一种米曲霉蛋白酶的酶学性质及其在酪蛋白磷酸肽制备中的应用

Characterization of a protease from Aspergillus oryzae and application in casein phosphopeptides preparation

【目的】分离纯化米曲霉蛋白酶的主要组分,分析其酶学特性,并应用于酪蛋白磷酸肽(Casein phosphopeptides,CPPs)的制备。【方法】采用硫酸铵盐析、DEAE-Sepharose FF阴离子交换层析和Butyl-sepharose HP疏水层析对米曲霉蛋白酶进行分离纯化,SDS-PAGE检测分子量与纯度,MALDI-TOF-MS检测酶切位点。【结果】得到一种蛋白酶组分(命名为PE),分子量大小为58 k D左右。该酶最适反应条件为55°C,p H 8.0,酶活被Fe3+抑制,被Mn2+激活。以酪蛋白为底物时,Km=0.36 g/L,最大反应速率Vm=18.18 mg/(L·min)。蛋白酶PE对牛胰岛素B链上-Leu-Cys-、-Val-Glu-、-Tyr-Leu-和-Arg-Gly-组成的肽键有较高的切割能力,酶切位点较多。利用其水解酪蛋白,通过钡-乙醇沉淀法得到CPPs,产率为15.87%,摩尔氮磷比r(N/P)为6.17,得到的CPPs可以使钙沉淀推迟35 min。【结论】利用米曲霉蛋白酶水解酪蛋白产生CPPs,为其在功能性食品加工方面的应用提供有利的参考。

[Objective] This study aimed to purify and characterize protease from Aspergillus oryzae, and to apply in casein phosphopeptides（CPPs） preparation. [Methods] Ammonium sulfate precipitation, DEAE-Sepharose FF anion exchange chromatography and Butyl-Sepharose HP hydrophobic chromatography were performed to purify the enzyme. The molecular weight and purity were determined by SDS-PAGE, and cleavage sites were detected by MALDI-TOF-MS. [Results] A protease named PE with the weight about 58 k D was obtained from Aspergillus oryzae. Protease PE shows maximal activity at p H 8.0 and 55 °C. It was inhibited by Fe3＋, and activitied by Mn2＋. With casein as the substrate, Km and Vm of the protease were 0.36 g/L and 18.18 mg/（L·min）, respectively. Protease PE has cleavage ability in-Leu-Cys-,-Val-Glu-,-Tyr-Leu- and-Arg-Gly- residues of bovine insulin chain B, showing a wide range of residue specificity. CPPs were obtained after proteolysis of casein using PE by barium-ethanol precipitation. Yield and r（N/P） of the CPPs were 15.87% and 6.17, respectively, and delayed calcium deposit for 35 min. [Conclusion] The research provided a reference for Aspergillus oryzae protease using in the functional food industry.