Department of Bioscience and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China.
World J Microbiol Biotechnol. 2012 May;28(5):2087-93. doi: 10.1007/s11274-012-1012-7. Epub 2012 Feb 10.
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 Mn(2+), Fe(3+), Mg(2+) and Na(+), but inhibited by K(+), Co(2+), Zn(2+), Ni(2+), and to a lesser extent by Cu(2+). 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.
凝乳酶可以特异性地切断κ-酪蛋白的 Phe105-Met106 肽键，形成不溶性的κ-酪蛋白副产物，导致牛奶凝结，这一过程常用于制作奶酪。在这项研究中，为了获得一种安全、高效且能同时生产出美味奶酪的替代牛奶凝固剂，选择并在毕赤酵母中组成型高水平表达牛前凝乳酶 B。重组凝乳酶主要以分泌形式表达，并表现出凝乳活性。它通过硫酸铵分级沉淀、阴离子交换和阳离子交换层析进行纯化。纯化酶的最终收率为 24.2%，在 SDS-PAGE 中呈现出一条约 36 kDa 的单带，分子量。蛋白水解试验表明它特异性地水解κ-酪蛋白。它在 25-50°C 下稳定，在 37°C 和 pH 4.0 时具有最佳活性。重组凝乳酶的活性被 Mn(2+)、Fe(3+)、Mg(2+)和 Na(+)等阳离子激活，但被 K(+)、Co(2+)、Zn(2+)、Ni(2+)和 Cu(2+)抑制，抑制程度较小。这些结果表明，重组牛凝乳酶是一种酸性牛奶凝固酶，可考虑将其作为一种安全、高效的酶，适用于奶酪生产。
