Comparison of Biochemical Properties of Recombinant Alpaca ( Vicugna pacos ) Chymosins Produced in Pro- and Eukaryotic Expression Systems

S. V. Belenkaya; Беленькая С. В.; V. V. Elchaninov; Ельчанинов В. В.; V. Y. Chirkova; Чиркова В. Ю.; D. N. Shcherbakov; Щербаков Д. Н.

doi:10.31857/S0555109923050033

Comparison of Biochemical Properties of Recombinant Alpaca (Vicugna pacos) Chymosins Produced in Pro- and Eukaryotic Expression Systems

Authors: Belenkaya S.V.¹^,2, Elchaninov V.V.³, Chirkova V.Y.⁴, Shcherbakov D.N.¹^,4
Affiliations:
1. State Research Center of Virology and Biotechnology “Vector”
2. Novosibirsk State University
3. Federal Altai Scientific Center for Agrobiotechnologies, Siberian Research Institute of Cheese Making
4. Altai State University
Issue: Vol 59, No 5 (2023)
Pages: 465-471
Section: Articles
URL: https://journals.rcsi.science/0555-1099/article/view/138813
DOI: https://doi.org/10.31857/S0555109923050033
EDN: https://elibrary.ru/NMCTLZ
ID: 138813

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Abstract

Based on the yeast Kluyveromyces lactis, a strain-producer of recombinant alpaca prochymosin (Vicugna pacos) was developed. A comparative analysis of the biochemical properties of recombinant alpaca chymosin obtained in the expression systems of K. lactis and Escherichia coli was carried out. It was found that the recombinant alpaca chymosin synthesized in K. lactis exceeds the analog obtained in E. coli by 12.9 times in the number of enzyme turnovers, and by 2.9 times in catalytic efficiency. Compared to chymosin expressed in E. coli, the enzyme obtained in a eukaryotic producer has a thermal stability threshold increased by 5°C. Replacing a prokaryotic producer with a eukaryotic one enhances the negative sensitivity of the milk-clotting activity of recombinant alpaca chymosin to an increase in substrate pH in the range of 6.1–6.9, which is accompanied by an increase in the duration of coagulation by 8–35%. With an increase in the concentration of CaCl2 in the substrate, the coagulation activity of the target enzyme synthesized in E. coli was 12–14% higher than that of its analogue obtained in K. lactis.

Keywords

recombinant chymosin alpaca, prokaryotic expression system, eukaryotic expression system, Kluyveromyces lactis, biochemical properties, Michaelis–Menten kinetics

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