一种来自乳酸乳球菌的新型氨肽酶A的制备及特性分析  

Production and characterization of a novel aminopeptidase A from Lactococcus lactis

作　　者：田鑫 刘金洲 何忠会 陈琳方 刘梦元[1] TIAN Xin;LIU Jinzhou;HE Zhonghui;CHEN Linfang;LIU Mengyuan(State Key Laboratory of Biocatalysis and Enzymatic Engineering,School of Life Sciences,Hubei University,Wuhan 430062,Hubei,China;School of Laboratory Medicine,Hubei University of Chinese Medicine,Wuhan 430065,Hubei,China)

机构地区：[1]湖北大学生命科学学院生物催化与酶工程国家重点实验室,湖北武汉430062 [2]湖北中医药大学检验学院,湖北武汉430065

出　　处：《生物工程学报》2023年第8期3494-3507,共14页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(30973669);省部共建生物催化与酶工程国家重点实验室开放基金(SKLBEE2020023)。

摘　　要：氨肽酶A(aminopeptidase A,Pep A)能特异性地水解N末端为谷氨酸(glutamic acid,Glu)或天冬氨酸(asparticacid,Asp)的肽链,提高蛋白质的水溶性和食物的风味,在食品工业和肉类加工中具有一定的应用前景。本研究采用全基因合成的方式获得了乳酸乳球菌(Lactococcus lactis ssp.lactis)IL1403氨肽酶A(Lactococcus lactis-Pep A,Lc-Pep A)的编码基因,将该基因克隆并导入毕赤酵母(Pichia pastoris)GS115(His4),在毕赤酵母中实现了Lc-Pep A的高效分泌表达,表达产物经鉴定和纯化制备后,进行了生物学特性的分析。结果表明,Lc-Pep A具有较强的底物特异性,对2种底物谷氨酸对硝基苯胺(glutamicacid-p-nitroaniline,Glu-pNA)和天冬氨酸对硝基苯胺(aspartic acid-p-nitroaniline,Asp-pNA)具有相似的催化活力和酶动力学参数。Lc-Pep A是一种金属蛋白酶,最适反应温度为60℃,最适pH为8.0,具有较宽的热稳定性和酸碱稳定性。金属离子Co^(2+)、Mn^(2+)及Zn^(2+)等对酶活力具有不同程度的激活作用,而Ni^(2+)和Cu^(2+)对酶活力具有强烈的抑制作用。Lc-Pep A对常规蛋白酶抑制剂不敏感,但能被金属蛋白酶抑制剂、EDTA及二硫键还原剂抑制。这些研究为Lc-Pep A的生产和指导该酶的应用打下了坚实的基础。Aminopeptidase A(Pep A)is a metal-dependent enzyme that specifically hydrolyze peptides with the N-terminal amino acids glutamic acid(Glu)and aspartic acid(Asp).A possible application of PepA is the hydrolysis of Glu/Asp-rich food proteins such as wheat gluten and casein,increasing the flavor and solubility of food protein.In the present study,the gene encoding a Pep A from Lactococcus lactis ssp.lactis IL1403 was synthesized and introduced into Pichia pastoris GS115(His4).Lc-Pep A was successfully expressed and secreted to the culture medium,followed by identification and purification to homogeneity.Characteristics study demonstrated that Lc-Pep A could specifically hydrolyze the substrates Glu-pNA and Asp-pNA with similar catalytic activity,and this was further confirmed by the kinetics parameters measured.Additionally,Lc-Pep A showed a broad thermostability and pH stability with an optimum temperature of 60℃and an optimum pH of 8.0.The enzyme activity of Lc-Pep A was activated by metal ions Co^(2+),Mn^(2+),and Zn^(2+)but was strongly inhibited by Ni^(2+)and Cu^(2+).The routine proteinase inhibitor had no effect on the activity of Lc-Pep A.However,Lc-Pep A was strongly inhibited by the metallopeptidase inhibitor,EDTA,and disulfide bond-reducing agents.The study may facilitate production and application of Lc-Pep A.

关 键 词：氨肽酶A 乳酸乳球菌 食品工业 肉类加工 

分 类 号：TS201.25[轻工技术与工程—食品科学]