화학공학소재연구정보센터
홈 로그인 로그아웃 연락처 사이트맵
Macromolecular Research, Vol.21, No.1, 5-9, January, 2013
DOI10.1007/s13233-013-1007-8  Export Citation
One-step immobilization and purification of his-tagged enzyme using poly(2-acetamidoacrylic acid) hydrogel
Eun-Ju Ha, Kyeong Kyu Kim1, Hyung Soon Park2, Sun-Gu Lee3, Jang-Oo Lee, Seong Soo A. An [email protected]4, and Hyun-jong Paik†
  • Department of Polymer Science and Engineering, Pusan National University, Busan, 60935, Korea
  • 1Department of Molecular and Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan School of Medicine, Gyeonggi, 440-746, Korea
  • 2Probiond Co., Ltd., Gwanjin VBS Center, Seoul, 143-834, Korea
  • 3Department of Chemical Engineering, Pusan National University, Busan, 60935, Korea
  • 4Department of BioNano Technology, Gachon University, Gyeonggi, 461-701, Korea
E-mail:
Ni2+-Complexed poly(2-acetamidoacrylic acid) (PAAA) hydrogel support was developed for the one-step immobilization and purification of recombinant histidine-tagged glutamyl aminopeptidase (His-tagged GAP). Ni2+-PAAA hydrogel was prepared from the polymerization of 2-acetamidoacrylic acid (AAA) and 2,2-[(1,4-dioxo-1,4-butanediyl) diamino] bis(2-propenoic acid) (DBDBPA) with potassium persulfate in dimethyl sulfoxide (DMSO), followed by Ni2+complexation. His-tagged GAP was immobilized directly from the cell lysate onto the Ni2+-PAAA hydrogel support and then purified. Catalytic activity of immobilized His-tagged GAP for the hydrolysis of alanylpara-nitroanilide revealed 90% conversion after 30 min of incubation, indicating sustained catalytic activity. The hydrogel-immobilized enzyme also exhibited enhanced thermal stability of sustained 70% activity after 1 h incubation at 60 °C, while the free enzyme activity was reduced to 50% at the same condition. After four cycles of hydrogel regeneration, the immobilized enzyme lost only 20% of its initial activity. Ni2+-PAAA hydrogel provided a new and convenient immobilization/purification system for His-tag enzymes through easy and simple procedures.
Keywords:poly(2-acetamidoacrylic acid) hydrogel;purification;immobilization;his-tagged enzyme
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