Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4

  • 236 Accesses

  • 25 Citations

Abstract

The milk-clotting enzyme from Bacillus amyloliquefaciens D4 was purified to 17.2-fold with 20% recovery by precipitation in ammonium sulfate and ion-exchange chromatography. The molecular mass of the enzyme was 58.2 kDa as determined by SDS-PAGE, and it was proved to be a metalloprotease by EDTA inhibition. The enzyme was active in the pH range 5.5–7.0 and was inactivated completely by heating at 55 °C for 20 min. The highest level of enzyme activity was obtained at 65 °C, pH 5.5, in the presence of 25mM CaCl2. The milk-clotting activity was inhibited only slightly by Na+ and K+ and significantly by Cu2+, Zn2+ and Sn2+. The K m value of this enzyme was 0.471 mg/mL. The high level of milk-clotting activity coupled with a low level of thermal stability suggested that the milk-clotting enzyme from B. amyloliquefaciens D4 should be considered as a potential substitute for calf rennet.

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

References

  1. 1.

    S. Kumar, N. S. Sharma, M. R. Saharan and R. Singh, Process. Biochem., 40, 1701 (2005).

  2. 2.

    M. T. H. Cavalcanti, M. F. S. Teixeira, J. L. Lima Filho and A. L. F. Porto, Bioresour. Technol., 93, 29 (2004).

  3. 3.

    S. Raposo and A. Domingos, Process. Biochem., 43, 139 (2008).

  4. 4.

    J. M. Ageitos, J. A. Vallejo, A. B. F. Sestelo, M. Poza and T.G. Villa, J. Appl. Microbiol., 103, 2205 (2007).

  5. 5.

    S. Neelakantan, A.K. Mohanty and J.K. Kaushik, Curr. Sci., 77, 143 (1999).

  6. 6.

    J. D. Breccia, F. Siñeriz, M.D. Baigori, G. R. Castro and R. Hatti-Kaul, Enzyme Microb. Technol., 22, 42 (1998).

  7. 7.

    H. Chung and F. Friedberg, Biochem. J., 180, 387 (1985).

  8. 8.

    N. P. Balaban, L.A. Malikova, A. M. Mardanova, G.N. Rudenskaya and M. R. Sharipova, Biochemistry (Moscow), 4, 459 (2007).

  9. 9.

    Y.-J. Lee, B.-K. Kim, B.-H. Lee, K.-I. Jo, N.-K. Lee, C.-H. Chung, Y.-C. Lee and J.-W. Lee, Bioresour. Technol., 99, 378 (2008).

  10. 10.

    U. Laemmli, Nature, 227, 680 (1970).

  11. 11.

    M.M. Bradford, Anal. Biochem., 72, 248 (1976).

  12. 12.

    K. Arima, J. Yu and S. Iwasaki, Methods. Enzymol., 19, 446 (1970).

  13. 13.

    H. Lineweaver and D. Burk, J. Am. Chem. Soc., 56, 658 (1934).

  14. 14.

    A. Nouani, N. Belhamiche, R. Slamani, S. Belbraouet, F. Fazouane and M. M. Bellal, Int. J. Dairy. Technol., 112 (2009).

  15. 15.

    S. Preetha and R. Boopathy, World. J. Microbiol. Biotechnol., 13, 573 (1997).

  16. 16.

    J. L. Sardinas, Appl. Microbiol., 16, 248 (1968).

  17. 17.

    A.G. Vishwanatha, Rao. Appu and S.A. Singh, Appl. Microbiol. Biotechnol., 1, 1 (2009).

  18. 18.

    A. Magda, EI Bendary, E. Maysa, H. Moharam and H.A. Thanaa, J. Appl. Sci. Res., 3, 695 (2007).

  19. 19.

    L.B. Areces, M. B. D. J. Bonino, M. A. A. Parry, E. R. Fraile, H.M. Fernández and O. Cascone, Appl. Biochem. Biotechnol., 37, 283 (1992).

  20. 20.

    A. M. Hashem, Bioresour. Technol., 75, 219 (2000).

  21. 21.

    M. Yasuda, M. Aoyama, M. Sakaguchi, K. Nakachi and N. Kobamoto, Appl. Microbiol. Biotechnol., 51, 474 (1999).

  22. 22.

    M. Z. Sternberg, J. Dairy. Sci., 54, 159 (1971).

  23. 23.

    Y. Wang, Q. Cheng, Z. Ahmed, X. Jiang and X. Bai, Korean. J. Chem. Eng., 1, 1 (2009).

  24. 24.

    M. J. Sousa, Y. Ardo and P. L.H. McSweeney, Int. Dairy. J., 11, 327 (2001).

  25. 25.

    M. Harboe and P. Budtz, Technology of Cheesemaking. In: Law, Barry A. Ed., New York, Academic Press (1999).

  26. 26.

    H. S. Kim, M.D. Legoy and D. Thomas, Korean J. Chem. Eng., 1, 35 (1989).

  27. 27.

    H.M. Fernandez-Lahore, R. M. Auday, E. R. Fraile, M. Biscoglio de Jimenez Bonino, L. Pirpignani, C. Machalinski and O. Cascone, J. Peptide Res., 53, 599 (1999).

  28. 28.

    S.G. Anema, S. Kim Lee and H. Klostermeyer., LWT-Food Sci. Technol., 40, 99 (2007).

  29. 29.

    C. Merheb-Dini, E. Gomes, M. Boscolo and R. da Silva, Food Chem., 120, 87 (2010).

  30. 30.

    M. S. Pires, G. A. Orellana and C.A. Gatti, Food Hydrocolloids, 13, 235 (1999).

  31. 31.

    H. Matta and V. Punj, Int. J. Food Microbiol., 42, 139 (1998).

  32. 32.

    M. R. Payne and K. R. Morison, Int. Dairy. J., 9, 887 (1999).

  33. 33.

    H. Yamazaki, Y. Ohnishi and S. Horinouchi. Published Only in Database of NCBI (1999).

  34. 34.

    G.L. Gray, K. Hayenga, D. Cullen, L. J. Wilson and S. Norton, Gene., 48, 41 (1986).

  35. 35.

    H. Shimada, M. Honjo, I. Mita, A. Nakayama, A. Akaoka, K. Manabe and Y. Furutani, J. Biotechnol., 2, 75 (1985).

Download references

Author information

Correspondence to Bozhong Gan.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

He, X., Ren, F., Guo, H. et al. Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4. Korean J. Chem. Eng. 28, 203–208 (2011). https://doi.org/10.1007/s11814-010-0347-8

Download citation

Key words

  • Milk-clotting Enzyme
  • Rennet
  • Purification
  • Enzyme Properties
  • Bacillus amyloliquefaciens