TY - JOUR

T1 - Comparison of the Biochemical Properties of Recombinant Alpaca (Vicugna pacos) Chymosins Produced in Pro- and Eukaryotic Expression Systems

AU - Belenkaya, S. V.

AU - Elchaninov, V. V.

AU - Chirkova, V. Y.

AU - Shcherbakov, D. N.

N1 - The work was performed with the financial support of the Ministry of Science and Higher Education of the Russian Federation (agreement dated October 12, 2021 no. 075-15-2021-1355) within the framework of the implementation of certain measures of the Federal Scientific and Technical Program for the Development of Synchrotron and Neutron Research and Research Infrastructure for 2019–2027. Публикация для корректировки.

PY - 2023/10

Y1 - 2023/10

N2 - Based on the yeast Kluyveromyces lactis, a strain-producer of recombinant alpaca (Vicugna pacos) prochymosin has been developed. A comparative analysis of the biochemical properties of recombinant alpaca chymosin obtained in the expression systems of K. lactis and Escherichia coli has been carried out. It was found that the recombinant alpaca chymosin synthesized in K. lactis exceeds the analogue obtained in E. coli by 12.9 times in the turnover number of the enzyme, 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 increase of 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.

AB - Based on the yeast Kluyveromyces lactis, a strain-producer of recombinant alpaca (Vicugna pacos) prochymosin has been developed. A comparative analysis of the biochemical properties of recombinant alpaca chymosin obtained in the expression systems of K. lactis and Escherichia coli has been carried out. It was found that the recombinant alpaca chymosin synthesized in K. lactis exceeds the analogue obtained in E. coli by 12.9 times in the turnover number of the enzyme, 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 increase of 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.

KW - Kluyveromyces lactis

KW - Michaelis-Menten kinetics

KW - biochemical properties

KW - eukaryotic expression system

KW - prokaryotic expression system

KW - recombinant alpaca chymosin

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85173776641&origin=inward&txGid=443f24ed6b1bcacf58629594a524b670

UR - https://www.mendeley.com/catalogue/e1b0169f-eb39-3167-9f3d-9d445b48ccde/

U2 - 10.1134/S0003683823050034

DO - 10.1134/S0003683823050034

M3 - Article

VL - 59

SP - 630

EP - 635

JO - Applied Biochemistry and Microbiology

JF - Applied Biochemistry and Microbiology

SN - 0003-6838

IS - 5

ER -