兰州大学机构库 >生命科学学院
N-糖基化对Aspergillus niger 963植酸酶phyA2酶学性质的影响
Alternative TitleEffects of N-Glycosylation on characterizationof Aspergillus niger 963 phytase phyA2
殷亮
Thesis Advisor王新宇 ; 陈茹梅
2010-05-23
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword糖基化 植酸酶 定点突变 毕赤酵母 热稳定性 酶学性质
Abstract糖基化(Glycosylation)是真核细胞蛋白质翻译后修饰的方式之一,对于蛋白质的结构和功能具有重要的影响。曲霉来源的植酸酶是一种重要的磷酸酯酶,属于组氨酸酸性磷酸酶家族(EC.3.1.3.2),具有典型的该家族植酸酶活性位点保守序列RHGARYP。研究表明,这类植酸酶都是糖蛋白,氨基酸序列中存在10个左右的糖基化位点(N-X-T/S),必须经过糖基化修饰才具有生物学功能, 但这种修饰对于植酸酶蛋白的意义及其不同的糖基化位点在催化反应中扮演的角色目前尚不清楚。本研究中我们利用Megaprimer PCR介导的基因定点突变技术,构建了植酸酶phyA2基因两个N-糖基化突变体,来研究不同位点糖基化修饰的缺失对植酸酶蛋白的影响。主要研究结果如下:(1)以已克隆的黑曲霉Aspergillus niger 963的植酸酶phyA2基因为模板,利用Megaprimer PCR介导的定点突变技术在核酸水平上成功地实现了两个N糖基化位点的诱变,并构建了毕赤酵母表达载体pPIC9-N87Q和pPIC9-N102Q。转化酵母工程菌株GS115, 在发酵罐水平上成功表达出了两个重组蛋白。对重组蛋白进行SDS-PAGE检测和Western-blotting免疫印记分析,发现突变体蛋白表观分子量降低了约10kD左右。(2)对野生型和突变体蛋白酶活性的最适温度及其热稳定性进行了研究,结果表明:野生型植酸酶phyA2、突变体N87Q 和N102Q的最适温度都在50℃左右,而与野生型相比,突变体N87Q的酶活在45℃~50℃保持一个相对较高的水平。60℃处理1h后,N87Q剩余约50%的酶活性, 与野生型(WT)相比稳定性降低了约20% 。 N102Q处理10min后,剩余约10%的酶活性,15min后完全丧失活性。 70℃处理2min后,野生型WT和突变体N87Q剩余80%左右酶活,而N102Q活性则迅速降低到25%,其后它们的酶活基本保持不变。80℃处理2 min后,突变体和野生型酶活迅速降低到一个较低的水平,4min又升高约10%,其后基本保持在一个相对稳定水平。且N102Q随着处理温度的升高,稳定性反而增强。(3)对野生型和突变体蛋白最适pH和pH稳定性研究发现:突变体N87Q与野生型WT最适pH基本一致,在pH2.0和pH 6.0处出现两个峰,而N102Q峰值出现在pH2.5和pH 6.0,但pH2.5处的酶活力明显降低。pH稳定性研究表明:与野生型相比,N87Q稳定性变化不大,在整个pH范围内均保留了约70%的酶活,而N102Q在pH<3的环境下酶活损失约50%,而在pH>8的环境下完全失活。(4)金属离子、EDTA和SDS对突变体和野生型酶活的影响差异不大。其中1mmol/L的Fe2+、Fe3+、Ca2+、Co2+、Mn2+、K+和EDTA对酶具有显著地激活作用,而Mg2+ 、Zn2+ 、Cu2+ 对酶促反应具有极强的地抑制作用,Ag+ 轻微的抑制了酶促反应,而作为较强变性剂的SDS对酶活的影响反而不大。(5)突变体和野生型的比活,酶促反应动力学常数Km和Vmax基本没有差异。比活=100,000IU/mg左右,Km=0.435mmol/L,Vmax=3,547,357.218IU/mg.min。
Other AbstractGlycosylation is one of the ways for the post-translational modifications of Eukaryotic cells,which is extremely important for structure and function of proteins.The phytase from Aspergillus belongs to Histidine Acid Phosphatase family(EC.3.1.3.2),including a typical conserved motif of RHGARYP,which was related to catalytic activity of the enzyme.Previous results showed that the phytase from this family was a glycosylated protein with approximately 10 potential glycosylation sites.The activity of the phytase depends on the glycosylation modification,However the significance of this modification to the enzyme and the roles of different glycosylation sites in the catalytic reaction has not been reported.To study the glycosylation sites of the phytase from Aspergillus niger 963 in the significance of its enzymatic properties,we deleted two N-glycosylation sites of phyA2 gene by Megaprimer PCR-mediated gene site-directed mutagenesis techniques,and the mutants were expressed in the Pichia pastoris successfully.Main results follow as:(1)Using the cloned phyA2 gene from Aspergillus niger 963 as a template,we deleted two glycosylation sites by Megaprimer PCR-mediated gene site-directed mutagenesis techniques in the nucleic acid level.We also constructed expression vector pPIC9-N87Q,pPIC9-N102Q.The recombinant protein were expressed in Pichia pastoris GS115 by fermentation.The SDS-PAGE and Western-blotting analysis of recombiant proteins showed that the apparent molecular weight of the mutants were dropped about 10kD.(2)The study of the optimum temperature and the thermalstability revealed that the optimum temperature of wild type phytase phyA2 and its mutants N87Q and N102Q were 50℃,but the N87Q had a higher activity between 45℃ and 50℃ compared with the WT.The N87Q retained more than 50% of its initial activity after incubation at 60℃ for 1h,which was 20% lower than WT.N102Q retained more than 10% of its initial activity after 10 mins, but its activity was completely lost after 15 mins;after incubation at 70℃ for 2 mins,both WT and N87Q retained more than 80% of its initial activity,but N102Q was only 25%.Afterward their activity was maintained at this level;Pre-treatment 2 mins at 80℃,the activity of the WT and mutants were decreased to a lower level,but increased by 10% after 4 mins,and then,maintained a stable level.N102Q was more stable with the increased temperature.This is a surprising phenomenon.(3) The study of the optimum pH and pH-stabi...
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221909
Collection生命科学学院
Recommended Citation
GB/T 7714
殷亮. N-糖基化对Aspergillus niger 963植酸酶phyA2酶学性质的影响[D]. 兰州. 兰州大学,2010.
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