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

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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

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

CLC Number: