粪肠球菌源EndoE的重组表达及酶学性质研究  

Recombinant Expression and Catalytic Properties of EndoE from Enterococcus faecalis

作　　者：韩小韦 黄云娜 牛毅男 李学俊[1] 徐全乐[1] 陈鹏[1] HAN Xiao-Wei;HUANG Yun-Na;NIU Yi-Nan;LI Xue-Jun;XU Quan-Le;CHEN Peng(College of Life Sciences, Northwest A&F University, Yangling 712100, China;Conghua Middle School, Guangzhou 510900, China)

机构地区：[1]西北农林科技大学生命科学学院,杨凌712100 [2]广州市从化区从化中学,广州510900

出　　处：《农业生物技术学报》2019年第6期1118-1125,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.30400282和No.31171606);陕西省重点研发计划(No.2017NY-033)

摘　　要：内切-β-N-乙酰氨基葡萄糖苷酶(Endo-β-N-acetylglucosaminidase, ENGase, EC3.2.1.96)是糖蛋白去糖基化重要的工具酶,其可以切割糖蛋白上N-连接的聚糖。本研究基于粪肠球菌(Enterococcus faecalis)基因组数据,通过PCR扩增ENGase基因endoE (endoglycosidase E),并以同源重组方式构建表达载体pET-28a-endoE,实现了EndoE在大肠杆菌(Escherichia coli) BL21 Star (DE3)中的高效表达,并对EndoE去糖化的催化特性等进行了较为系统的分析。研究结果显示,重组EndoE在大肠杆菌中以可溶形式表达,经钴离子螯合层析与阴离子交换层析可获得高纯度的目标蛋白,产量为45.6 mg/L。去糖基化活性分析表明,重组EndoE能去除天然及变性核糖核苷酸酶B (ribonuclease B, RNase B)、鸡卵白蛋白(ovalbumin, Ova)和免疫球蛋白G (immune globulin G, IgG) N-连接的糖基侧链。基质辅助激光解吸/电离串联飞行时间质谱证实了EndoE对RNase B N-连接糖链的水解活性。EndoE在20~50 ℃和pH 4.0~6.0的范围内均具有良好的水解活性,并对100 mmol/L二硫苏糖醇(DL-dithiothreitol, DTT)、1 mol/L NaCl和2% Triton X-100具有耐受性。EndoE较已报道的粪肠球菌来源的ENGase具有更为广泛的底物作用范围,是蛋白质糖基化分析中有潜在应用价值的工具酶。Endo-β-N-acetylglucosaminidase (ENGase) can cleave the N-linked oligosaccharides of glycoprotein and is an essential tool enzyme for protein deglycosylation. This study obtained the genome data of Enterococcus faecalis from the NCBI database and amplified the ENGase gene endoE (endoglycosidase E) by PCR. Then the expression vector pET-28a-endoE was constructed by homologous recombination and transformed into Escherichia coli BL21 star (DE3) competent cells. The recombinant protein was efficiently expressed in E. coli, and the deglycosylation properties of EndoE was systematically analyzed. The results showed that recombinant protein was expressed in soluble form in E. coli, and the yield was 45.6 mg/L after purified by Co2+ affinity chromatography and anion-exchange chromatography. The deglycosylation activity analysis indicated that the recombinant EndoE could hydrolyze the N-linked glycan of both natural and denatured ribonuclease (RNase B), ovalbumin (Ova) and immune globulin G (IgG). Matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS) proved that recombinant EndoE could remove the N-linked oligosaccharides in RNase B. In addition, in the temperature range of 20~50 ℃ or pH range of 4.0~6.0, EndoE had ideal hydrolytic activity. It also had tolerance up to 100 mmol/L DL-dithiothreitol (DTT), 1 mol/L NaCl, and 2% Triton X-100. Compared to the other reported ENGase from Enterococcus faecalis, EndoE has a broader range of substrates and is a prospective tool enzyme in protein deglycosylation study.

关 键 词：内切-β-N-乙酰氨基葡萄糖苷酶(ENGase) N-糖基化 重组表达 酶学性质 

分 类 号：S182[农业科学—农业基础科学] Q936[生物学—微生物学]