Comparison of the Biochemical Properties of Recombinant Alpaca (Vicugna pacos) Chymosins Produced in Pro- and Eukaryotic Expression Systems

IF 1 4区生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Applied Biochemistry and Microbiology Pub Date : 2023-10-02 DOI:10.1134/S0003683823050034

S. V. Belenkaya, V. V. Elchaninov, V. Y. Chirkova, D. N. Shcherbakov

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Abstract

Based on the yeast Kluyveromyces lactis, a strain-producer of recombinant alpaca (Vicugna pacos) prochymosin has been developed. A comparative analysis of the biochemical properties of recombinant alpaca chymosin obtained in the expression systems of K. lactis and Escherichia coli has been carried out. It was found that the recombinant alpaca chymosin synthesized in K. lactis exceeds the analogue obtained in E. coli by 12.9 times in the turnover number of the enzyme, and by 2.9 times in catalytic efficiency. Compared to chymosin expressed in E. coli, the enzyme obtained in a eukaryotic producer has a thermal stability threshold increase of 5°C. Replacing a prokaryotic producer with a eukaryotic one enhances the negative sensitivity of the milk-clotting activity of recombinant alpaca chymosin to an increase in substrate pH in the range of 6.1–6.9, which is accompanied by an increase in the duration of coagulation by 8–35%. With an increase in the concentration of CaCl₂ in the substrate, the coagulation activity of the target enzyme synthesized in E. coli was 12–14% higher than that of its analogue obtained in K. lactis.

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重组羊驼Chymosis在原核和真核表达系统中的生化特性比较

在乳酸克鲁维酵母的基础上，开发了一株重组羊驼（Vicugna pacos）前膜菌素的生产菌。对在乳酸双歧杆菌和大肠杆菌表达系统中获得的重组羊驼凝乳酶的生化特性进行了比较分析。研究发现，在乳酸双歧杆菌中合成的重组羊驼凝乳酶的酶周转数比在大肠杆菌中获得的类似物高12.9倍，催化效率高2.9倍。与在大肠杆菌中表达的凝乳酶相比，在真核生物生产商中获得的酶的热稳定性阈值增加了5°C。用真核生物替代原核生物生产者可以增强重组羊驼凝乳酶对底物pH值在6.1–6.9范围内增加的牛奶凝固活性的负敏感性，同时凝固时间增加8–35%。随着基质中CaCl2浓度的增加，在大肠杆菌中合成的目标酶的凝血活性比在乳酸双歧杆菌中获得的类似物高12-14%。

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来源期刊

Applied Biochemistry and Microbiology 生物-生物工程与应用微生物

CiteScore

1.70

自引率

12.50%

发文量

审稿时长

6-12 weeks

期刊介绍： Applied Biochemistry and Microbiology is an international peer reviewed journal that publishes original articles on biochemistry and microbiology that have or may have practical applications. The studies include: enzymes and mechanisms of enzymatic reactions, biosynthesis of low and high molecular physiologically active compounds; the studies of their structure and properties; biogenesis and pathways of their regulation; metabolism of producers of biologically active compounds, biocatalysis in organic synthesis, applied genetics of microorganisms, applied enzymology; protein and metabolic engineering, biochemical bases of phytoimmunity, applied aspects of biochemical and immunochemical analysis; biodegradation of xenobiotics; biosensors; biomedical research (without clinical studies). Along with experimental works, the journal publishes descriptions of novel research techniques and reviews on selected topics.