食品科学 ›› 2013, Vol. 34 ›› Issue (23): 250-255.doi: 10.7506/spkx1002-6630-201323051

• 生物工程 • 上一篇    下一篇

磷酸三酯酶突变体H23A的真核表达及性质表征

詹冬玲1,任玉雪1,李克剑2,闵伟红1,刘 洋3,张英文1,刘景圣1,*   

  1. 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
  • 收稿日期:2013-08-06 修回日期:2013-11-17 出版日期:2013-12-15 发布日期:2014-01-03
  • 通讯作者: 刘景圣 E-mail:1833801176@qq.com
  • 基金资助:

    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)

Eukaryotic Expression and Characterization of the Mutant H23A of Phosphotriesterase

ZHAN Dong-ling1,REN Yu-xue1,LI Ke-jian2,MIN Wei-hong1,LIU Yang3,ZHANG Ying-wen1,LIU Jing-sheng1,*   

  1. 1. College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China;2. Pharmacological
    Department, General Hospital of China National Petroleum Corporation in Jilin, Jilin 132022, China;3. College of Chemical and
    Pharmaceutical Engineering, Jilin Institute of Chemical Technology, Jilin 132022, China
  • Received:2013-08-06 Revised:2013-11-17 Online:2013-12-15 Published:2014-01-03
  • Contact: LIU Jing-sheng E-mail:1833801176@qq.com

摘要:

通过同源序列比对和晶体结构分析,嗜热菌Geobacillus kaustophilus HTA426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,Vmax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。

关键词: 磷酸三酯酶, 突变体, 真核表达, 性质

Abstract:

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 GS115. 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.

Key words: phosphotriesterase (PTE), mutant, eukaryotic expression, properties

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