E.I. Antonova1, A.B. Achilov2, N.V. Firsova3, D.A. Victorov4, P.S. Torutanov5, N.A. Lengesova6

1–6 Scientific Research Center for Fundamental and Applied Problems of Bioecology and Biotechnology Ulyanovsk State Pedagogical University named after I.N. Ulyanov (Ulyanovsk, Russia)

1 antonov_67@mail.ru, 2 a.achilow@inbox.ru, 3 n-firsova@mail.ru, 4 viktorov.da@gmail.com, 5 pavel.toru2012@yandex.ru, 6 lengesova@yandex.ru

The isolation of natural chymosin is associated with ethical problems and high economic costs. Its genetically engineered analogues have replaced the natural one using recombinant technologies in microorganisms. Camel chymosin is more promising as it has higher thermostability and milk curdling activity, making it attractive for commercial cheese production.

Therefore, the aim of our study was to purify and analyse the enzymatic activity of recombinant chymosin from Camelus dromedarius (rChn-Cam) produced in P.rastoris expression system.

A study on the synthesis, purification and analysis of the enzymatic properties of rChn-Cam Camelus dromedarius in the P.pastoris expression system revealed that strains producing P.pastoris GS115/his4, before and after transformation by shuttle plasmid pPICZ(alpha)B/proCYM_camel_pp_IDT with AOXI promoter, of Mut+ phenotype are more appropriately grown, regardless of the nutrient medium composition, with additional addition of 0.5% methanol as a carbon source. When grown in YPD enriched medium (biotin 0.00004% and 1% glycerol), the optimal zeocin concentration is 50 μg/ml. Purification of recombinant His-Tag - labelled chymosin on Ni-NTA Sepharose (metal-affinity chromatography), with SDS-PAGE, Western-blot and immunoassay revealed its presence on a MALDI-TOF time-of-flight mass spectrometer FLEX series (MALDI-TOF MS) using a MALDI BioTyper panel, with a mass of 35.673 kDa. When the pH of the substrate was increased from 5.0 to 6.5, the coagulation activity of rChn-Cam decreased by 24%, with a thermal inactivation threshold of 40-45°C. The coagulation activity unit of rChn-Cam decreased with increasing concentration of zeocin (50, 100, 200 μg/ml); an opposite dependence of rChn-Cam concentration on the time of onset of substrate coagulation was observed.

Thus the obtained data will allow in practical activity to optimise the obtaining of highly productive strain-producer P.rastoris for further high yield of rChn-Cam and is a good alternative to rChn, which are currently used in the industry for cheese production.

Antonova E.I., Achilov A.B., Firsova N.V., Viktorov D.A., Torutanov P.S., Lengesova N.A. Microbiological synthesis, purification and enzymatic properties of recombinant chymosin Camelus dromedarius in the expression system of Pichia pastoris. Technologies of Living Systems. 2025. V. 22. № 1. Р. 87-98. DOI: https://doi.org/10.18127/j20700997-202501-07 (In Russian).

1. Malik A., Gupta A., Dahiya S. et al. A review on Pichia pastoris: A successful tool for expression of recombinant proteins. The Pharma Innovation Journal. 2021. № 10 (11). P. 550-556.
2. Akishev Z., Aktayeva S., Kiribayeva A. Obtaining of recombinant camel chymosin and testing its milk-clotting activity on cow’s, goat’s, ewes’, camel’s and mare’s milk. Biology. 2022. № 11. Р. e1545.
3. Antonova E.I., Abbyazova A.N., Firsova N.V. i dr. Geneticheskie konstrukcii kak istochnik polucheniya rekombinantnogo himozina. Fundamental'nye i prikladnye issledovaniya po prioritetnym napravleniyam bioekologii i biotekhnologii. Materialy VII Vseross. nauchno-prakt. konferencii s mezhdunar. uchastiem, Ul'yanovsk, 2024 g.. Pod red. E. I. Antonovoj. CHeboksary: Izd-vo «Sreda». 2024. S. 49-55. (in Russian).
4. Balabova D.V., Rudometov A.P., Belenkaya S.V. Biochemical and technological properties of moose (Alces alces) recombinant chymosin. Vavilovskii Zhurnal Genet. (Vavilov. J. Genet. Breed.). 2022. V. 26. P. 240-249.
5. da Silva R.R., Souto T.B., Gonsales da Rosa N. et al. Evaluation of the milk clotting properties of an aspartic peptidase secreted by Rhizopus microspores. Preparative Biochemistry and Biotechnology. 2020. V. 50. P. 226-233.
6. Balabova D.V., Belash E.A., Belenkaya S.V. et al. Biochemical properties of a promising milk-clotting enzyme, Moose (Alces alces) recombinant chymosin. Foods. 2023. № 12 (20). P. e3772. https://pubmed.ncbi.nlm.nih.gov/37893665/
7. Fox P.F., Guinee T.P., Cogan T.M. et al. Enzymatic coagulation of milk. Fundamentals of Cheese Science. Eds.: Springer: New York. NY, USA. 2017. Chap. 7. P. 185-229.
8. González-Velázquez D.A., Mazorra-Manzano M.A., Martínez-Porchas M. et al. Exploring the milk-clotting and proteolytic activities in different tissues of Vallesia glabra: a new source of plant proteolytic enzymes. Applied Biochemistry and Biotechnology. 2021. V. 193. P. 389-404.
9. Sbhatu D.B., Tekle H.T., Tesfamariam K.H. Ficus palmata ForskaL (BELES ADGI) as a source of milk clotting agent: a preliminary research. BMC Research Notes. 2020. V. 13. P. 446.
10. Murashkin D.E., Belen'kaya S.V., Bondar' A.A. i dr. Analiz nekotoryh biohimicheskih svojstv rekombinantnogo himozina sibirskoj kosuli (Capreolus pygargus), poluchennogo v kul'ture kletok mlekopitayushchih (CHO-K1). Biohimiya. 2023. T. 88 (9). S. 1556-1569. (in Russian).
11. Akishev Z., Kiribayeva A., Mussakhmetov A. et al. Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris. Heliyon. 2021. V. 7 (5). P. e07137. https://doi.org/10.1016/j.heliyon.2021.e07137
12. Belenkaya S.V., Elchaninov V.V., Shcherbakov D.N. Development of a producer of recombinant maral chimosine based on the Kluyveromyces lactis yeast. Biotekhnologiya. 2021. V. 37 (5). P. 20-27.
13. Belen'kaya S.V., Balabova D.V., Belov A.N. i dr. Osnovnye biohimicheskie svojstva rekombinantnyh himozinov (obzor). Prikladnaya biohimiya i mikrobiologiya. 2020. T. 56 (4). S. 315-326. (in Russian).
14. Al-Zoreky N.S., Almathen F.S. Using recombinant camel chymosin to make white soft cheese from camel milk. Food Chemistry. 2021. V. 337. P. e127994.
15. Jensen J.L., Mølgaard A., Poulsen J.-C.N. et al. Camel and bovine chymosin: the relationship between their structures and cheese-making properties. Acta Crystallographica Section D: Biological Crystallography. 2013. V. 69. P. 901-913.
16. Obst U., Lu T.K., Sieber V. A modular toolkit for generating Pichia pastoris secretion libraries. ACS Synthetic Biology. 2017. V. 6. P. 1016-1025.
17. Patra P., Das M., Kundu P. et al. Recent advances in systems and synthetic biology approaches for developing novel cell-factories in non-conventional yeasts. Biotechnology Advances. 2021. V. 47. P. e107695. https://pubmed.ncbi.nlm.nih.gov/33465474/
18. Sinicyn A.P., Sinicyna O.A., Rozhkova A.M. Poluchenie promyshlenno vazhnyh fermentov na osnove ekspressionnoj sistemy griba Penicillium verruculosum. Biotekhnologiya. 2020. T. 36 (6). S. 17-34. (in Russian).
19. Saitua F., Torres P., Pérez-Correa J.R. et al. Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris. BMC systems biology. 2017. V. 11. Iss. 1. Article 27. https://bmcsystbiol.biomedcentral.com/articles/10.1186/s12918-017-0408-2
20. Jungo C., Marison I., Von Stockar U. Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures:
    A quantitative study based on concentration gradients in transient continuous cultures. Journal of Biotechnology. 2007. V. 128 (4). P. 824-837.
21. Myagkonosov D.S., Abramov D.V., Delickaya I.N., Ovchinnikova E.G. Proteoliticheskaya aktivnost' molokosvertyvayushchih fermentov raznogo proiskhozhdeniya. Pishchevye sistemy. 2022. T. 5 (1). S. 47-54. (in Russian).
22. Akishev Zh.D., Tursunbekova A.E., Hasenov B.B. Molokosvertyvayushchaya rekombinantnogo verblyuzh'ego himozina. Exp. Biol. 2022. T. 1 (90). S. 39-49. (in Russian).
23. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970. V. 227. P. 680-685.
24. Rudometov A.P., Belen'kaya S.V., Shcherbakov D.N. i dr. Issledovanie fermentativnoj stabil'nosti zhidkih preparatov rekombinantnogo himozina korovy (Bos taurus taurus L). Syrodelie i maslodelie. 2017. № 6. S. 40-43. (in Russian).
25. Warburg O., Christian W.Z. Isolierung und Kristallisation des Garungsterments Enolase. Biochemistry. 1942. V. 310. P. 384-421.
26. Ageitos J.M., Vallejo J.A., Sestelo A.B. et al. Purification and characterization of a milk-clotting protease from Bacillus licheniformis strain USC13. Journal of Applied Microbiology. 2007. V. 103 (6). P. 2205-2213.