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Purification and characterization of Bacillus cereus protease suitable for detergent industry

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Abstract

An extracellular alkaline protease from an alkalophilic bacterium, Bacillus cereus, was produced in a large amount by the method of extractive fermentation. The protease is thermostable, pH tolerant, and compatible with commercial laundry detergerts. The protease purified and characterized in this study was found to be saperior to endogenous protease already present in commercial laundry detergents. The enzyme was purified to homogeneity by ammonium sulfate precipitation, concentration by ultrafiltration, anionexchange chromatography, and gel filtration. The purified enzyme had a specific activity of 3256.05 U/mg and was found to be amonomeric protein with a molecular mass of 28 and 31 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE, respectively. Its maximum protease activity against casein was found to be at pH 10.5 and 50°C. Proteolytic activity of the enzyme was detected by casein and gelatin zymography, which gave a very clear protease activity zone on gel that corresponded to the band obtained on SDS-PAGE and nondenaturing PAGE with a molecular mass of nearly 31 kDa. The purified enzyme was analyzed through matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and identified as a subtilisin class of protease. Specific serine protease inhibitors, suggesting the presence of serine residues at the active site, inhibited the enzme significantly.

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References

  1. North, M. J. (1982), Microbiol. Rev. 46, 308–340.

    CAS  Google Scholar 

  2. Walsh, K. A. and Wilcox, P. E. (1970), in Methods in Enzymology, vol. 19, Perlmann, G. E. and Lorand, L., eds., Academic, New York, pp. 31–226.

    Google Scholar 

  3. Ward, O. P. (1985), in Comprehensive Biotechnology, vol. 3, Moo-Young, M., ed., Pergamon, Oxford, pp. 789–818.

    Google Scholar 

  4. Kalisz, H. M. (1988), in Advances in Biochemical Engineering/Biotechnology, vol. 36, Fiechter, A., ed., Springer-Verlag, Berlin, pp. 1–65.

    Google Scholar 

  5. Outtrup, H. (1990), in Microbial Enzymes and Biotechnology, Fogarty, W. M. and Kelly, C. T., eds., Elsevier Science, New York, pp. 227–254.

    Google Scholar 

  6. Godfrey, T. and Reichelt, J. (1985), in Industrial Enzymology, Godfrey, T. and West, S., eds., Nature Press, London.

    Google Scholar 

  7. Rao, M. B., Tanksale, A. M., Ghatge, M. S., and Deshpande, V. V. (1998), Microbiol. Mol. Biol. Rev. 62, 597–635.

    CAS  Google Scholar 

  8. Wolff, A. M., Showell, M. S., Venegas, M. G., Barnett, H. L., and Wertz, W. C. (1993), in Subtilisin Enzymes: Practical Protein Enginerring, Boltz, R. and Betzel, C., eds., Plenum, New York, pp. 113–120.

    Google Scholar 

  9. Banerjee, U. C., Sani, R. K., Azmi, W., and Sani, R. (1999), Process Biochem. 35, 213–219.

    Article  CAS  Google Scholar 

  10. Singh, J., Batra, N., and Sobti, R. C. (2001), Process Biochem. 36, 781–785.

    Article  CAS  Google Scholar 

  11. Dhandapani, R. and Vijayaragvan, R. (1994), World J. Microbiol. Biotechnol. 10, 33–35.

    Article  CAS  Google Scholar 

  12. Feng, Y. Y., Yang, W. B., Ong, S. L., Hu, J. Y., and Ng, W. J. (2001), Appl. Microbiol. Biotechnol. 57, 153–160.

    Article  CAS  Google Scholar 

  13. Kobayashi, T., Hakamada, J., Hitomi, J., Koike, K., and Ito, S. (1996), Appl. Microbiol. Biotechnol. 45, 63–71.

    Article  CAS  Google Scholar 

  14. Banik, R. M. and Prakash, M. (2004), Microbiol. Res. 159, 135–140.

    Article  CAS  Google Scholar 

  15. Lee, Y. H. and Chang, H. N. (1990), J. Ferment. Bioeng. 69, 89–92.

    Article  CAS  Google Scholar 

  16. Hotha, S. and Banik, R. M. (1997), J. Chem. Tech. Biotechnol. 69, 5–10.

    Article  CAS  Google Scholar 

  17. Hagihara, B, Matsubara, H., Nakai, M., and Okumuki, K. (1958), J. Biochem. 45, 185–194.

    CAS  Google Scholar 

  18. Bradford, M. M. (1976), Anal. Biochem. 72, 248–254.

    Article  CAS  Google Scholar 

  19. Laemmli, U. K. (1970), Nature 227, 680–685.

    Article  CAS  Google Scholar 

  20. Oliver, G. W., Stetler-Stevenson, W. G., and Kleiner, D. E. (1999), in Proteolytic Enzymes: Tools and Targets, Sterchi, E. and Stöcker, W., eds., Springer-Verlag, Berlin, pp. 63–76.

    Google Scholar 

  21. Leber, T. M. and Balkwill, F. R. (1997), Anal. Biochem. 249, 24–28.

    Article  CAS  Google Scholar 

  22. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996), Anal. Chem. 68 (5), 850–858.

    Article  CAS  Google Scholar 

  23. Jobin, M. C. and Grenier, D. (2003), FEMS Microbiol. Lett. 220, 113–119.

    Article  CAS  Google Scholar 

  24. Thangam, E. B. and Rajkumar, G. S. (2002), Biotechnol. Appl. Biochem. 35, 149–154.

    Article  CAS  Google Scholar 

  25. Kuhn, P., Knapp, S., Michael, S., Ganshaw, G., Thoene, M., and Bott, R. (1998), Biochemistry 37, 13,445–13,452.

    Article  Google Scholar 

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Author notes

  1. Monika Prakash

    Present address: Department of Life Sciences, Mangalmay Institute of Management and Technology, Plot No. 8&9, Knowledge Park-II, Greater Noida, India

Authors and Affiliations

  1. Institute of Biochemistry, Johannes Gutenberg University, Becherweg 30, D-55099, Mainz, Germany

    Monika Prakash & Claudia Koch-Brandt

  2. School of Biochemical Engineering, Institute of Technology, Banaras Hindu University, Varanasi-05, India

    Rathindra Mohan Banik

Authors
  1. Monika Prakash
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  2. Rathindra Mohan Banik
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  3. Claudia Koch-Brandt
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Correspondence to Monika Prakash.

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Prakash, M., Banik, R.M. & Koch-Brandt, C. Purification and characterization of Bacillus cereus protease suitable for detergent industry. Appl Biochem Biotechnol 127, 143–155 (2005). https://doi.org/10.1385/ABAB:127:3:143

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  • DOI: https://doi.org/10.1385/ABAB:127:3:143

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