猪血管紧张素转换酶2的分离纯化及性质研究  

Purification and Characterization of Porcine Angiotensin Converting Enzyme 2

作　　者：刘维维 张凌晶[1] 纪梦雅 翁凌[1] 张志刚 刘光明[1] 曹敏杰[1] LIU Weiwei;ZHANG Lingjing;JI Mengya;WENG Ling;ZHANG Zhigang;LIU Guangming;CAO Minjie(College of Ocean Food and Biological Engineering,Jimei University,Xiamen 361021,China;State Key Laboratory of Meat Production Safety Technology,Xiamen 361100,China)

机构地区：[1]集美大学海洋食品与生物工程学院,福建厦门361021 [2]肉食品安全生产技术国家重点实验室,福建厦门361100

出　　处：《食品工业科技》2022年第18期89-96,共8页Science and Technology of Food Industry

基　　金：国家重点研发计划项目(2018YFD0901004)。

摘　　要：血管紧张素转换酶2(Angiotensin converting enzyme 2,ACE2)是参与血压调节的关键酶之一,由于传统的ACE2纯化方法难度大、效率低,因此,有必要优化其分离纯化方法,为ACE2的基础研究提供保障。本研究以猪肾为原材料,通过酸沉淀、硫酸铵盐析,结合DEAE-Sepharose、Q-Sepharose及Phenyl Sepharose柱层析对ACE2进行分离纯化。探究了温度、pH、金属离子、抑制剂对纯化的ACE2的活性影响,并通过PAS染色验证其是否为糖蛋白。实验结果表明,纯化的ACE2分子量约为98 kDa,纯化倍数为149.8,得率为0.1%。对ACE进行质谱鉴定,得到41个肽段,与猪ACE2的氨基酸序列高度一致。酶学性质研究结果表明,ACE2为金属蛋白酶,最适pH为7.0,最适温度为40℃。Zn^(2+)能激活其活性,而金属离子Cu^(2+)、Fe^(3+)和Cd^(2+)对ACE2的活性有抑制作用。圆二色谱分析发现,ACE2的热变性温度为67.5℃。PAS染色结果显示猪ACE2为糖蛋白。本研究结果可为天然ACE2的高效纯化提供参考,也有助于推进以ACE2为靶点的功能性食品开发。Angiotensin converting enzyme 2(ACE2)is one of the key enzymes involved in blood pressure regulation.Conventional purification method for ACE2 was complicated and with low efficacy.Thus,it is necessary to optimize the purification method to provide reference for the research of ACE2.In this study,porcine kidney was used as raw material,and ACE2 was purified by acid precipitation,ammonium sulfate precipitation,column chromatographies of DEAE-Sepharose,Q-Sepharose and Phenyl Sepharose.The effects of temperature,pH,metal ions,and inhibitors on the activity of ACE2 were explored,and PAS staining was adopted to verify whether it is glycoprotein or not.The results showed that the molecular weight of purified ACE2 was about 98 kDa,with purification folds of 149.8,and yield of 0.1%.Purified protein was identified by mass spectrometry,and 41 peptides were obtained which were identical to the amino acid sequence of porcine ACE2.ACE2 was a metalloproteinase,its optimum pH was 7.0 and optimum temperature was 40℃.Metal ion Zn^(2+) could activate its activity while Cu^(2+),Fe^(3+) and Cd^(2+) inhibited it.The results of circular dichroism spectrum showed that the denaturation temperature of ACE2 was 67.5℃,and its secondary structure irreversibly changed after heating.Porcine ACE2 was a glycoprotein as revealed by PAS staining.This study would provide a reference for effective purification of natural ACE2 and is also helpful to promote the development of functional foods targeting ACE2.

关 键 词：血管紧张素转换酶2(ACE2) 组织分布 纯化 酶学性质 

分 类 号：Q556[生物学—生物化学]