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Production and optimization of a commercially viable alkaline protease from a haloalkaliphilic bacterium

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Abstract

Twenty five haloalkaliphilic bacterial strains were isolated from sea water along the Coastal Gujarat (India) and screened for their ability to secret alkaline proteases. Among them, a potent strain S-20-9 (GenBank accession number EU118360), resembling to Halophilic Bacterium MBIC3303 on the basis of 16S rRNA gene sequencing, was selected for the optimization of enzyme production. S-20-9 produced protease optimally, under aerobic conditions during mid-stationary phase over a broad range of salt (5∼25%, w/v) and pH (7∼10). The optimum production was at pH 9 and 15% (w/v) NaCl. The production was suppressed by lactose, maltose, sucrose, and inorganic nitrogen sources, especially ammonium ions. Further, the production was significantly stimulated by KH2PO4 and suppressed by glucose. Similarly, the production was also suppressed at higher concentrations of gelatin, yeast extract, peptone, and casamino acids, indicating towards a threshold value for nitrogen requirement. The growth and protease production were enhanced by mono-valent cation (KCl), while the divalent cations acted as inhibitors. The study holds significance as only few reports are available on the alkaline proteases from haloalkaliphilic bacteria, particularly those from moderate saline habitats.

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References

  1. Horikoshi, K. (1999) Alkaliphiles: some applications of their products for biotechnology. Microbiol. Mol. Biol. Rev. 63: 735–750.

    CAS  Google Scholar 

  2. Schiraldi, C. and M. De Rosa (2002) The production of biocatalysts and biomolecules from extremophiles. Trends Biotechnol. 20: 515–521.

    Article  CAS  Google Scholar 

  3. Gupta, R., Q. K. Beg, and P. Lorenz (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol. Biotechnol. 59: 15–32.

    Article  CAS  Google Scholar 

  4. Najafi, M. F., D. Deobagkar, and D. Deobagkar (2005) Potential application of protease isolated from Pseudomonas aeruginosa PD100. Electron. J. Biotechnol. 8: 197–203.

    Article  CAS  Google Scholar 

  5. Hameed, A., T. Keshavarz, and C. S. Evans (1999) Effect of dissolved oxygen tension and pH on the production of extracellular protease from a new isolate of Bacillus subtilis K2, for use in leather processing. J. Chem. Technol. Biotechnol. 74: 5–8.

    Article  CAS  Google Scholar 

  6. Dayanandan, A., J. Kanagaraj, L. Sounderraj, R. Govindaraju, and G. S. Rajkumar (2003) Application of an alkaline protease in leather processing: an ecofriendly approach. J. Cleaner Prod. 11: 533–536.

    Article  Google Scholar 

  7. Fujiwara, N., K. Yamamoto, and A. Masui (1991) Utilization of a thermostable alkaline protease from an alkalophilic thermophile for the recovery of silver from used X-ray film. J. Ferment. Bioeng. 72: 306–308.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Neklyudov, A. D., A. N. Ivankin, and A. V. Berdutina (2000) Properties and uses of protein hydrolysates (review). Appl. Biochem. Microbiol. 36: 452–459.

    Article  Google Scholar 

  10. Pedersen, N. R., R. Wimmer, R. Matthiesen, L. H. Pedersen, and A. Gessesse (2003) Synthesis of sucrose laurate using a new alkaline protease. Tetrahedron Asymmetry 14: 667–673.

    Article  CAS  Google Scholar 

  11. Morihara, K. (1974) Comparative specificity of microbial proteinases. Adv. Enzymol. Relat. Areas Mol. Biol. 41: 179–243.

    Article  CAS  Google Scholar 

  12. Suh, H. J. and H. K. Lee (2001) Characterization of a keratinolytic serine protease from Bacillus subtilis KS-1. J. Protein Chem. 20: 165–169.

    Article  CAS  Google Scholar 

  13. Gessesse, A., R. Hatti-Kaul, B. A. Gashe, and B. Mattiasson (2003) Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather. Enzyme Microb. Technol. 32: 519–524.

    Article  CAS  Google Scholar 

  14. Ryu, K., J. Kim, and J. S. Dordick (1994) Catalytic properties and potential of an extracellular protease from an extreme halophile. Enzyme Microb. Technol. 16: 266–275.

    Article  CAS  Google Scholar 

  15. Studdert, C. A., R. E. De Castro, K. H. Seitz, and J. J. Sanchez (1997) Detection and preliminary characterization of extracellular proteolytic activities of the haloalkaliphilic archaeon Natronococcus occultus. Arch. Microbiol. 168: 532–535.

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Alva, V. A. and B. M. Peyton (2003) Phenol and catechol biodegradation by the haloalkaliphile Halomonas campisalis: influence of pH and salinity. Environ. Sci. Technol. 37: 4397–4402.

    Article  CAS  Google Scholar 

  19. Sanchez-Porro, C., E. Mellado, C. Bertoldo, G. Antranikian, and A. Ventosa (2003) Screening and characterization of the protease CP1 produced by the moderately halophilic bacterium Pseudoalteromonas sp. strain CP76. Extremophiles 7: 221–228.

    CAS  Google Scholar 

  20. Lama, L., I. Romano, V. Calandrelli, B. Nicolaus, and A. Gambacorta (2005) Purification and characterization of a protease produced by an aerobic haloalkaliphilic species belonging to the Salinivibrio genus. Res. Microbiol. 156: 478–484.

    Article  CAS  Google Scholar 

  21. Jogi, C., R. H. Joshi, M. S. Dodia, and S. P. Singh (2005) Extracellular alkaline protease from haloalkaliphilic bacteria isolated from sea water along coastal Gujarat. J. Cell Tissue Res. 5: 439–444.

    CAS  Google Scholar 

  22. Patel, R. K., M. S. Dodia, and S. P. Singh (2005) Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: Production and optimization. Process Biochem. 40: 3569–3575.

    Article  CAS  Google Scholar 

  23. Patel, R. K., M. S. Dodia, R. H. Joshi, and S. P. Singh (2006) Production of extracellular halo-alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. isolated from seawater in Western India. World J. Microbiol. Biotechnol. 22: 375–382.

    Article  CAS  Google Scholar 

  24. Dodia, M. S., R. H. Joshi, R. K. Patel, and S. P. Singh (2006) Characterization and stability of extracellular alkaline proteases from halophilic and alkaliphilic bacteria isolated from saline habitat of coastal Gujarat, India. Braz. J. Microbiol. 37: 276–282.

    Article  CAS  Google Scholar 

  25. Dodia, M. S., C. M. Rawal, H. G. Bhimani, R. H. Joshi, S. K. Khare, and S. P. Singh (2008) Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6. J. Ind. Microbiol. Biotechnol. 35: 121–131.

    Article  CAS  Google Scholar 

  26. Hagihara, B. (1958) The Enzymes. Vol. 4. Academic Press Inc., New York, NY, USA.

    Google Scholar 

  27. Kole, M. M., I. Draper, and D. F. Gerson (1988) Production of protease by Bacillus subtilis using simultaneous control of glucose and ammonium concentrations. J. Chem. Technol. Biotechnol. 41: 197–206.

    CAS  Google Scholar 

  28. Beg, Q. K., R. K. Saxena, and R. Gupta (2002) Derepression and subsequent induction of protease synthesis by Bacillus mojavensis under fed-batch operations. Process Biochem. 37: 1103–1109.

    Article  CAS  Google Scholar 

  29. Gupta, R., Q. K. Beg, S. Khan, and B. Chauhan (2002) An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Appl. Microbiol. Biotechnol. 60: 381–395.

    Article  CAS  Google Scholar 

  30. Varela, H., M. D. Ferrari, L. Belobradjic, R. Wehrauch, and M. L. Loperena (1996) Effect of medium composition on the production by a new Bacillus subtilis isolate of protease with promising unhairing activity. World J. Microbiol. Biotechnol. 12: 643–645.

    Article  CAS  Google Scholar 

  31. Nehete, P. N., V. D. Shah, and R. M. Kothari (1985) Profiles of alkaline protease production as a function of composition of the slant, age, transfer and isolate number and physiological state of culture. Biotechnol. Lett. 7: 413–418.

    Article  CAS  Google Scholar 

  32. Hamada, T. (1998) Use of gyrB gene and 16S rRNA sequence analysis to investigate phylogeny of halophilic bacterium MBIC3303. NCBI (Unpublished data).

  33. Martins, R. F., W. Davids, W. Abu Al-Soud, F. Levander, P. Radstrom, and R. Hatti-Kaul (2001) Starchhydrolyzing bacteria from Ethiopian soda lakes. Extremophiles 5: 135–144.

    Article  CAS  Google Scholar 

  34. Bïrbïr, M. and C. Sesal (2003) Extremely halophilic bacterial communities in Şereflikoçhisar Salt Lake in Turkey. Turk. J. Biol. 27: 7–22.

    Google Scholar 

  35. Hirasawa, K., K. Uchimura, M. Kashiwa, W. D. Grant, S. Ito, T. Kobayashi, and K. Horikoshi (2006) Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens. Antonie Van Leeuwenhoek 89: 211–219.

    Article  CAS  Google Scholar 

  36. Chu, I. M., C. Lee, and T. S. Li (1992) Production and degradation of alkaline protease in batch cultures of Bacillus subtilis ATCC 14416. Enzyme Microb. Technol. 14: 755–761.

    Article  CAS  Google Scholar 

  37. Puri, S., Q. K. Beg, and R. Gupta (2002) Optimization of alkaline protease production from Bacillus sp. by response surface methodology. Curr. Microbiol. 44: 286–290.

    Article  CAS  Google Scholar 

  38. Uyar, F. and Z. Baysal (2004) Production and optimization of process parameters for alkaline protease production by a newly isolated Bacillus sp. under solid state fermentation. Process Biochem. 39: 1893–1898.

    Article  CAS  Google Scholar 

  39. Stepanov, V. M., G. N. Rudenskaya, L. P. Revina, Y. B. Gryaznova, E. N. Lysogorskaya, I. Y. Filippova, and I. I. Ivanova (1992) A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins. Biochem. J. 285: 281–286.

    CAS  Google Scholar 

  40. Tian, X., Y. Xu, H. Liu, and P. Zhou (1997) New species of Natronobacterium. Wei Sheng Wu Xue Bao 37: 1–6.

    CAS  Google Scholar 

  41. Romano, I., A. Giordano, L. Lama, B. Nicolaus, and A. Gambacorta (2005) Halomonas campaniensis sp. nov., a haloalkaliphilic bacterium isolated from a mineral pool of Campania Region, Italy. Syst. Appl. Microbiol. 28: 610–618.

    Article  CAS  Google Scholar 

  42. O’Reilly, T. and D. F. Day (1983) Effect of cultural conditions on protease production by Aeromonas hydrophila. Appl. Environ. Microbiol. 45: 1132–1135.

    Google Scholar 

  43. Mao, W., R. Pan, and D. Freedman (1992) High production of alkaline protease by Bacillus licheniformis in a fed-batch fermentation using a synthetic medium. J. Ind. Microbiol. 11: 1–6.

    Article  CAS  Google Scholar 

  44. Chaphalkar, S. and S. Dey (1994) Some aspects of production of extracellular protease from Streptomyces diastaticus. J. Microbiol. Biotechnol. 9: 85–100.

    CAS  Google Scholar 

  45. Chauhan, B. and R. Gupta (2004) Application of statistical experimental design for optimization of alkaline protease production from Bacillus sp. RGR-14. Process Biochem. 39: 2115–2122.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  47. Anwar, A. and M. Saleemuddin (1998) Alkaline proteases: A review. Bioresour. Technol. 64: 175–183.

    Article  CAS  Google Scholar 

  48. Kunert, J. and P. Kopecek (2000) Multiple forms of the serine protease Alp of Aspergillus fumigatus. Mycoses 43: 339–347.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Laboratory of Marine Microbiology, Department of Biosciences, Saurashtra University, Rajkot, 360 005, Gujarat, India

    R. H. Joshi, M. S. Dodia & S. P. Singh

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  1. R. H. Joshi
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  2. M. S. Dodia
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  3. S. P. Singh
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Correspondence to S. P. Singh.

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Joshi, R.H., Dodia, M.S. & Singh, S.P. Production and optimization of a commercially viable alkaline protease from a haloalkaliphilic bacterium. Biotechnol Bioproc E 13, 552–559 (2008). https://doi.org/10.1007/s12257-007-0211-9

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  • DOI: https://doi.org/10.1007/s12257-007-0211-9

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