磷酸三酯酶突变体H23A的真核表达及性质表征  

Eukaryotic Expression and Characterization of the Mutant H23A of Phosphotriesterase

作　　者：詹冬玲[1] 任玉雪[1] 李克剑 闵伟红[1] 刘洋[3] 张英文[1] 刘景圣[1] 

机构地区：[1]吉林农业大学食品科学与工程学院,吉林长春130118 [2]中油吉化集团总医院药剂科,吉林吉林132022 [3]吉林化工学院化学与制药工程学院,吉林吉林132022

出　　处：《食品科学》2013年第23期250-255,共6页Food Science

基　　金：吉林农业大学博士启动基金项目(201223);国家自然科学基金项目(31070638);吉林省自然科学基金项目(20105109)

摘　　要：通过同源序列比对和晶体结构分析,嗜热菌Geobacillus kaustophilusHTA426磷酸三酯酶的H23位点高度保守,并且位于金属离子结合位点附近。结合Rosseta design程序设计,本实验将突变体H23A在毕赤酵母GS115中高效表达。通过组氨酸标签镍柱分离纯化、非变性蛋白电泳和Western blotting鉴定表达产物,证明该突变体酶主要是以介于单体和二聚体之间的寡聚形式存在。初步酶学性质研究表明,突变体酶是别构酶,V max为92.45U/mg,Hill系数h为1.98;最适温度为70℃,最适pH值为10.0;70℃的热稳定性良好,半衰期为5.1h,而且大多数二价金属离子对突变体H23A都有激活作用。Through homologous sequence alignment and crystal structure analysis, it＇s found that the site of histidine 23 （H23） from the phosphotriesterase （PTE）-encoding gene of Geobacillus kaustophilus HTA426 PTE was highly conservative and was located near the metal ion binding sites. By using the Rosseta design program, the mutant H23A was overexpressed in Pichia pastoris GS 115. Through His-tagged ni-sepharose chromatography, non-denaturing electrophoresis and westernblotting, the recombinant enzyme was identified as an intermediate form between a monomer and a dimmer. The primary enzymatic properties indicated that the expressed recombinant enzyme was an allosteric enzyme with Vmax and hill coefficient （h） of 92.45 U/mg and 1.98, respectively. The optimal temperature and pH were 70 ℃ and 10.0, respectively. The recombinant PYE enzyme had a better heat tolerance with half-time of 5.1 h. Most divalent metal ions showed activating effects on the enzyme activity.

关 键 词：磷酸三酯酶 突变体 真核表达 性质 

分 类 号：Q786[生物学—分子生物学]