一种米曲霉蛋白酶的分离纯化及酶解特性研究

Purification and characterization of a protease from Aspergillus oryzae

对米曲霉固态发酵所产蛋白酶分离纯化,采用硫酸铵盐析、DEAE-FF层析、Butyl-HP层析和Superdux 7510/300GL凝胶层析得到一种电泳纯的蛋白酶,SDS-PAGE显示分子量大小为27 ku左右。以酪蛋白为底物时,该蛋白酶Km=1.23 g·L-1,Vm=27.03μg·m L-1·min-1,最适反应条件为50℃,p H9.0。该蛋白酶对酪蛋白水解活性最高,而对牛血清蛋白的水解活性很低;对牛胰岛素B链上-Phe-Val-,-Cys-Gly-,-Glu-Ala-和-Arg-Gly-组成的肽键有较强的切割能力,酶切位点较多,对疏水性氨基酸具有较高的选择性,为米曲霉所产蛋白酶在食品上的应用提供有力的参考。

This study aimed to purify and characterize protease from Aspergillus oryzae. Ammonium sulfate precipitation, DEAE-FF anion exchange chromatography, ButyI-HP chromatography and Superdux 75 10/300GL gel filtration chromatography were used to purify the protease from A. oryzae. The molecular weights of the protease was approximately 27 ku. Using casein as a substrate,the Km was 1.23g·L-1,and the Vm was 27.03 μg·mL-1·min-L Moreover,the optimum conditions of the protease were 50 13 and prig.0. The protease had the highest hydrolytic activity to casein while lowest activity to BSA（Bull Serum Albumin）. The protease had cleavage ability between-Phe-Val-,-Cys-Gly-,-Glu-Ala and-Arg-Gly-residues in bovine insulin chain B,showing a wide range of residue specificity and high selectivity to hydrophobic amino acids. The results provided a reference for use of proteases from A. oryzae in food industry.