Skip to main content
SpringerLink
Log in

Characterization of the Altai Maral Chymosin Gene, Production of a Chymosin Recombinant Analog in the Prokaryotic Expression System, and Analysis of Its Several Biochemical Properties

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

For the first time, the chymosin gene (CYM) of a maral was characterized. Its exon/intron organization was established using comparative analysis of the nucleotide sequence. The CYM mRNA sequence encoding a maral preprochymosin was reconstructed. Nucleotide sequence of the CYM maral mRNA allowed developing an expression vector to ensure production of a recombinant enzyme. Recombinant maral prochymosin was obtained in the expression system of Escherichiacoli [strain BL21 (DE3)]. Total milk-coagulation activity (MCA) of the recombinant maral chymosin was 2330 AU/ml. The recombinant maral prochymosin relative activity was 52955 AU/mg. The recombinant maral chymosin showed 100-81% MCA in the temperature range 30-50°C, thermal stability (TS) threshold was 50°C, and the enzyme was completely inactivated at 70°C. Preparations of the recombinant chymosin of a single-humped camel and recombinant bovine chymosin were used as reference samples. Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of the recombinant maral chymosin, were 1.18 ± 0.1 µM, 2.68 ± 0.08 s−1 and 2.27± 0.10 µm M−1·s−1, respectively.

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.

Similar content being viewed by others

Abbreviations

Chn:

rChn, chymosin and recombinant chymosin

CYM :

chymosin gene

κ-CS:

κ-casein

ME:

milk-clotting enzyme

MEP:

milk-clotting enzyme preparation

rProChn:

recombinant prochymosin

rChn Bos:

recombinant bovine chymosin

rChn Cam:

recombinant single-humped camel chymosin

TS:

thermal stability

REFERENCES

  1. Harboe, M., Broe, M. L., and Qvist, K. B. (2010) The production, action and application of rennet and coagulants, in Technology of Cheesemaking (Law, B. A., and Tamime, A. Y., eds.) 2nd Edn., pp. 98-129.

  2. Belov, A. N., Koval, A. D., Avdonina, E. A., and Yelchaninov, V. V. (2009) Influence of milk-converting preparation on organoleptic indicators of cheese, Cheese-Making and Butter-Making, 1, 22-24.

    Google Scholar 

  3. Jacob, M., Jaros, D., and Rohm, H. (2011) Recent advances in milk clotting enzymes, Int. J. Dairy Technol., 64, 14-33.

    Article  CAS  Google Scholar 

  4. Feijoo-Siota, L., Blasco, L., Rodriguez-Rama, J. L., Barros-Velázquez, J., de Miguel, T., Sánchez-Pérez, A., and Villa, T. G. (2014) Recent patents on microbial proteases for the dairy industry, Recent Adv. DNA Gene Seq., 8, 44-55.

    CAS  PubMed  Google Scholar 

  5. Kimberlin, R. H. (1996). Bovine spongiform encephalopathy and public health: some problems and solutions in assessing the risk, in 3rd International Symposium on Transmissible Subacute Spongiform Encephalopathies: Prion Diseases, Paris (Court, L., and Dodet, B., eds.) Elsevier, Amsterdam, pp. 487-502.

  6. Lebedev, L. R., Kosogova, T. A., Teplyakova, T. V., Kriger, A. V., Elchaninov, V. V., Belov, A. N., and Koval, A. D. (2016) Study of technological properties of milk-clotting enzyme from Irpex lacteus (Irpexlacteus (fr.) fr.), Foods Raw Mater., 4, 58-65.

    Article  CAS  Google Scholar 

  7. Yelchaninov, V. V. (2006) Milk-clotting enzyme from reindeer rennet, Cheese-Making and Butter-Making, 4, 42-44.

    Google Scholar 

  8. Yelchaninov, V. V. (2006) Investigation of a milk-clotting enzyme from reindeer rennet, Author’s abstract of Candidate’s dissertation, Kemerovo technological Institute of food industry, Kemerovo, p. 172.

  9. Uchiyama, H., Uozumi, T., Beppu, T., and Arima, K. (1980) Purification of prorennin mRNA and its translation in vitro, Agricult. Biol. Chem., 44, 1373-1381.

    CAS  Google Scholar 

  10. Flamm, E. L. (1991) How FDA approved chymosin: a case history, Biotechnology, 9, 349.

    CAS  PubMed  Google Scholar 

  11. Pungercar, J., Strukelj, B., Gubensek, F., Turk, V., and Kregar, I. (1990) Complete primary structure of lamb preprochymosin deduced from cDNA, Nucleic Acids Res., 18, 4602.

    Article  CAS  Google Scholar 

  12. Rogelj, I., Perko, B., Francky, A., Penca, V., and Pungerčar, J. (2001) Recombinant lamb chymosin as an alternative coagulating enzyme in cheese production, J. Dairy Sci., 84, 1020-1026.

    Article  CAS  Google Scholar 

  13. Vega-Hernández, M. C., Gómez-Coello, A., Villar, J., and Claverie-Martı́n, F. (2004) Molecular cloning and expression in yeast of caprine prochymosin, J. Biotechnol., 114, 69-79.

    Article  Google Scholar 

  14. Eskandari, M. H., Hosseini, A., and Aminlari, M. (2008) Nucleotide sequence of cDNA encoding for preprochymosin in native goat (Caprahircus) from Iran, Iranian J. Veterinary Res., 9, 262-265.

    Google Scholar 

  15. Kappeler, S., Farah, Z., van den Brink, J. M., Rahbek-Nielsen, H., and Budtz, P. (2016) U.S. Patent No. 9,307,775, Washington, DC, U.S. Patent and Trademark Office.

  16. Kappeler, S. R., Rahbek-Nielsen, H., Farah, Z., Puhan, Z., Hansen, E. B., and Johansen, E. (2006) Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk, Biochem. Biophys. Res. Commun., 342, 647-654.

    Article  CAS  Google Scholar 

  17. Ersöz, F., and İnan, M. (2019) Large-scale production of yak (Bos grunniens) chymosin A in Pichia pastoris, Protein Expr. Purif., 154, 126-133.

    Article  Google Scholar 

  18. Luo, F., Jiang, W. H., Yang, Y. X., Li, J., and Jiang, M. F. (2016) Cloning and expression of yak active chymosin in Pichia pastoris, Asian-AustralasJ. Anim. Sci., 29, 1363.

    CAS  Google Scholar 

  19. Vallejo, J. A., Ageitos, J. M., Poza, M., and Villa, T. G. (2008) Cloning and expression of buffalo active chymosin in Pichia pastoris, J. Agric. Food Chem., 56, 10606-10610.

    Article  CAS  Google Scholar 

  20. Belenkaya, S. V., Rudometov, A. P., Shcherbakov, D. N., Balabova, D. V., Krieger, A. V., Belov, A. N., Koval, A. D., and Yelchaninov, V. V. (2018) Some biochemical properties of recombinant Alpaca chymosin (Vicugna pacos L.), Appl. Biochem. Microbiol., 54, 585-593.

    Article  Google Scholar 

  21. Vallejo, J. A., Ageitos, J. M., Poza, M., and Villa, T. G. (2012) A comparative analysis of recombinant chymosins, J. Dairy Sci., 95, 609-613.

    Article  CAS  Google Scholar 

  22. Bansal, N., Drake, M. A., Piraino, P., Broe, M. L., Harboe, M., Fox, P. F., and McSweeney, P. L. H. (2009) Suitability of recombinant camel (Camelusdromedarius) chymosin as a coagulant for Cheddar cheese, Int. Dairy J., 19, 510-517.

    Article  CAS  Google Scholar 

  23. Wei, C., Tang, B., Zhang, Y., and Yang, K. (1999) Oxidative refolding of recombinant prochymosin, Biochem. J., 340, 345.

    Article  CAS  Google Scholar 

  24. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680.

    Article  CAS  Google Scholar 

  25. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248-254.

    Article  CAS  Google Scholar 

  26. Rudometov, A. P., Belenkaya, S. V., Shcherbakov, D. N., Balabova, D. V., Krieger, A. V., and Yelchaninov, V. V. (2017) Study of enzymatic stability of the liquid preparations of the recombinant bovine (Bos taurus taurus L.) chymosin, Cheese-Making and Butter-Making, 6, 40-43.

    Google Scholar 

  27. Hall, T. A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT, in Nucleic Acids Symposium Series, Information Retrieval Ltd., London, UK, 41, 95-98.

  28. Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., and Lepore, R. (2018) SWISS-MODEL: homology modelling of protein structures and complexes, Nucleic Acids Res., 46, W296-W303.

    Article  CAS  Google Scholar 

  29. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and Ferrin, T. E. (2004) UCSF Chimera – a visualization system for exploratory research and analysis, J. Comput. Chem., 25, 1605-1612.

    Article  CAS  Google Scholar 

  30. Johnson, K. A., and Goody, R. S. (2011) The original Michaelis constant: translation of the 1913 Michaelis–Menten paper, Biochemistry, 50, 8264-8269.

    Article  CAS  Google Scholar 

  31. Naidu, A., Fitak, R. R., Munguia-Vega, A., and Culver, M. (2011) Novel primers for complete mitochondrial cytochrome b gene sequencing in mammals, Mol. Ecol. Res., 12, 191-196.

    Article  Google Scholar 

  32. Pavlinov, I. Y. (2006) Systematics of Recent Mammals, Moscow Univ. Publ., Moscow.

  33. Shcherbakov, D. N., Rudometov, A. P., Yelchaninov, V. V., Belenkaya, S. V., Krieger, A. V., and Ilichev, A. A. (2006) Recombinant plasmid ret21a-ProChym providing the synthesis of the chimeric protein prochymosin in Bos Taurus, and Escherichia coli strain BL21(DE3)pLysE ret21a-ProChym – producer of the chimeric protein Prochymosin in Bos Taurus, Russian Patent No. 2670071, SPK S12N 9/6483 (2006.01); S12N 15/64 (2006.01); S12N 15/70 (2006.01); S12Y 304/23004 (2006.01).

  34. Pedersen, V. B., Christensen, K. A., and Foltmann, B. (1979) Investigations on the activation of bovine prochymosin, Eur. J. Biochem., 94, 573-580.

    Article  CAS  Google Scholar 

  35. Wang, N., Wang, K. Y., Li, G., Guo, W., and Liu, D. (2015) Expression and characterization of camel chymosin in Pichia pastoris, Protein Expr. Purif., 111, 75-81.

    Article  CAS  Google Scholar 

  36. Jiang, X. P., Yin, M. L., Chen, P., and Yang, Q. (2012) Constitutive expression, purification and characterization of bovine prochymosin in Pichia pastoris GS115, World J. Microbiol. Biotechnol., 28, 2087-2093.

    Article  CAS  Google Scholar 

  37. Noseda, D. G., Recúpero, M. N., Blasco, M., Ortiz, G. E., and Galvagno, M. A. (2013) Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter, Protein Expr. Purif., 9, 235-244.

    Article  Google Scholar 

  38. Jensen, J. L., Mølgaard, A., Navarro Poulsen, J. C., Harboe, M. K., Simonsen, J. B., Lorentzen, A. M., and Larsen, S. (2013) Camel and bovine chymosin: the relationship between their structures and cheese-making properties, Acta Crystallogr. D Biol. Crystallogr., 69, 901-913.

    Article  Google Scholar 

  39. Jensen, J. L., Jacobsen, J., Moss, M. L., Rasmussen, F., Qvist, K. B., Larsen, S., and van den Brink, J. M. (2015) The function of the milk-clotting enzymes bovine and camel chymosin studied by a fluorescence resonance energy transfer assay, J. Dairy Sci., 98, 2853-2860.

    Article  CAS  Google Scholar 

  40. Zhou, X. X., Wang, Y. B., Pan, Y. J., and Li, W. F. (2008) Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins, Amino Acids, 34, 25-33.

    Article  CAS  Google Scholar 

  41. Bobkova, E. V., Gedrovich, A. V., Ankilova, V. N., Lavrik, O. I., Baratova, L. A., and Shishkov, A. V. (1990) Comparative study of phenylalanyl-tRNA synthetases from Escherichia coli and Thermusthermophilus by tritium topography, Biochemistry, 55, 1570-1577.

    CAS  Google Scholar 

Download references

Acknowledgements

The authors are grateful to the Genomics Core Facility (ICBFM SB) of the Siberian Branch of the Russian Academy of Sciences for DNA sequencing.

Funding

The work was financially supported by the Ministry of Science and Higher Education of the Russian Federation (project No. FZMW-2020-0002 “Development of recombinant enzyme producers for cheese making”).

Author information

Authors and Affiliations

  1. State Research Center of Virology and Biotechnology “Vector”, 630559, Koltsovo, Novosibirsk Region, Russia

    S. V. Belenkaya, A. Yu. Bakulina, A. A. Ilyichev & D. N. Shcherbakov

  2. Novosibirsk State University, 630090, Novosibirsk, Russia

    S. V. Belenkaya, T. A. Kurgina, A. Yu. Bakulina & E. A. Rukhlova

  3. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090, Novosibirsk, Russia

    A. A. Bondar, T. A. Kurgina & O. I. Lavrik

  4. Federal Altai Scientific Center of Agrobiotechnologies, Siberian Research Institute of Cheese Making, 656910, Barnaul, Russia

    V. V. Elchaninov

  5. Altai State University, 656049, Barnaul, Russia

    D. N. Shcherbakov

Authors
  1. S. V. Belenkaya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. A. Bondar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T. A. Kurgina
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. V. V. Elchaninov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A. Yu. Bakulina
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. E. A. Rukhlova
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. O. I. Lavrik
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A. A. Ilyichev
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. D. N. Shcherbakov
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S. V. Belenkaya.

Ethics declarations

The authors declare no conflict of interests in financial or any other sphere. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Belenkaya, S., Bondar, A., Kurgina, T. et al. Characterization of the Altai Maral Chymosin Gene, Production of a Chymosin Recombinant Analog in the Prokaryotic Expression System, and Analysis of Its Several Biochemical Properties. Biochemistry Moscow 85, 781–791 (2020). https://doi.org/10.1134/S0006297920070068

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297920070068

Keywords

Search

Navigation