| Autor: |
Belenkaya SV; State Research Center of Virology and Biotechnology 'Vector', Koltsovo, Novosibirsk Region, 630559, Russia. belenkaya.sveta@gmail.com.; Novosibirsk State University, Novosibirsk, 630090, Russia., Bondar AA; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia., Kurgina TA; Novosibirsk State University, Novosibirsk, 630090, Russia.; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia., Elchaninov VV; Federal Altai Scientific Center of Agrobiotechnologies, Siberian Research Institute of Cheese Making, Barnaul, 656910, Russia., Bakulina AY; State Research Center of Virology and Biotechnology 'Vector', Koltsovo, Novosibirsk Region, 630559, Russia.; Novosibirsk State University, Novosibirsk, 630090, Russia., Rukhlova EA; Novosibirsk State University, Novosibirsk, 630090, Russia., Lavrik OI; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia., Ilyichev AA; State Research Center of Virology and Biotechnology 'Vector', Koltsovo, Novosibirsk Region, 630559, Russia., Shcherbakov DN; State Research Center of Virology and Biotechnology 'Vector', Koltsovo, Novosibirsk Region, 630559, Russia.; Altai State University, Barnaul, 656049, Russia. |
| Abstrakt: |
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. |
