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J. Microbiol. Biotechnol. 2004; 14(2): 231-236

Published online April 28, 2004

Copyright © The Korean Society for Microbiology and Biotechnology.

One-step Purification of Poly-His Tagged Penicillin G Acylase Expressed in E. coli

Kim Jin Hee , Hye Jin Kang , Eung Soo Kim , Jeong Ho Kim 1 and Yoon Mo Koo 1*

Department of Biological Engineering ERC for the Advanced Bioseparation Technology Inha University Incheon 402-751 Korea, 1Department of Biology Inha University Incheon 402-751 Korea

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: PenicillinGacylase, E.coliATCC11105, IMAC, one-steppurification, osmoticshock