–31.

    Article  CAS  PubMed  Google Scholar 

  7. Bressollier, P., Letourneau, F., Urdaci, M., and Verneuil, B., Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus, Appl. Environ. Microbiol., 1999, vol. 65, pp. 2570–2576.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chamroensaksri, N., Akaracharanya, A., Visessanguan, W., and Tanasupawat, S., Characterization of halophilic bacterium Nb2-1 from Pla-Ra and its protease production, J. Food. Biochem., 2008, vol. 32, pp. 536–555.

    Article  CAS  Google Scholar 

  9. Da Costa, M.S., Santos, H., and Galinski, E.A., An overview of the role and diversity of compatible solutes in Bacteria and Archaea, Adv. Biochem. Eng. Biotechnol., 1998, vol. 61, pp. 117–153.

    CAS  PubMed  Google Scholar 

  10. Danson, M.J. and Hough, D.W., The structural basis of protein halphilicity, Comp. Biochem. Physiol., 1997, vol. 117, pp. 307–312.

    Article  Google Scholar 

  11. DelMar, E.G., Largman, C., Brodrick, J.W., and Geokas, M.C., A sensitive new substrate for chymotrypsin, Anal. Biochem., 1979, vol. 99, pp. 316–320.

    Article  CAS  PubMed  Google Scholar 

  12. Fang, Z., Zhang, J., Liu, B., Jiang, L., Du, G., and Chen, J., Cloning, heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1, Process. Biochem., 2014, vol. 49, pp. 647–654.

    Article  CAS  Google Scholar 

  13. Galinski, E.A. and Trüper, H.G., Microbial behaviour in salt-stressed ecosystems, FEMS Microbiol. Rev., 1994, vol. 15, pp. 95–108.

    Article  CAS  Google Scholar 

  14. Ghordel, S., Kammoun, M., Soltana, H., Nasri, M., and Hmidet, N., Streptomyces flavogriseus HS1: isolation and characterization of extracellular proteases and their compatibility with laundry detergents, Biomed. Res. Int., 2014, vol. 2014, p. 345980.

    Google Scholar 

  15. Godfrey, T. and West, S., Introduction to industrial enzymology, in Industrial Enzymology, Godfrey, T. and West, S., Eds., London: Macmillan, 1996, pp. 1–8.

    Google Scholar 

  16. Gong, J., Wang, Y., Zhang, D., Zhang, R., Su, C., Li, H., Zhang, X., Xu, Z., and Shi, J., Biochemical characterization of an extreme alkaline and surfactant-stable keratinase derived from a newly isolated actinomycete Streptomyces aureofaciens K13, RSC Adv., 2015, vol. 5, pp. 24691–24699.

    Article  CAS  Google Scholar 

  17. Gupta, R., Beg, Q.K., and Lorenz, P., Bacterial alkaline peptidases: molecular approaches and industrial applications, Appl. Microbiol. Biotechnol., 2002a, vol. 59, pp. 15–32.

    Article  CAS  PubMed  Google Scholar 

  18. Gupta, R., Beg, Q.K., Khan, S., and Chauhan, B., An overview on fermentation, downstream processing and properties of microbial alkaline proteases, Appl. Microbiol. Biotechnol., 2002b, vol. 60, pp. 381–395.

    Article  CAS  PubMed  Google Scholar 

  19. Habbeche, A., Saoudi, B., Jaouadi, B., Haberra, S., Kerouaz, B., Boudelaa, M., Badis, A., and Ladjama, A., Purification and biochemical characterization of a detergent-stable keratinase from a newly thermophilic actinomycete Actinomadura keratinilytica strain Cpt29 isolated from poultry compost, J. Biosci. Bioeng., 2014, vol. 117, pp. 413–421.

    Article  CAS  PubMed  Google Scholar 

  20. Hasan, A.K.M.Q. and Tamiya, E., Patent WO 9727313, 1997.

  21. Heussen, C. and Dowdle, E.B., Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates, Anal. Biochem., 1980, vol. 102, pp. 196–202.

    Article  CAS  PubMed  Google Scholar 

  22. Hmidet, N., Ali, N.E., Haddar, A., Kanoun, S., Alya, S., and Nasri, M., Alkaline proteases and thermostable α-amylase co-produced by Bacillus licheniformis NH1: characterization and potential application as detergent additive, Biochem. Eng. J., 2009, vol. 47, pp. 71–79.

    Article  CAS  Google Scholar 

  23. Horikoshi, K., Alkaliphiles: some applications of their products for biotechnology, Microbiol. Mol. Biol. Rev., 1999, vol. 63, pp. 735–750.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Jaouadi, B., Ellouz-Chaabouni, S., Rhimi, M. and Bejar, S., Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency, Biochimie, 2008, vol. 90, pp. 1291–1305.

    Article  CAS  PubMed  Google Scholar 

  25. Joo, H.S., Kumar, C.G., Park, G.C., Paik, R.S., and Chang, C.S., Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties, J. Appl. Microbiol., 2003, vol. 95, pp. 267–272.

    Article  CAS  PubMed  Google Scholar 

  26. Joo, H.S., Park, G.C., Kim, K.T., Paik, S.R., and Chang, C.S., Simple methods for alkaline protease purification from the polychaeta, Periserrula leucophryna, Process. Biochem., 2001, vol. 37, pp. 299–303.

    Article  CAS  Google Scholar 

  27. Karan, R.E. and Khare, S.K., Stability of haloalkaliphilic Geomicrobium sp. protease modulated by salt, Biochemistry (Moscow), 2011, vol. 76, pp. 686–693.

    CAS  PubMed  Google Scholar 

  28. Karan, R. and Khare, S.K., Purification and characterization of a solvent-stable protease from Geomicrobium sp. EMB2, Environ. Technol., 2010, vol. 31, pp. 1061–1072.

    Article  CAS  PubMed  Google Scholar 

  29. Kitayama, M., Patent JP 4271781 1992, 1992.

  30. Kumar, C.G., Malik, K., and Tiwari, M.P., Novel enzyme-based detergents: an indian perspective, Curr. Sci., 1998, vol. 75, pp. 1312–1318.

    CAS  Google Scholar 

  31. Li, Q., Yi, L., Marek, P., and Iverson, B.L, Commercial proteases: present and future, FEBS Lett., 2013, vol. 587, pp. 1155–1163.

    Article  CAS  PubMed  Google Scholar 

  32. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 1951, vol. 193, pp. 265–275.

    Article  CAS  PubMed  Google Scholar 

  33. Maurer, K.H., Detergent proteases, Curr. Opin. Biotechnol., 2004, vol. 15, pp. 330–334.

    Article  CAS  PubMed  Google Scholar 

  34. Mevarech, M., Frolow, F., and Gloss, L.M., Halophilic enzymes: proteins with a grain of salt, Biophys. Chem., 2000, vol. 86, pp. 155–164.

    Article  CAS  PubMed  Google Scholar 

  35. Ng, T.K. and Kenealy, W.R., Industrial applications of thermostable enzymes, in TGM and Applied Microbiology, Brock, T.D., Ed., New Jersey: Wiley, 1986, pp. 197–205.

    Google Scholar 

  36. Niyonzima, F.N. and More, S.S., Concomitant production of detergent compatible enzymes by Bacillus flexus XJU-1, Braz. J. Microbiol., 2014, vol. 45, pp. 903–910.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Phadatare, S.U., Deshpande, V.V., and Srinivasan, M.C., High activity alkaline protease from Conidiobolus coronatus (NCL 86.8.20): enzyme production and compatibility with commercial detergents, Enzyme Microb. Technol., 1993, vol. 15, pp. 72–76.

    Article  CAS  Google Scholar 

  38. Phrommao, E., Rodtong, S., and Yongsawatdigul, J., Identification of novel halotolerant bacillopeptidase F-like proteinases from a moderately halophilic bacterium, Virgibacillus sp. SK37, J. Appl. Microbiol., 2011, vol. 110, pp. 191–201.

    Article  CAS  PubMed  Google Scholar 

  39. Rao, M.B., Tanksale, A.M., Ghatge, M.S., and Deshpande, V.V., Molecular and biotechnological aspects of microbial proteases, Microbiol. Mol. Biol. Rev., 1998, vol. 62, pp. 597–635.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Saeki, K., Hitomi, J., Okuda, M., Hatada, Y., Kageyama, Y., Takaiwa, M., Kubota, H., Hagihara, H., Kobayashi, T., Kawai, S., and Ito, S., A novel species of alkalophilic Bacillus that produces an oxidatively stable alkaline serine protease, Extremophiles, 2002, vol. 6, pp. 65–72.

    Article  CAS  PubMed  Google Scholar 

  41. Santos, A.F., Pacheco, C.A., Valle, R.S., Seldin, L., and Santos, A.L.S., Enzymes produced by halotolerant spore-forming gram-positive bacterial strains isolated from a resting habitat (Restinga de Jurubatiba) in Rio de Janeiro, Brazil: focus on proteases, Appl. Biochem. Biotechnol., 2014, vol. 174, pp. 2748–2761.

    Article  Google Scholar 

  42. Santos, A.F., Valle, R.S., Pacheco, C.S., Alvarez, V.M., Seldin, L., and Santos, A.L.S., Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium, Braz. J. Microbiol., 2013, vol. 44, pp. 1299–1304.

    Article  PubMed  Google Scholar 

  43. Seldin, L., Van Elsas, J.D., and Penido, E.G.C., Bacillus nitrogen fixers from Brazilian soils, Plant and Soil, 1983, vol. 70, pp. 243–255.

    Article  Google Scholar 

  44. Showell, M.S., Enzymes, detergent, in Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation, Flickinger, M.C. and Drew, S.W., Eds., New York: Wiley, 1999, pp. 958–971.

    Google Scholar 

  45. Sinha, R. and Khare, S.K., Characterization of detergent compatible protease of a halophilic Bacillus sp. EMB9: differential role of metal ions in stability and activity, Bioresour. Technol., 2013, vol. 145, pp. 357–361.

    Article  CAS  PubMed  Google Scholar 

  46. Syed, D.G., Lee, J.C., Li, W.J., Kim, C.J., and Agasar, D., Production, characterization and application of keratinase from Streptomyces gulbargensis, Bioresour. Technol., 2009, vol. 100, pp. 1868–1871.

    Article  CAS  PubMed  Google Scholar 

  47. Tatineri, R., Doddapaneni, K.K., Potumarthi, R.C., Vellanki, R.N., Kandathil, M.T., Kolli, N., and Mangamoori, L.N., Purification and characterization of an alkaline keratinase from Streptomyces sp., Bioresour. Technol., 2008, vol. 99, pp. 1596–1602.

    Article  Google Scholar 

  48. Vidyasagar, M., Prakash, S., Litchfield, C., and Sreeramulu, K., Purification and characterization of a thermostable, haloalkaliphilic extracellular serine protease from extreme halophilic archaeon Halogeometricum borinquense strain TSS101, Archaea, 2006, vol. 2, pp. 51–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. World Enzyme–Industry Market Research, Cleveland: The Freedonia Group, 2014.

Download references

Funding

This study was supported by grants from the following Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa no Estado do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES—Financial code 001).

Author information

Authors and Affiliations

  1. Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, RJ, Brazil

    A. F. Santos, T. F. O. Souza, L. Seldin, M. H. Branquinha & A. L. S. Santos

  2. Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, 21941-909, Rio de Janeiro, RJ, Brazil

    A. F. Santos, T. F. O. Souza & D. M. G. Freire

  3. Programa de Pós-Graduação em Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, 21941-909, Rio de Janeiro, RJ, Brazil

    A. F. Santos, T. F. O. Souza, D. M. G. Freire & A. L. S. Santos

Authors
  1. A. F. Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. T. F. O. Souza
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D. M. G. Freire
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L. Seldin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. H. Branquinha
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. L. S. Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to T. F. O. Souza or A. L. S. Santos.

Ethics declarations

The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Santos, A.F., Souza, T.F., Freire, D.M. et al. Halobacillus blutaparonensis Strain M9 as a Source of Extracellular Serine Peptidases with Properties for Biotechnological Purposes. Microbiology 90, 124–132 (2021). https://doi.org/10.1134/S0026261721010094

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0026261721010094

Keywords:

Search

Navigation