Abstract

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

S. V. Belenkaya^1,2,a*, A. A. Bondar³, T. A. Kurgina^2,3, V. V. Elchaninov⁴, A. Yu. Bakulina^1,2, E. A. Rukhlova², O. I. Lavrik³, A. A. Ilyichev¹, and D. N. Shcherbakov^1,5

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

²Novosibirsk State University, 630090 Novosibirsk, Russia

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

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

⁵Altai State University, 656049 Barnaul, Russia

^* To whom correspondence should be addressed.

Received May 5, 2020; Revised May 5, 2020; Accepted May 19, 2020
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 Escherichia coli [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 (K_m), turnover number (k_cat), and catalytic efficiency (k_cat/K_m) 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.
KEY WORDS: recombinant chymosin, milk-clotting activity, thermal stability, Michaelis–Menten kinetics parameters
DOI: 10.1134/S0006297920070068

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

S. V. Belenkaya1,2,a*, A. A. Bondar3, T. A. Kurgina2,3, V. V. Elchaninov4, A. Yu. Bakulina1,2, E. A. Rukhlova2, O. I. Lavrik3, A. A. Ilyichev1, and D. N. Shcherbakov1,5

S. V. Belenkaya^1,2,a*, A. A. Bondar³, T. A. Kurgina^2,3, V. V. Elchaninov⁴, A. Yu. Bakulina^1,2, E. A. Rukhlova², O. I. Lavrik³, A. A. Ilyichev¹, and D. N. Shcherbakov^1,5