S. V. Belenkaya, V. V. Elchaninov, V. Y. Chirkova, D. N. Shcherbakov
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Comparison of the Biochemical Properties of Recombinant Alpaca (Vicugna pacos) Chymosins Produced in Pro- and Eukaryotic Expression Systems
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.
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.
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