摘要
酶法去糖基化是研究糖蛋白结构与功能的重要手段.传统的酶法去糖基多使用游离酶,反应体系残留的去糖基化酶成为后续糖蛋白分析(如质谱)潜在的污染信号来源,探索去糖基化酶新的利用方式对于糖蛋白糖链结构的分析具有重要价值.本研究对粪肠球菌(Enterococcus faecalis)来源的内切β-N-乙酰氨基葡萄糖苷酶(endo-β-N-acetylglucosaminidase,EndoEf)进行了重组表达和纯化,以Sepharose CL 6B为固定化载体,采用环氧氯丙烷法活化,对EndoEf的固定化方法进行优化,并对固定化EndoEf的催化特性进行分析.研究结果表明,EndoEf可在大肠杆菌(Escherichia coli)BL21 Star(DE3)中高效表达,每升菌液可纯化186.5 mg EndoEf.EndoEf固定化的最优条件:在pH 8.0的50 mmol/L磷酸缓冲液中,按每克载体加入4.0 mg酶蛋白,4℃偶联12 h;固定化酶能去除核糖核苷酸酶B(ribonuclease B,RNase B)和鸡卵白蛋白(ovalbumin,Ova)的N-糖链,在4℃保存60d仍具有活性;固定化EndoEf在30～40℃和pH 6.0～7.0的范围内具有良好的水解活性,对蛋白质变性剂二硫苏糖醇(dithiothreitol,DTT)及十二烷基硫酸钠(sodium dodecyl sulfate,SDS)的耐受性较游离酶明显提高,可耐受12.0 mmol/L DTT和2.0%SDS的变性条件,并耐受1.0 mol/L NaCl高盐条件.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)分析显示,离心可完全去除反应体系中的固定化EndoEf.固定化体系的建立及酶学性质的分析为该酶在糖蛋白研究中的实践应用提供了基础资料.
Abstract
Enzymatic deglycosylation is an important means to study the structure and function of glycoproteins.Traditional enzymatic deglycosylation mostly uses free enzymes,so that the residual deglycosylase becomes a potential source of contamination signals for subsequent glycoprotein analysis(such as mass spectrometry analysis).To explore new utilization methods of deglycosylation is of great value for the analysis of glycoprotein glycan chain structure.In this study,the recombinant endo-β-N-acetylglucosaminidase(EndoEf)of Enterococcus faecaliswas expressed and purified,using sepharose CL 6B activated by epichlorohydrin as the carrier to immobilize,and the catalytic characteristics of immobilized EndoEf were analyzed.The results showed that EndoEf could be efficiently expressed in Escherichia coli BL21 Star(DE3),and 186.5 mg of enzyme protein could be purified per liter of bacterial solution.The optimal conditions for immobilization of EndoEf were 4.0 mg enzyme per gram of carrier in 50 mmol/L phosphate buffer at pH 8.0,12 h conjugated at 4℃.Immobilized enzymes could remove N-glycan chains of ribonuclease B(RNase B)and ovalbumin(Ova),remaining active when stored at 4℃for 60 d.Immobilized EndoEf had higher activity in the range of 30～40℃and pH 6.0～7.0.The tolerance to DL-dithiothreitol(DTT)and sodium dodecyl sulfate(SDS)was significantly improved than that of the free enzyme,which could tolerate denaturation conditions of 12.0 mmol/L DTT and 2.0%SDS,and tolerate 1.0 mol/L NaCl.Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS)analysis showed that centrifugation completely removed immobilized EndoEf from the reaction system.The establishment of immobilization method and the analysis of catalytic characteristics could provide a basis for the application of immobilized EndoEf in glycoprotein research.
国家科技期刊平台
NSTL
万方数据