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
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摘要: 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。Abstract: This study aimed to purify and characterize protease from Aspergillus oryzae. Ammonium sulfate precipitation,DEAE-FF anion exchange chromatography,Butyl-HP chromatography and Superdux 75 10/300 GL 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 Kmwas 1.23g·L-1, and the Vmwas27.03 μg·m L-1·min-1. Moreover,the optimum conditions of the protease were 50 ℃ and p H9.0. 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.
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