Skip to main content
Download PDF
  • Industrial Microbiology
  • Original Articles
  • Published:

Isolation and identification of alkaline protease producer halotolerantBacillus licheniformis strain BA17

Annals of Microbiology volume 57, pages 369–375 (2007)Cite this article

Abstract

An alkaline protease producerBacillus licheniformis strain was isolated from Van Lake in Turkey. The strain is Gram positive, aerobic, motile, sporulating rod-shaped bacterium. Spores were ellipsoidal and positioned central in nonswollen sporangium. The cells were able to grow well at a pH range of 5.7–10. The optimal growth temperature was found to be 37 °C. Growth at a wide range of NaCl concentration (from 0 to 20%) showed that BA17 is halotolerant. Main fatty acid composition of BA17 was anteiso-C15:0 and iso-C15∶0. The strain was presumptively identified asB. licheniformis according to 16S rDNA gene sequence analysis. The most appropriate medium for the growth and protease production is composed of 0.5% yeast extract, 0.5% NaNO3, 0.02% MgSO4\7H2O, 0.1% K2HPO4 and 0.5% maltose. The optimum temperature and pH of the alkaline protease of strain BA17 were found to be 60 °C and pH 11, respectively. The activity was completely lost in the presence of PMSF, suggesting that the preparation contains serine-alkaline protease(s).

References

  • Al-Shehri M.A., Mostafa S.Y. (2004). Production and some properties of protease produced byBacillus licheniformis isolated from Tihamet Aseer, Saudi Arabia. Pakistan J. Biol. Sci., 7: 1631–1635.

    Article  Google Scholar 

  • Altschul S.F., Gish W., Miller M., Myers E.W., Lipman D.J. (1990). Basic local alignment search tool. J. Mol. Biol., 215: 403–410.

    PubMed  CAS  Google Scholar 

  • Amoozegar M.A., Fatemi A.Z., Karbalaei-Heidari H.R., Razavi M.R. (2006). Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile,Salinivibrio sp. strain AF-2004. Microbiol. Res., (In press).

  • Banerjee U.C., Sani R.K., Azmi W., Soni R. (1999). Thermostable alkaline protease fromBacillus brevis and its characterization as a laundry detergent additive. Proc. Biochem., 35: 213–219.

    Article  CAS  Google Scholar 

  • Banik M.R., Prakash M. (2004). Laundry detergent compatibility of the alkaline protease fromBacillus cereus. Microbiol. Res., 159: 135–140.

    Article  PubMed  CAS  Google Scholar 

  • Berber I., Yenidünya E. (2005). Identification of alkaliphilicBacillus species isolated from Lake Van and its surroundings by computerized analysis of extracellular protein profiles. Turk J. Biol., 29: 181–188.

    Google Scholar 

  • Britschgi T.B., Giovannoni S.J. (1991). Phylogenetic analysis of a natural marine bacterioplankton population by rRNA gene cloning and sequencing. Appl. Environ. Microbiol., 57: 1707–1713.

    PubMed  CAS  Google Scholar 

  • Chauhan B., Gupta R. (2004). Application of statistical experimental design for optimization of alkaline protease production fromBacillus sp.. RGR-14. Proc. Biochem., 39: 2115–2122.

    Article  CAS  Google Scholar 

  • Claus D., Berkeley R.C.W. (1986). The GenusBacillus. In: Bergey’s Manual of Systematic Bacteriology, vol. 2, pp. 1105–1129.

  • Danson M.J., Hough D.W. (1997). The structural basis of protein halophilicity. Comp. Biochem. Physiol., 117A: 307–312.

    Article  CAS  Google Scholar 

  • Degens E.T., Wong H.K., Kepme S., Kurtman F. (1984). A geological study of Lake Van, Eastern Turkey. Int. J. Earth Sci. (Historical Archive), 73: 701–734.

    CAS  Google Scholar 

  • Demirjian D.C., Moris-Varas F., Cassidy C.S. (2001). Enzymes from extremophiles. Curr. Opin. Chem. Biol. 5: 144–151.

    Article  PubMed  CAS  Google Scholar 

  • Denizci A.A., Kazan D., Abeln E.C.A., Erarslan A. (2004). Newly isolatedBacillus clausii GMBAE42: an alkaline protease producer capable to grow under higly alkaline conditions. J. Appl. Microbiol., 96: 320–327.

    Article  PubMed  CAS  Google Scholar 

  • Edgcomb V.P., Kysela D.T., Tekse A., Gomez A.V., Sogin M.L. (2002). Bentic eukaryotic diversity in the Guaymas Basin Hydrothermal Vent Environment. PNAS, 99: 7658–7662.

    Article  PubMed  CAS  Google Scholar 

  • Ferrero M.A., Gastro G.R., Abate C.M., Baigori M.D., Sineriz F. (1996). Thermostable alkaline proteases ofBacillus licheniformis MIR29 isolation, production and characterization. Appl. Microbiol. Biotechnol., 45: 327–332.

    Article  CAS  Google Scholar 

  • Folmsbee M., Ducan K., Han O.S., Nagle D., Jennings E., McInerney M. (2006). Re-identification of the halkotolerant, biosurfactant-producingBacillus licheniformis strain JF-2 asBacillus mojavensis strain JF-2. Syst. Appl. Microbiol., 29: 645–649.

    Article  PubMed  CAS  Google Scholar 

  • Gessesse A. (1997). The use of nug meal as a low-cost substrate for the production of alkaline protease by the alkaliphilicBacillus sp. AR-009 and some properties of the enzyme. Biores. Technol., 62: 59–61.

    Article  CAS  Google Scholar 

  • Ghorbel B., Sellami-Kamoun A., Nasri M. (2003). Stability studies of protease fromBacillus cereus BG1. Enzyme Microb. Tech., 32: 513–518.

    Article  CAS  Google Scholar 

  • Gimènez M.I., Studdert C.A., Sanchez J.J., De Castro R.E., (2000). Extracellular protease foNatrialba magadii: purification and biochemical characterization. Extremophiles, 4: 181–188.

    Article  PubMed  Google Scholar 

  • Grant W.D., Jones B.E., Mwatha W.E. (1990). Alkaliphiles: ecology, diversity and applications. FEMS Microbiol., 75: 255–270.

    Article  CAS  Google Scholar 

  • Grant W.D., Jones B.E., Eds (2000). Alkaline environments. In: Encyclopaedia of Microbiology, 2nd edn., Academic Press, 1: 126–133.

  • Gomes J., Steiner W., (2004). The biocatalytic potential of extromophiles and extremozymes. Foot Technol. Biotechnol., 4: 223–235.

    Google Scholar 

  • Hadj-Ali N.E., Agrebi R., Ghorbel-Frikha B., Sellami-Kamoun A., Kanoun S., Nasri M. (2007). Biochemical and molecular characterization of a detergent stable alkaline serine-protease from a newly isolatedB. licheniformis NH1. Enzyme Microb. Tech., 40: 515–523.

    Article  CAS  Google Scholar 

  • Horikoshi K. (1999). Alkaliphiles: Some applications of their products for biotechnology. Microbiol. Mol. Biol., December: 735–750.

    Google Scholar 

  • Joo H.-S., Kumar C.G., Park G.-C., Kim K.T., Paik S.R., Chang C.-S. (2002). Optimization of the production of an extracellular alkaline protease fromBacillus horikosii. Proc. Biochem., 38: 155–159.

    Article  CAS  Google Scholar 

  • Joo H.-S., Kumar C.G., Park G.-C., Paik S.R., Chang C.-S. (2003). Oxidant and SDS-stable alkaline protease fromBacillus clausii I-52: Production and some properties. J. Appl. Microbiol., 95: 267–272.

    Article  PubMed  CAS  Google Scholar 

  • Joo H.-S., Chang C.-S. (2005). Oxidant and SDS-stable alkaline protease from a halo-tolerantBacillus clausii I-52: enhanced production and simple purification. J. Appl. Microbiol., 98: 491–497.

    Article  PubMed  CAS  Google Scholar 

  • Kamoun S.A., Haddar A., Ali N.E., Frikha G.B., Kanoun S., Nasri M. (2006). Stability of thermostable alkaline protease fromBacillus licheniformis RP1 in commercial solid laundry detergent formulations. Microbiol. Res., (In press).

  • Kazan D., Denizci A.A, Öner M.N.K, Erarslan A. (2005). Purification and characterisation of a serine alkaline protease fromBacillus clausii GMBAE 42. J. Ind. Microbiol. Biotechnol., 32: 335–344.

    Article  PubMed  CAS  Google Scholar 

  • Kempe S., Degens E.T. (1985). An early soda ocean. Chem. Geol., 53: 95–108.

    Article  CAS  Google Scholar 

  • Kim S.S., Kim Y.J., Rhee I. (2001). Purification and characterization of a novel extracellular alkaline protease fromBacillus cereus KCTC3674. Arch. Microbiol., 175: 458–461.

    Article  PubMed  CAS  Google Scholar 

  • Kumar C.G., Takagi H. (1999). Microbial alkaline proteases: a bioindustrial viewpoint. Biotechnol Adv., 17: 561–594.

    Article  PubMed  CAS  Google Scholar 

  • Kumar S., Tamura K., Nei M. (2004). MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform., 5: 150–163.

    Article  PubMed  CAS  Google Scholar 

  • López-García P., Benzerara K., Menguy N., Kazmierczak J., Kempe S., Guyot F., Moreira D. (2005). Microbial diversity and spectroscopic study of the aragonite microbialites from the alkaline Lake Van (Turkey). Geophys. Res. Abstr., 7: 3648.

    Google Scholar 

  • Mabrouk S.S., Hashem A.M., El-Shayeb N.M.A., Ismail A.M.S., Abdel-Fattah A.F. (1999). Optimization of alkaline protease productivity byBacillus licheniformis ATCC 21415. Biores. Technol., 69: 155–159.

    Article  CAS  Google Scholar 

  • Manachini P.L., Fortina G.M., Levati L., Parini C. (1998). Contribution to phenptypic and genotypic characterization ofB. licheniformis and description of new genomovars. Syst. Appl. Micryobiol., 21: 520–529.

    CAS  Google Scholar 

  • Margesin R., Schinner F. (2001). Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles, 5: 73–83.

    Article  PubMed  CAS  Google Scholar 

  • Manczinger L., Rozs M., Vagvilgyi C.S., Kevei F. (2003). Isolation and characterization of a new keratinolyticBacillus licheniformis strain. World J. Microbiol. Biotechnol., 19: 35–39.

    Article  CAS  Google Scholar 

  • Mehrotra S., Pandey P.K., Gaur R., Darmwall N.S. (1999). The production of alkaline protease by aBacillus species isolate. Biores. Technol., 67: 201–203.

    Article  CAS  Google Scholar 

  • Moon S.H., Parulekar S.J. (1991). A parametric study of protease production in batch and fed-batch cultures ofBacillus firmus. Biotechnol. Bioeng., 37: 467–483.

    Article  PubMed  CAS  Google Scholar 

  • Nascimento W.C.A., Martins M.L.L. (2004). Production and properties of an extracellular protease from thermophilicBacillus sp. Braz. J. Microbiol., 35: 91–96.

    Article  Google Scholar 

  • North M.J. (1982). Comparative biochemistry of the proteinases of eukaryotic microorganisms. Microbiol. Rev., 46: 308–340.

    PubMed  CAS  Google Scholar 

  • Öner M.N., Denizci A.A., Kazan D., Erarslan A. (2006). A serine alkaline protease from a newly isolated obligate alkaliphilic Bacillus sp. GMBAE 72. FEBS J. Suppl. 1 June 2006, 273: 335–335.

    Google Scholar 

  • Patel R., Dodia M., Singh S.P. (2005). Extracellular alkaline protease from a newly isolated haloalkaliphilicBacillus sp.: Production and optimization. Proc. Biochem., 40: 3569–3575.

    Article  CAS  Google Scholar 

  • Prakasham R.S., Rao C.S., Sarma P.N. (2006). Green gram husk-an inexpensive substrate for alkaline protease production byBacillus sp. in solid-state fermentation. Bioresource Technol., 97: 1449–1454.

    Article  CAS  Google Scholar 

  • Puri S., Beg Q.K., Gupta R. (2002). Optimization of alkaline protease production fromBacillus sp. by Responce Surface Methodology. Curr. Microbiol., 44: 286–290.

    Article  PubMed  CAS  Google Scholar 

  • Saitou N., Nei M. (1987). A neighbor-joining method: new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 44: 406–425.

    Google Scholar 

  • Silva M.T.S.L., Santo F.E., Pereira P.T., Poseiro C.P. (2006). Phenotypic characterization of food waste degradingBacillus strains isolated from aerobic bioreactors. J. Basic Microbiol., 46: 34–46.

    Article  PubMed  CAS  Google Scholar 

  • Song Y., Yang R., Guo Z., Zhang M., Wang X., Zhou F. (2000). Distinctnessical of spore and vegetative cellular fatty acid profiles of some aerobic endospore-forming bacilli. J. Microbiol. Methods, 39: 225–241.

    Article  PubMed  CAS  Google Scholar 

  • Sorokin D.Y., Kuenen J.G. (2005). Haloalkaliphilic sulfur-oxidizing bacteria in soda lakes. FEMS Microbiol. Rev., 29: 685–702.

    Article  PubMed  CAS  Google Scholar 

  • Takami H., Akiba T., Horikoshi K. (1989). Production of extremely thermo stable alkaline protease fromBacillus sp. no. AH-101. Appl. Microbiol. Biotechnol., 30: 120–124.

    Article  CAS  Google Scholar 

  • Thompson J.D., Higgins D.G., Gibson T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence weighting position specific gap penalties and weight matric choice. Nucleic Acid Res., 22: 4673–4680.

    Article  PubMed  CAS  Google Scholar 

  • van den Burg B. (2003). Extremophiles as a source for novel enzymes. Curr. Opin. Microbiol., 6: 213–218.

    Article  PubMed  CAS  Google Scholar 

  • Ventosa A., Nieto J.J., Oren A. (1998). Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev., 62: 504–544.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Faculty of Engineering, Department of Chemical Engineering, Marmara University, Göztepe Campus, 34722, Kadiköy, Istanbul

    Özlem Ateş, Ebru Toksoy Oner & Dilek Kazan

  2. Faculty of Art and Science, Department of Biology, Çukurova University, Balcalt, Adana

    Burhan Arikan

  3. The Scientific and Technological Research Council of Turkey, Marmara Research Center, Genetic Engineering and Biotechnology Institute, P.O. Box 21, 41470, Gebze-Kocaeli, Turkey

    Aziz Akin Denizci & Dilek Kazan

Authors
  1. Özlem Ateş
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ebru Toksoy Oner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Burhan Arikan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Aziz Akin Denizci
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dilek Kazan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dilek Kazan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ateş, Ö., Oner, E.T., Arikan, B. et al. Isolation and identification of alkaline protease producer halotolerantBacillus licheniformis strain BA17. Ann. Microbiol. 57, 369–375 (2007). https://doi.org/10.1007/BF03175075

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03175075

Key words

Annals of Microbiology

ISSN: 1869-2044

Contact us