雅致放射毛霉AS3.2778碱性蛋白酶的纯化及性质研究(英文)

Purification and Characterization of One Alkaline Protease from Actinomucor elegans AS3.2778

利用盐析、离子交换、疏水层析及凝胶过滤的方法从雅致放射毛霉AS3.2778的发酵麸曲中分离纯化出一碱性蛋白酶,其纯化提高了22.7倍,酶活回收率16.1%,最终比酶活可达到6094μ/mg。电泳分析发现,该蛋白酶是一单体蛋白,其分子量大约在32kDa。性质分析表明:该蛋白酶在60℃,pH8.5～10.5具有最大催化活性;在40℃以下,pH6.0 ～9.0的范围有很好的稳定性;1mmol/L的PMSF可以完全抑制其活性,显示该蛋白酶属于丝氨酸蛋白酶家族。底物专一性的研究发现,该蛋白酶有相当广泛的肽键选择性,对绝大多数由疏水性氨基酸(尤其是亮氨酸)构成的肽键有很强的水解能力。

One alkaline protease from Actinomucor elegans AS3. 2778 was purified 22.7 fold with a total yield of 16.1% and a final specific activity of 6094u/mg protein. The enzyme was purified using ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography method, and its properties were also investigated. The molecular weight of this enzyme is 32 kDa with SDS-PAGE method, optimum temperature is 60℃, optimum pH is 8.5 to 10.5, it is stable in the pH range of 6.0 to 9.0 at 〈 40℃ temperature, and being completely inhibited by the serine protease inhibitor, PMSF, indicated that it belongs to the serine protease family. Specificity test indicated this protease has extensive selectivity to peptide bones, especially to peptide bones composed of Leucine residue.