Improvement of the catalytic properties of penicillin G acylase from Escherichia coli ATCC 11105 by selection of a new substrate specificity.
Cloned penicillin G acylase (PGA) from Escherichia coli ATCC 11105 was mutagenized in vivo using N-methyl-N'-nitro-N-nitrosoguanidine. Mutants of PGA were selected by their ability to allow growth of the host strain E. coli M8820 with the new substrates phenylacetyl-beta-alanyl-L-proline (PhAc-beta Ala-Pro) phthalyl-L-leucine (Pht-Leu) or phthalylglycyl-L-proline (Pht-Gly-Pro) as sole source of proline and leucine respectively. PGA mutants were purified and immobilized onto spherical methacrylate (G-gel). The immobilized form of mutant PGA selected with (PhAc-beta Ala-Pro) hydrolyzed 95% of 9 mmol penicillin G 30% faster than wild-type PGA using the same specific activities. The specific activity of the soluble enzyme was 2.7-fold, and inhibition by phenylacetic acid was halved. Immobilized PGA mutant selected with Pht-Gly-Pro hydrolyzed penicillin G 20% faster than wild-type PGA. The Km of the soluble enzyme was increased 1.7-fold. Furthermore, the latter two mutants were also 3.6-fold more stable at 45 degrees C than wild-type PGA. The specific activity of the mutant selected with Pht-Leu was 6.3-fold lower, and inhibition by phenylacetic acid was increased 13-fold.
Niersbach
H
H
University of Kaiserslautern, Department of Microbiology, Germany.
Kühne
A
A
Tischer
W
W
Weber
M
M
Wedekind
F
F
Plapp
R
R
D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D016296:Mutagenesis; D010399:Penicillin Amidase / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D010399:Penicillin Amidase