米曲霉蛋白酶的分离纯化及酶学性质研究

Purification and characterization of proteases from Aspergillus oryzae

摘要:

【目的】获得米曲霉蛋白酶主要成分及其酶学性质。【方法】利用硫酸铵盐析，DEAE-Sepharose FF阴离子交换层析、Phenyl-Sepharose HP疏水层析和Superdex-G75/200凝胶层析对米曲霉所产蛋白酶系进行分离纯化，SDS-PAGE检测蛋白酶纯度和分子量，采用高效液相凝胶色谱分析两种蛋白酶酶解产物。【结果】从米曲霉所产蛋白酶系中分离纯化获得两种蛋白酶组分P1和P2，分子质量分别约为37 kD和45 kD。以酪蛋白为底物时，P1的Km=8.36 g/L，Vm=12.95 μg/(mL·min)，最适反应条件为pH 8.0、45 °C；P2的Km=4.11 g/L，Vm=4.86 μg/(mL·min)，最适反应条件为pH 7.0、45 °C。两种蛋白酶均对酪蛋白水解活性最高，而对牛血清蛋白的水解活性很低。P1和P2分别酶解大豆分离蛋白后水解产物中肽分子质量分布呈现出一定的差异。【结论】两种蛋白酶的酶学性质存在差异；两者对疏水氨基酸构成的肽键具有选择性，但其作用基团存在特异性。这些研究结果将为米曲霉所产蛋白酶在食品上的应用提供指导。

Abstract:

[Objective] This study aimed to purify and characterize proteases from Aspergillus oryzae. [Methods] Ammonium sulfate precipitation, DEAE-Sepharose FF anion exchange chromatography, Phenyl-Sepharose HP hydrophobic chromatography and Superdex-G75/200 gel filtration chromatography were used to purify the proteases from A. oryzae. The molecular weight and purity were determined by SDS-PAGE, and the enzymatic hydrolyzates of soy protein isolated were assayed by HPLC. [Results] Two proteases (P1 and P2) were obtained from Aspergillus oryzae and their molecular weights were approximately 37 kD and 45 kD, respectively. Using casein as a substrate, the optimum conditions of P1 and P2 were pH 8.0, 45 °C and pH 7.0, 45 °C, respectively. The Km of P1 and P2 were 8.36 g/L and 4.11 g/L, respectively. Moreover, the Vm of P1 and P2 were 12.95 μg/(mL·min) and 4.86 μg/(mL·min), respectively. Both P1 and P2 had the highest hydrolytic activity to casein while lowest activity to BSA (Bull Serum Albumin). Meanwhile, the molecular weight of peptides in soy protein isolated hydrolyzates had different distributions. [Conclusion] Purified P1 and P2 have some differences in enzymatic properties. And they both have strong cutting capacity towards the hydrophobic peptide bond, but there are also differences in the specificity of bond groups. These results have a great significance for the application of proteases from Aspergillus oryzae in the food industry.