Biochemical properties of commercial recombinant chymosin preparations

This study investigates the biochemical properties of commercial recombinant chymosin samples available on the Russian market. One of the most critical steps in the production of natural cheese is the coagulation of milk into a curd under the action of rennet or other milk-clotting enzymes. While milk coagulation can be induced by various proteases capable of hydrolyzing κ-casein, the quality of the final product significantly depends on the biochemical properties of the enzyme. These properties influence the proteolytic processes occurring in the cheese from production to maturation. In this work, we examined the effects of hydrogen ions, calcium, and temperature on the specific (milk-clotting) activity of the enzyme preparations. The activity was determined in accordance with the industry standard OST 10288-2001 (“Milk-Clotting Enzyme Preparations”). The non-specific (proteolytic) activity was measured using a standard method of E.D. Kaverzneva, adapted for laboratory studies of milk-clotting enzymes. The results demonstrated that the biochemical properties of the tested commercial recombinant chymosin samples were equivalent to those of the industry reference standard for rennet. This finding supports their suitability for producing cheeses with a high second heating temperature, for which the conventional technology uses rennet. In Russia, the production of genetically engineered chymosins is not currently undertaken. Consequently, the development of domestic production technologies for recombinant chymosin remains highly relevant. Such studies are being conducted by the Siberian Research Institute of Cheese Making in collaboration with the State Research Center of Virology and Biotechnology “Vector” (Rospotrebnadzor).

1. Musina O.N., Grishkova A.V., Prosekov A.Yu. Domestic self-sufficiency in milk-converting enzymes. Dairy Industry. 2024;1:24-27. (In Russian). DOI: 10.21603/1019-8946-2024-1-10. EDN: KINKYI.

2. Sviridenko G.M. Requirements to the bacterial starters for fermented milk products manufacturing. Cheesemaking and buttermaking. 2014;4:24-27. (In Russian). EDN: SJDTGT.

3. Abramov D.V., Myagkonosov D.S., Delitskaya I.N., Mordvinova V.A., Municheva T.E., Ovchinnikova E.G. Perspectives of using complex milk-clotting enzyme preparations for ripening rennet cheeses production. Cheesemaking and buttermaking. 2019;1:24-26. (In Russian). DOI: 10.31515/2073-4018-2019-1-24-26. EDN: YYIIWT.

4. Abramov D.V., Myagkonosov D.S., Mordvinova V.A., Delitskaya I.N., Ovchinnikova E.G. Current market trends of milk-coagulating enzyme preparations. Cheesemaking and buttermaking. 2018;6:7-11. (In Russian). EDN: YZKXZR.

5. Myagkonosov D.S., Abramov D.V., Delitskaya I.N., Ovchinnikova E.G. Proteolytic activity of milk-clotting enzymes of different origin. Food Systems. 2022;5(1):47-54. (In Russian). DOI: 10.21323/2618-9771-2022-5-1-47-54. EDN: FKAOJB.

6. Nestorovski T., Velkoska – Markovska L., Srbinovska S., Miskoska – Milevska E., Petanovska – Ilievska B., Popovski Z.T. Different approaches in analyzing chymosin purity. Journal of Agricultural, Food and Environmental Sciences. 2019;73(3):24-29. DOI: 10.55302/JAFES19733024n.

7. Kurbanova M.G., Bondarchuk O.N., Maslennikova S.M. Practical aspects of hydrolysis of milk casein by endopeptidases. Food Processing: Techniques and Technology. 2013;2:34-39. (In Russian). EDN: QANQOF.

8. Illarionova E.E., Kruchinin A.G., Kalugina D.N., Turovskaya S.N. The research on the effect of the milk-clotting ferments of different origin on the milk clots formation. Food Industry. 2021;9:27-29. (In Russian). DOI: 10.52653/PPI.2021.9.9.008. EDN: HPZWKT.

9. Kruchinin A.G., Illarionova E.E. Fermented milk concentrates structural and mechanical characteristics comparison. Bulletin of KSAU. 2022;3:162-170. (In Russian). DOI: 10.36718/1819-4036-2022-3-162-170. EDN: SNYSSA.

10. Belenkaya S.V., Shcherbakov D.N., Chapoval A.I., Esina T.I., Elchaninov V.V. The effect of thioredoxin and prochymosin coexpression on the refolding of recombinant alpaca chymosin. Vavilov Journal of Genetics and Breeding. 2023;27(4):421-427. DOI: 10.18699/VJGB-23-50. EDN: CCPBRD.

11. Hayat S.M.G., Farahani N., Golichenari B., Sahebkar A. Recombinant protein expression in Escherichia coli (E. coli): what we need to know. Current Pharmaceutical Design. 2018;24(6):718-725. DOI: 10.2174/1381612824666180131121940.

12. Rudometov A.P., Belenkaya S.V., Shcherbakov D.N., Balabova D.V., Krieger A.V., Elchaninov V.V. Study of enzymatic stability of the liquid preparations of the recombinant bovine (Bos taurus taurus L.) chymosin. Cheesemaking and buttermaking. 2017;6:40-43. (In Russian). EDN: ZUDMCR.

13. Myagkonosov D.S., Abramov D.V., Delitskaya I.N., Ovchinnikova E.G. Proteolytic activity of milk-clotting enzymes of different origin. Food Systems. 2022;5(1):47-54. (In Russian). DOI: 10.21323/2618-9771-2022-5-1-47-54. EDN: FKAOJB.

14. Sankar Р.L., Cho М.K. Engineering values into genetic engineering: a proposed analytic framework for scientific social responsibility. The American Journal of Bioethics. 2015;15(12):18-24. DOI: 10.1080/15265161.2015.1104169.

15. Vallejo J.A., Ageitos J.M., Poza M., Villa T.G. Short communication: a comparative analysis of recombinant chymosins. Journal of Dairy Science. 2012;95(2):609-613. DOI: 10.3168/jds.2011-4445.

16. Kaverzneva E.D. Standard method for determining proteolytic activity for complex protease preparations. Prikladnaya biokhimiya i mikrobiologiya. 1971;7(2):225-228. (In Russian).

17. Shcherbakov D.N., Rudometov A.P., Elchaninov V.V., Belenkaya S.V., Kriger A.V., Ilichev A.A. pET21a-ProChym recombinate plasmid, providing synthesis of chemeric B Bos taurus proximosin protein, as well as Escherichia coli BL21(DE3)pLysE pET21a-ProChym – producer of chemeric B Bos taurus proximosin protein. Patent RF, no. 2670071; 2018. (In Russian).

18. Taheri-Kafrani A., Kharazmi S., Nasrollahzadeh M., Soozanipour A., Ejeian F., Etedali P., et al. Recent developments in enzyme immobilization technology for high-throughput processing in food industries. Critical Reviews in Food Science and Nutrition. 2021;61(19):3160-3196. DOI: 10.1080/10408398.2020.1793726.

19. Lepilkina O.V., Grigorieva A.I. Enzymatic proteolysis during the conversion of milk into cheese. Food Systems. 2023;6(1):36-45. (In Russian). DOI: 10.21323/2618-9771-2023-6-1-36-45. EDN: MYSJFF.

20. Dimitrov B.G. Special issues of “Mozarella” type cheeses manufacturing. Cheesemaking and buttermaking. 2016;2:34-35. (In Russian). EDN: VQDGUN.