Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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One-step Purification of Poly-His Tagged Penicillin G Acylase Expressed in E. coli

  • Kim, Jin-Hee (Department of Biologicla Engineering, ERC for the Advanced Bioseparation Technology, Inha University) ;
  • Kang, Hye-Jin (Department of Biologicla Engineering, ERC for the Advanced Bioseparation Technology, Inha University) ;
  • Kim, Eung-Soo (Department of Biologicla Engineering, ERC for the Advanced Bioseparation Technology, Inha University) ;
  • Kim, Jeong-Ho (Department of Biology, Inha University) ;
  • Koo, Yoon-Mo (Department of Biologicla Engineering, ERC for the Advanced Bioseparation Technology, Inha University)
  • Published : 2004.04.01
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Abstract

The inexpensive large-scale production of pure PGA (Penicillin G Acylase) has been a commercial goal. PGA has been used as a model enzyme in the development of simple one-step purification methods. In this study, the purification of poly-His tagged PGA protein secreted into the periplasmic space was carried out by using immobilized metal-ion affinity chromatography (IMAC). The PGA gene was obtained from E. coli ATCC 11105. Codons encoding histidines were fused at the C-terminus of the PGA gene by PCR. E. coli JM109 harboring pPGA-HIS6 vector produced active his-tagged acylases in the presence of lac promoter during cultivation at $26^{\circ}C$. The maximum specific activity of the acylase purified by using one-step chromatography after osmotic shock was 38.5 U/mg and was recovered with the yield of 70％. Both 23 kDa ($\alpha$) and 62 kDa ($\beta$) subunits were recovered by using IMAC with just C-terminus tagging of the $\beta$ subunit. The purification of the periplasmic fraction by osmotic shock and that of purified acylase was increased by 2.6-fold and 19-fold, respectively, compared to the crude extract.

Keywords

References

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