College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
Phytochemistry. 2011 Apr;72(6):458-64. doi: 10.1016/j.phytochem.2010.12.002. Epub 2011 Feb 24.
A milk coagulating protease was purified ∼10.2-fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, together with ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was estimated to be ∼36kDa by SDS-PAGE, and exhibited a pI of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of ∼60°C. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg(2+) and Cu(2+). Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher for α(s)-casein followed by β- and κ-casein. The purified enzyme differed in molecular mass, pI, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry.
从生姜根茎中分离得到一种凝乳蛋白酶，通过硫酸铵分级沉淀、离子交换和凝胶过滤层析等方法可将其纯化约 10.2 倍，回收率为 34.9%。SDS-PAGE 估计该蛋白酶的相对分子质量约为 36kDa，等电点为 4.3。它是一种糖蛋白，含糖量为 3%。纯化酶在 pH5.5 和温度约 60°C 时表现出最大活性。碘乙酰胺、E-64、PCMB、Hg(2+)和 Cu(2+)强烈抑制其蛋白酶活性。抑制研究和 N 端序列将该酶归类为半胱氨酸蛋白酶。分离出的酶对 α(s)-酪蛋白的水解能力高于 β-和 κ-酪蛋白。与先前报道的生姜蛋白酶相比，该纯化酶的相对分子质量、等电点、糖含量和 N 端序列不同。这些结果表明，该纯化酶可能具有作为乳制品工业中凝乳酶替代品的潜在应用。
