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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

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 (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) 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