摘要：
Endo-N-acetyl-β-glucosaminidase (Endo/ENGase,EC3.2.1.96) is a kind of glycohydrolase which recognizes and cleaves the β-1,4-glycosidic bonds of N,N'-acetylglucosamine in core structure of N-linked glycans.It is widely used in analyzing N-glycosylation of protein.In order to characterize the enzymatic properties of endo-β-N-acetylglucosaminidase from Enterococcus faecalis,the endo-N-acetyl-β-glucosaminidase gene endoEf of Enterococcus faecalis was cloned by PCR and the prokaryotic expression vector pET-28a-endoEf was constructed using seamless cloning technology.The catalytic properties and pivotal catalytic residues of EndoEf were analyzed on the basis of recombinant expression and purification.The results showed that EndoEf was efficiently expressed in Escherichia coli BL21 Star (DE3) in soluble form,and that the target protein was successfully purified from bacteria solution by one step Co2+ affinity chromatography with a yield of 202.1 mg per liter.The specific activity of EndoEfwas measured as 1.0×104 U/mg using ribonuclease B (RNaseB) as substrate.EndoEf belonged to the glycoside hydrolase family 18 (GH18) which contained conservative motifs DXDXE.In addition,both natural or denatured RNaseB and ovalbumin (Ova) were able to be hydrolyzed by EndoEf.Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS) further confirmed the hydrolysis on N-linked sugar chains in RNaseB.Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weight of EndoEf was 30.2 kD.Enzymatic properties analysis showed that the optimum temperature and pH range of EndoEf were 40 °C and 5.0～7.0,and EndoEfwas relatively more stable at 40～50 °C and pH 7.0～9.0.EndoEf had salt-tolerance up to 1 mol/L NaC1,and remained full activity under either condition of 100 mmol/L DTT,2％ SDS and Triton X-100.Three mutants D 125N,D 127N and E 129Q of EndoEf were constructed by site-directed mutagenesis method.Activity analysis showed that E129Q was almost inactive,and D127N lost most of its activity,while D125N had no significant change in activity.It could be concluded that the glutamic acid at position 129 was essential for catalytic reactions.The establishment of recombinant expression system and analysis of enzyme properties may lay a foundation for the practical application of EndoEf on glycoprotein study.%内切-β-N-乙酰氨基葡萄糖苷酶(endo-β-N-acetylglucosaminidase,Endo/ENGase,EC3.2.1.96)是一类特异性识别并切割N-连接聚糖核心结构中两个N-乙酰葡萄糖胺(N-acetylglucosamine,GlcNAc)之间的β-1.4-糖苷键的糖苷酶,广泛应用于蛋白质N-糖基化分析.为表征粪肠球菌(Enterococcus aecalis)来源的内切-β-N-乙酰氨基葡萄糖苷酶的酶学特性,本研究通过PCR扩增粪肠球菌内切-β-N-乙酰氨基葡萄糖苷酶基因endoEf并通过无缝克隆的方式构建原核表达载体pET-28a-endoEf在实现重组表达和纯化的基础上对EndoEf蛋白的催化特性及关键催化残基进行分析.结果表明,EndoEf可在大肠杆菌中以可溶形式高效表达,经钴离子亲和层析可获得高纯度的目标蛋白,每升菌液可纯化202.1 mg目标蛋白;以RNaseB 为底物,其比活力为1.0×104 U/mg.EndoEf包含保守基序DXDXE,属于糖苷水解酶18(glycoside hydrolases 18,GH18)家族,可以酶切天然及变性状态下的核糖核酸酶B(ribonuclease B,RNaseB)和鸡卵清蛋白(ovalbumin,Ova);基质辅助激光解吸/电离串联飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF-MS)进一步证明了EndoEf对RNaseB上N-连接糖链的水解.十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electropheresis,SDS-PAGE)显示,EndoEf表观分子量为30.2 kD.酶学性质分析表明,EndoEf最适温度为40°C,最适pH范围为5.0～7.0;在温度为40～50°C、pH 7.0～9.0之间具有较好的稳定性,可耐受1 mol/LNaC1、1 00 mmol/L DTT、2％ SDS和2％ Triton X-100.定点突变技术构建EndoEf的3个突变体D125N、D127N和E129Q,并对突变体酶活性进行分析表明,E129Q活性几乎完全丧失,D127N丧失大部分水解活性,而D125N没有明显活性改变,说明EndoEf的129位谷氨酸是催化必须氨基酸.EndoEf重组表达体系的建立和酶学性质的分析为其在糖蛋白研究中的实践应用提供了科学依据.
