Topic
Penicillin amidase
About: Penicillin amidase is a research topic. Over the lifetime, 576 publications have been published within this topic receiving 15563 citations. The topic is also known as: penicillin amidohydrolase & ampicillin acylase.
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TL;DR: Penicillin G acylase CLEAs had the same activity in the synthesis of ampicillin as cross-linked crystals of the same enzyme, but the accompanying hydrolysis of the side-chain donor was much less.
450 citations
TL;DR: The analysis shows that the environment of the catalytically active N-terminal serine of the B chain contains no adjacent histidine equivalent to that found in the serine proteases, indicating that this must be an important recognition site for cleavage.
Abstract: PENICILLIN acylase (penicillin amidohydrolase, EC 3.5.1.11) is widely distributed among microorganisms, including bacteria, yeast and filamentous fungi. It is used on an industrial scale for the production of 6-aminopenicillanic acid, the starting material for the synthesis of semi-synthetic penicillins. Its in vivo role remains unclear, however, and the observation that expression of the Escherichia coli enzyme in vivo is regulated by both temperature and phenylacetic acid has prompted speculation that the enzyme could be involved in the assimilation of aromatic compounds as carbon sources in the organism's free-living mode1. The mature E. coli enzyme is a periplasmic 80K heterodimer of A and B chains (209 and 566 amino acids, respectively2,3) synthesized as a single cytoplasmic precursor containing a 26-amino-acid signal sequence to direct export to the cytoplasm4 and a 54-amino-acid spacer between the A and B chains which may influence the final folding of the chains5. The N-terminal serine of the B chain reacts with phenylmethylsulphonyl fluoride, which is consistent with a catalytic role for the serine hydroxyl group. Modifying this serine to a cysteine6'7 inactivates the enzyme, whereas threonine, arginine or glycine substitution prevents in vivo processing of the enzyme7, indicating that this must be an important recognition site for cleavage. Here we report the crystal structure of penicillin acylase at 1.9 A resolution. Our analysis shows that the environment of the catalytically active N-terminal serine of the B chain contains no adjacent histidine equivalent to that found in the serine proteases. The nearest base to the hydroxyl of this serine is its own α-amino group, which may act by a new mechanism to endow the enzyme with its catalytic properties.
434 citations
TL;DR: Progressive establishment of new enzyme-support attachments was showed by the progressive irreversible covalent immobilization of several subunits of multi-subunits proteins, and the final stabilization factor become 100-fold comparing soluble penicillin G acylase and optimal derivative.
337 citations
TL;DR: The enzyme was shown to be inhibited by excess substrate, benzylpenicillin, and by both of the products of hydrolysis, which was found to be competitive and by 6-aminopenicillanic acid to be non-competitive.
282 citations
TL;DR: It is proposed that macromolecular cross‐linkers are too large to penetrate the protein active site and react with catalytically essential amino acid residues.
Abstract: Cross-linked enzyme aggregates (CLEAs) were prepared from several enzymes (penicillin G acylase, hydroxynitrile lyase, alcohol dehydrogenase, and two different nitrilases) by precipitation and subsequent cross-linking using dextran polyaldehyde. In most cases, higher immobilization yields were obtained using the latter cross-linker as compared with the commonly used glutaraldehyde. Active site titration of penicillin acylase CLEAs showed that the higher activity originated from a significantly lower loss in active sites using dextran polyaldehyde as a cross-linking agent. It is proposed that macromolecular cross-linkers are too large to penetrate the protein active site and react with catalytically essential amino acid residues.
273 citations