利用N-糖基化修饰对β-甘露聚糖酶Man47的稳定性改造  

The Stability Reconstruction of β-mannanase with N-glycosylation Modification

作　　者：谢春芳[1,2] 黎玉凤[3,2] 刘大岭[1,2] 姚冬生[3,2,4] 

机构地区：[1]暨南大学生物工程学系,广州510632 [2]广东省生物工程药物重点实验室,广州510632 [3]暨南大学生物医药研究院,广州510632 [4]基因工程药物国家工程研究中心,广州510632

出　　处：《中国生物工程杂志》2013年第12期79-85,共7页China Biotechnology

基　　金：国家"863"计划资助项目(2013AA102801)

摘　　要：N-糖基化是真核生物蛋白质最重要的翻译后修饰之一。以Armillariella tabescensβ-甘露聚糖酶Man47为研究对象,利用计算化学对A.tabescensβ-甘露聚糖酶Man47进行理性设计,经过分子对接、二级结构分析和糖基化的可行性分析后,构建具有EAS(enhanced aromatic sequence)序列的突变体g-123作为N-糖基化的突变位点。将其整合到毕赤酵母表达载体SMD1168上,通过电转化得到重组转化子。最后对突变体g-123的温度稳定性、酸碱稳定性、胃蛋白酶和胰蛋白酶抗性进行分析。结果表明:糖基化A.tabescensβ-甘露聚糖酶Man47突变体g-123与野生型相比,其热稳定性、酸碱稳定性、蛋白酶抗性均得到不同程度的改善。N-glycosylation is one of the most important posttranslational modification of proteins in eukaryotes. A rational strategy to introduce N-glycosylation site was proposed to ArmillarieUa tabescens beta mannose Man47. Then g-123 mutant with EAS （enhanced aromatic sequence） sequence was built through the molecular docking, secondary structure analysis and feasibility analysis of glycosylation. The sequence of g-123 mutant was inserted into SMDl168 with the yeast a-mating factor, then transformed it into Pichia by electroporation to obtain the recombinants. Finally the thermal stability, acid and alkali stability, pepsin- resistance and trypsin-resistance of g-123 and wild type were analyzed. The results showed that compared with wild type, the thermal stability, acid and alkali stability, protease resistance of the mutant g-123 with glycosylation was improved.

关 键 词：N-糖基化 翻译后修饰 蛋白质设计 生物信息学 

分 类 号：Q816[生物学—生物工程]