Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli

Abstract

The penicillin G acylase genes from the Proteus rettgeri wild type and from a hyperproducing mutant which is resistant to succinate repression were cloned in Escherichia coli K-12. Expression of both wild-type and mutant P. rettgeri acylase genes in E. coli K-12 was independent of orientation in the cloning vehicle and apparently resulted from recognition in E. coli of the P. rettgeri promoter sequences. The P. rettgeri acylase was secreted into the E. coli periplasmic space and was composed of subunits electrophoretically identical to those made in P. rettgeri. Expression of these genes in E. coli K-12 was not repressed by succinate as it is in P. rettgeri. Instead, expression of the enzymes was regulated by glucose catabolite repression.

Site‐directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105

“…This is supported by other partial (Bruns et al, 1985;Oliver et al, 1985) and complete (Oh et al, 1987;Guo et al, 1989) sequencing of DNA from the same or similar strains of E. coli. Similar processing has been reported for penicillin G acylase from other organisms (Daumy et al, 1985;Barber0 et al, 1986;Ohashi et al, 1988) and for cephalosporin acylases (Matsuda et al, 1987a, b) although a bacilliary penicillin V acylase appears to be an unrelated homotetramer (Olsson & U h l h , 1986;Olsson et al, 1985).…”

“…It is well established that phenylmethylsulphonyl fluoride (PhMeSO,F), which has a structural resemblance to the hydrolysis reaction product phenylacetic acid, reacts stoichiometrically with penicillin acylase (Kutzbach & Rauenbusch, 1974;Shvyadas et al, 1977;Siewinski et al, 1984) and sub-unit-complementation experiments with the enzyme from Proteus rettgeri (Daumy et al, 1985) point to reaction with a site or sites in the P-polypeptide. Cysteine residues are absent from the mature protein, so that serine may be suggested as the likely active-site target of PhMeS02F.…”

The folding and solution conformation of penicillin G acylase

“…The catalytic site is situated on the p peptide, which contains a phenylmethylsulphonyl-fluoride-sensitive residue required for enzymatic activity, and the binding site conferring specificity for the penicillin side chain has been reported to be located on the a peptide [4]. Both peptides are required for enzymatic activity [5].To produce PA in large amounts by recombinant DNA techniques it would be necessary to reproduce the activation and secretion process at high load of precursor. An alternative approach might be for DNA coding for the cy and fi chains to be cloned and expressed separately at high level, therefore probably leading to inclusion bodies which would have to be renatured and assembled to the active enzyme.…”

“…The catalytic site is situated on the p peptide, which contains a phenylmethylsulphonyl-fluoride-sensitive residue required for enzymatic activity, and the binding site conferring specificity for the penicillin side chain has been reported to be located on the a peptide [4]. Both peptides are required for enzymatic activity [5].…”

Cloning of E. coli penicillin G acylase gene with mini-Mu containing a plasmid replicon

“…pat gene transcription (and hence expression) is induced by phenyl acetic acid (PAA) or other aryl fatty acids and salts and is subjected to catabolic repression by glucose, fructose, lactose and other carbon sources (Kaufmann and Bauer, 1%4, Gang and Shaikh, 1976;Vojti?tek and Slezak, 1975;Shewale and Sivaraman, 1989;Merino et uZ., 1992). To overcome such inappropriate induction characteristics in native strain and to obtain higher yields, there have been a lot of attempts to construct recombinant strains producing PGA (Mayer et al, 1980, &homer et aZ., 1984, Daumy et al, 1986Garcia and Buesa, 1986;Schumacher et OZ., 1986;Meevotisom and Saunders, 1987;Oh et aL, 1987;Ohashi et aZ., 1989;Chang and Lee, 1990;Panbangred et aZ., 1990;Zhang et aL, 1990). In all these works, authors used "classical" in vitro cloning strategy.…”