One step purification of PGA and immobilized reaction using IMAC

초록

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 method. 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). Effective secretion and high expression were examined as well as potential usefulness as a purification method. 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. JM109 harboring pPGA-HIS6 vector produced active his-tagged acylases in the presence of lac promoter during cultivation at 26 ºC3). The specific activity of the purified acylase by using one-step chromatography after osmotic shock was 38.5 U/mg and recovered with the yield of 70%. Both 23kDa(a subunit) and 62kDa(b subunit)1,2) were recovered by using IMAC with just C-terminus tagging of the b subunit. The purification of the periplasmic fraction by osmotic shock was 2.6 fold and that of purified acylase was 19 compared to the crude extract. In addition, in-situ immobilization of the periplasmic poly-His tagged PGA was performed. Immobilized acylase was used for the production of 6-APA from penicillin G in batch reaction at 37°C for 15min. The portion of protein bound to IMAC resin was higher than 95%. The production yield was about 80% and maintained during 10 successive operations.