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On the production of α,β‐heterodimeric acyl‐coenzyme A: isopenicillin N ‐acyltransferase of Penicillium chrysogenum
Author(s) -
Aplin Robin T.,
Baldwin Jack E.,
Cole Stephen C.J.,
Sutherland John D.,
Tobin Matthew B.
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)80060-8
Subject(s) - penicillium chrysogenum , acyltransferase , biochemistry , enzyme , coenzyme a , biosynthesis , cofactor , overproduction , lac operon , biology , acyl carrier protein , chemistry , recombinant dna , gene , reductase
A high level E. coli expression system has been constructed for the Penicillium chrysogenum pen DE 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 Gly 102 /Cys 103 . 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.