【作者】 邹树平；

【导师】 李春；

【作者基本信息】 天津大学 ， 生物化工， 2010， 博士

【摘要】 糖基化是一种重要的蛋白质翻译后修饰方式,对蛋白质的结构和功能有着重要影响。本文以课题组前期构建的三种不同糖基化类型表达系统(产紫青霉P.purpurogenum Li-3自然进化糖基化型、大肠杆菌无糖基化型和毕赤巴斯德酵母高甘露糖糖基化型)表达的β-D-葡萄糖醛酸苷酶为对象,对不同系统表达的β-D-葡萄糖醛酸苷酶进行分离纯化;确定β-D-葡萄糖醛酸苷酶在不同表达系统中的糖基化水平;研究不同糖基化水平β-D-葡萄糖醛酸苷酶的酶学性质及催化反应动力学;分析和计算糖基化后酶的构象稳定性及热力学参数,探讨糖基化对β-D-葡萄糖醛酸苷酶催化特性和结构的影响。主要研究结果如下:采用硫酸铵分级沉淀、阴离子交换层析、凝胶层析和亲和层析等方法,分别纯化了产紫青霉、重组大肠杆菌和重组毕赤巴斯德酵母表达的β-D-葡萄糖醛酸苷酶(分别称为PGUS、PGUS-P和PGUS-E),获得了三种β-D-葡萄糖醛酸苷酶的电泳级纯品,HPLC检测纯度分别为92.1%、95.3%和98.3%,其纯度满足后续糖基化与质谱分析的要求。采用MALDI - TOF MS测定PGUS、PGUS-E和PGUS-P的精确分子量分别为69.72 kDa、67.93 kDa和78.83 kDa。肽质量指纹图谱结合串联质谱对氨基酸序列鉴定表明,三种酶均和β-D-葡萄糖醛酸苷酶基因pgus(EU095019)所编码的氨基酸序列相对应。糖基化分析结果表明,PGUS-P为N-糖基化类型翻译后修饰,其糖含量为14.42%,而PGUS和PGUS-E无明显糖基化修饰。酶学性质比较分析表明,三种β-D-葡萄糖醛酸苷酶最适pH、最适反应温度、对底物对硝基苯-葡萄糖醛酸苷(PNPG)和甘草酸的催化反应动力学参数及催化水解甘草酸模式均有显著差异。以纯化后的PGUS-P为研究对象,用糖苷酶F(PNGase F)对其进行去糖基化,比较了PGUS-P去糖基化前后的酶学性质和反应动力学,表明PGUS-P去糖基化后,其最适反应温度未明显变化,但最适pH值范围增大,对金属离子敏感性降低。去N-糖基化后的PGUS-P与底物PNPG和甘草酸的亲和力均增强。比较了去糖基化前后PGUS-P的稳定性及构象变化,表明去糖基化后的PGUS-P耐变性剂、有机溶剂及表面活性剂能力均下降,且更容易被胰蛋白酶水解。差示量热扫描(DSC)研究表明,去糖基化的PGUS-P热变性温度Td和ΔH均下降,酶蛋白的热稳定性降低。荧光光谱和圆二色谱分析表明,去糖基化处理未改变PGUS-P酶蛋白的二级结构,但诱导了酶蛋白分子伸展及其三级结构的改变。构象稳定性机理分析表明,N-糖基化有利于提高酶蛋白去折叠中间态的自由能变ΔG(H2O),从而增加其构象稳定性。更多还原

【Abstract】 Glycosylation, one of the major naturally occurring modifications of the covalent structure of proteins, has been found to effect the structure and function of protein. To research the effects of glycosylation on the catalytic characteristic and structure of enzyme, threeβ-D-glucuronidases from different glycosylation expression systems—Penicillium purpurogenum Li-3 (natural evolutionary type glycosylation, E.coli BL21(DE3) (non-glycosylation) and Pichia pastoris GS115 (high mannose type glycosylation ) were purified and their glycosylation level were analysis. The catalytic characteristic and kinetics of theβ-D-glucuronidases with different glycosylation level were investigated, and we also studied the effect of glycosylation on the conformation stability and structure ofβ-D-glucuronidase. Main results are as follows:Threeβ-D-glucuronidases from Penicillium purpurogenum Li-3, E.coli and P. pastoris were purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-100 chromatography and Ni-NTA Sepharose chromatography. The purity of the PGUS, PGUS-E and PGUS-P obtained were 92.1% %、95.3% and 98.3% by HPLC assay, respectively. These purities can meet the analysis requirement of glycosylation and mass spectrum in the next work.The molecular mass of PGUS, PGUS-E and PGUS-P were determined to be 69.72 kDa, 67.93 kDa and 78.83 kDa by MALDI-TOF MS, respectively. Segments of the amino acids sequence analysis ofβ-D-glucuronidases by combining peptide mass finger printing and tandem mass spectrometry matched well with the deduced amino acid sequence of pgus (EU095019). According to the difference between theoretical and practical molecular mases, PGUS-P expressed by P. pastoris was estimated to be N-glycosylated with a glycan content of 14.42%, but PGUS expressed by P.purpurogenum Li-3 and PGUS-E expressed by E.coli were non-glycosylated. The comparison of catalytic properties of threeβ-D-glucuronidases were carried out, and results showed the optimum pH, optimum temperature and the kinetic parameters of threeβ-D-glucuronidases were obviously different.The enzymatic deglycosylation of PGUS-P was investigated using peptide- N-glycosidase F (PNGase F), and then the comparison of catalytic properties and kinetic parameters of native and deglycosylated PGUS-P were studied. The results showed temperature-optima of both native and deglycosylated isoforms of PGUS-P remained unchanged. However, the deglycosylated PGUS-P showed a wider range of pH-optima, a lower sensitivity on ion metal, and a greater affinity for substrates p-nitrophenyl-β-D-glucuronide and glycyrrhizin compered to glycosylated enzyme.The possible role of carbohydrate moieties in the stabilization of enymes has been investigated by using PGUS-P as a model system. A comparative study of native and deglycosylated PGUS-P was performed at various water-miscible organic solvents, detergents and chaotropic agent like urea. The glycosylated form of PGUS-P retained greater fraction of enzyme activity against the exposure caused by various physical and chemical denaturants. A significantly less stability for the deglycosylated enzyme than for the native form can be found when treatment with chymotryptic enzyme. DSC analysis indicated the deglycosylated PGUS-P samples presented lower Td andΔH, relative to that of untreated PGUS-P, indicating the deglycosylated PGUS-P has a lower thermal stability. Near-UV CD spectra and intrinsic fluorescence spectrum analyses confirmed much loss of tertiary conformation of deglycosylated PGUS-P, relative to native form, but the secondary structure were nearly unaffected by the deglycosylation treatment.更多还原