【摘要】 为研究N-糖基化对黑曲霉Aspergillus niger963植酸酶蛋白酶学性质的影响,利用Megaprimer PCR介导基因定点突变的技术,构建了植酸酶phyA2基因两个N-糖基化突变体,即将该基因编码蛋白质N87位和N102位的天冬酰胺密码子置换为编码与其具有相似结构的谷氨酰胺密码子,两个突变体分别命名为N87Q、N102Q,经测序结果比对和图谱分析,表明在核酸水平上成功实现了点突变,构建了酵母表达载体pPIC9-N87Q,pPIC9-N102Q,转化毕赤酵母GS115,经发酵罐水平诱导表达后,获得了N-糖基化缺失突变蛋白,对突变体蛋白在60℃进行处理发现,突变体N87Q处理1h后剩余50%的酶活,N102Q处理10min后酶活完全丧失,在37℃,不同的pH缓冲体系(pH1~10)处理1h,N87Q剩余约大于70%的活性,而N102Q在pH>8的环境下,没有检测到酶活。更多还原

【Abstract】 To study N-linked glycosylation of the phytase from Aspergillus niger 963 in the significance of its enzymatic properties,two N-glycosylation mutants by Megaprimer PCR-mediated gene site-directed mutagenesis techniques were constructed,Make the codon of N87 and N102-asparagine was replaced by the codon of glutamine.according to the location,the two mutants were named N87Q、N102Q,sequencing alignment and analysis showed that site-directed mutagenesis was achieved in the nucleic acid level,the expression vector pPIC9-N87Q,pPIC9-N102Q were constructed also.The recombinant protein were expressed in Pichia pastoris GS115 by fermentation.The N87Q retained more than 50% of its initial activity after incubation at 60℃ for 1h,while the activity of N102Q was lost completely after ten minutes.The N87Q retained more than 70% of its initial activity after being incubated under varying pH conditions(pH 1.0～10.0)at 37℃ for 1h,however,the N102Q was not active above pH 8.0.更多还原