Abstract
Chymosin can specifically break down the Phe105–Met106 peptide bond of milk κ-casein to form insoluble para-κ-casein, resulting in milk coagulation, a process that is used in making cheese. In this study, in order to obtain an alternative milk coagulant which is safe and efficient, and simultaneously can produce cheese with a good taste, bovine prochymosin B was chosen and constitutively expressed to a high level in Pichia pastoris. The recombinant chymosin was expressed mainly as a secretory form, and it exhibited milk-clotting activity. It was purified by ammonium sulfate fractionation, anion exchange, followed by cation exchange chromatography. A final yield of 24.2% was obtained for the purified enzyme, which appeared as a single band in SDS–PAGE having a molecular mass of approximate 36 kDa. Proteolysis assay showed that it specifically hydrolyzed κ-casein. It was stable at 25–50°C and had optimal activity at 37°C and pH 4.0. The activity of the recombinant chymosin was activated by cations such as Mn2+, Fe3+, Mg2+ and Na+, but inhibited by K+, Co2+, Zn2+, Ni2+, and to a lesser extent by Cu2+. These results suggested that recombinant bovine chymosin is an acid milk coagulant, and it could be considered as a safe and efficient enzyme suitable for use in cheese production.
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Acknowledgments
This work is supported by the National Key Project for Basic Research (2010CB126102), the National Natural Science Foundation of China (31070715, 81102378), the National High Technology Research and Development Program of China (2011AA10A204), the National Key Technology R&D Program(2011BAE06B05), Scientific Research Foundation for the Returned Overseas Chinese Scholars, the Fundamental Research Funds for the Central Universities (DUT11SM02). The authors thank Dr Alan K. Chang for checking and revising the language of the manuscript.
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Jiang, X.P., Yin, M.L., Chen, P. et al. Constitutive expression, purification and characterization of bovine prochymosin in Pichia pastoris GS115. World J Microbiol Biotechnol 28, 2087–2093 (2012). https://doi.org/10.1007/s11274-012-1012-7
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DOI: https://doi.org/10.1007/s11274-012-1012-7