期刊论文详细信息
FEBS Letters
On the production of α,β‐heterodimeric acyl‐coenzyme A: isopenicillin N‐acyltransferase of Penicillium chrysogenum
Cole, Stephen C.J.1  Tobin, Matthew B.1  Aplin, Robin T.1  Baldwin, Jack E.1  Sutherland, John D.1 
[1] The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, South Parks Road, Oxford, 0X1 3QY, UK
关键词: Acyltransferase;    Penicillin biosynthesis;    Isopenicillin N;    Benzylpenicillin;    Penicillium;    Aspergillus. E. coli;   
DOI  :  10.1016/0014-5793(93)80060-8
学科分类:生物化学/生物物理
来源: John Wiley & Sons Ltd.
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【 摘 要 】

A high level E. coli expression system has been constructed for the Penicillium chrysogenum penDE gene, which encodes the acyl-coenzyme A: isopenicillin N-acyltransferase (AT) enzyme. Induction of overexpression of recombinant AT (recAT) by increasing the growth temperature of the host adversely affected solubility and activity of the AT enzyme. Addition of isopropylthio-β-d-galactopyranoside (IPTG) at decreased growth temperatures (less than 32°C) resulted in the overproduction of soluble, active recAT. When purified to homogeneity, recAT was an α,β-heterodimer, comprised of 11 kDa (α) and 29 kDa (β) subunits, derived from a 40 kDa precursor polypeptide by a posttranslational cleavage. The recAT enzyme contained both the acyl-coenzyme A: isopenicillin N-acyltransferase and the acyl-coenzyme A: 6-aminopenicillanic acid acyltransferase activities. The processing event that generated the two subunits of recAT from the 40 kDa precursor polypeptide occurred between Gly102/Cys103. This expression system produced a large amount of soluble, active recAT that is identical to native AT, making it a suitable source of AT enzyme for further characterization.

【 授权许可】

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