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, A. A. Bondar, T. A. Kurgina, V. V. Elchaninov, A. Yu Bakulina, E. A. Rukhlova, O. I. Lavrik, A. A. Ilyichev, D. N. Shcherbakov

Результат исследования: Научные публикации в периодических изданияхстатья

Аннотация

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 Escherichiacoli [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.

Язык оригиналаанглийский
Страницы (с-по)781-791
Число страниц11
ЖурналBiochemistry (Moscow)
Том85
Номер выпуска7
DOI
СостояниеОпубликовано - 1 июл 2020

Fingerprint Подробные сведения о темах исследования «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». Вместе они формируют уникальный семантический отпечаток (fingerprint).

Цитировать