Abstract 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.

中文翻译：

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Altai Maral 凝乳酶基因的表征、凝乳酶重组类似物在原核表达系统中的产生及其几种生化特性的分析

摘要 首次对一种海马的凝乳酶基因（CYM）进行了表征。其外显子/内含子组织是使用核苷酸序列的比较分析建立的。编码maral 前凝乳酶原的CYM mRNA 序列被重建。CYM maral mRNA 的核苷酸序列允许开发表达载体以确保产生重组酶。在大肠杆菌[菌株BL21 (DE3)]的表达系统中获得重组maral凝乳酶原。重组maral凝乳酶的总牛奶凝固活性(MCA)为2330 AU/ml。重组maral凝乳酶原相对活性为52955 AU/mg。重组maral凝乳酶在30-50°C的温度范围内表现出100-81%的MCA，热稳定性（TS）阈值为50°C，酶在70°C时完全失活。制备的单峰骆驼重组凝乳酶和重组牛凝乳酶作为参考样品。重组maral凝乳酶的Michaelis-Menten常数（ K m ）、周转数（ k cat ）和催化效率（ k cat /K m ）分别为1.18 ± 0.1 µM、2.68 ± 0.08 s -1 和2.27±0.10 µm M -1 ·s -1 ，分别。