Enzymes for Research, Diagnostic and Industrial Use

Penicillin Amidase

Official Full Name
Penicillin Amidase
The biosynthesis of Penicillin amidase in E. coli by hydrophobic protein chromatography is an inducible reaction which is regulated by metabolized carbon source (e.g. polyols, carboxylic acid etc.). It is also influenced by catabolite repression. It catalyzes the formation of amide bonds through an acyl-enzyme intermediate.
penicillin amidase; penicillin acylase; benzylpenicillin acylase; novozym 217; semacylase; α-acylamino-β-lactam acylhydrolase; ampicillin acylase; EC; 9014-06-6

Product Name
EC No.
CAS No.9014-06-6
CAS No.9014-06-6
SourceEscherichia col...
Related Protocols
penicillin amidase -Enzymatic Assay Protocol
penicillin amidase -Enzymatic Assay Protocol
Related Reading

Penicillin amidase (EC, PA, penicillin acylase) belongs to the class of hydrolases, a subclass of aminohydrolases, and represents a group of so-called N-terminal nucleophilic hydrolases. It catalytzes the penicillin and H2O to form carboxylate and 6-aminopenicillanate. The systematic name of this enzyme class is penicillin amidohydrolase. Other names in common use include penicillin acylase, benzylpenicillin acylase, alpha-acylamino-beta-lactam acylhydrolase, and ampicillin acylase. Penicillin amidase has been found in bacteria, yeast, and fungi. Its main function is in utilizing heterocyclic compounds as a source of carbon.


In order to categorize all different penicillin accepting enzymes, a classification of these enzyme has been proposed. The general name for this class of enzymes is alpha-acylamino-beta-lactam acylhydrolase with a division in several types according to the preferred acyl moiety.

Table 1. Classification of alpha-acylamino-beta-lactam acylhydrolase.

Type Preferred acyl moiety Names
I Phenylacetyl Penicillin acylase; Penicillin amidase; Benzylpenicillin acylase; Penicillin G acylase
II Phenoxyacetyl Penicillin V acylase
III Alpha-Aminoacyl Alpha-amino acid ester hydrolase; cephalexin synthetase; ampicillin acylase; D-phenylglycyl-beta-lactamide amidohydrolase
IV Glutaryl Glutaryl 7-ACA acylase; glutaryl acylase
V Adipyl Adipyl acylase; adipyl amidase


Both the cleavage and coupling reactions can be catalyzed by penicillin amidase. The enzyme is usually obtained from E. coli. It is a heterodimeric periplasmic protein consisting of a small a-subunit of 23 kD and a large b-subunit of 62 kD. The catalytic cycle starts with a nucleophilic attack by the hydroxyl group of the active-site serine that is located on the amino terminus of the β-chain (bS1) on the carbonyl carbon atom of the amide or ester bond of a substrate. Via a tetrahedral oxyanion intermediate an acyl-enzyme is formed concomitant with the departure of the leaving group. The acyl-enzyme can subsequently be deacylated by a b-lactam nucleophile yielding a semi-synthetic b-lactam antibiotic, or by H2O, resulting in the hydrolysis product.

Penicillin AmidaseFigure 1. Structure of penicillin amidase. (D.B. Janssen)

Catalytic Mechanism

Penicillin amidase catalyzes the transfer of an acyl group from one nucleophile to another. The hydrolysis of penicillin G constitutes the transfer of a phenylacetyl moiety from 6-aminopenicillanic acid to water. The mechanism of the acylation is similar to that involving other serine hydrolases, such as lipases and proteases, and proceeds via an acyl-enzyme intermediate. This covalent intermediate is formed by acylation of the hydroxyl group of an active serine residue by penicillin G upon liberation of 6-aminopenicillanic acid. Subsequently the acyl-enzyme can react with water to yield phenylacetic acid and the native enzyme. Since all of the steps are reversible, the reverse process—conversion of acids to amides—is possible, in principle, at low water activities.

Penicillin AmidaseFigure 2. Mechanism of penicillin acylase-catalyzed amide hydrolysis. (Van Langen, L.M. 2001)

In most serine hydrolases the nucleophilicity of the serine residue is enhanced by the coupled action of neighboring amino acid residues (the catalytic triad Asp-His-Ser). Inspection of the three-dimensional structure of penicillin acylase, however, revealed no basic residue near the catalytically active serine. Hence, a mechanism was postulated in which the hydroxyl function of the active serine of penicillin acylase is activated by its own N-terminal amino function via a bridging water molecule. The oxyanion tetrahedral intermediate is stabilized by interactions with the main chain amides of B23 and B69 and the Nδ of Asn241.

Physiological Function

Penicillin amidase is usually present in organisms that synthesize penicillin, but the physiological role of the enzyme is uncertain. The enzyme probably only accounts for a small degree of resistance to beta-lactam antibiotics compared with the major role of the beta-lactamases, but it may be significant in some organisms. For example, highly resistant strains of Francisella tularensis all possessed both beta-lactamase and penicillin acylase activity. In addition, an increase in PenG and PenV acylase activity has been observed during the autolysis of filamentous fungi.


Penicillin amidase has been extensively studied for more than 50 years. In practice, this enzyme is commonly used to produce 6-aminopenicillanic acid, which is the main synthon in the synthesis of penicillin antibiotics. Penicillin amidase is also used for the synthesis of various semi-synthetic β-lactam antibiotics. Broad substrate specificity and high regio-, chemo- and stereoselectivity of the enzyme are used for the production of chiral compounds (which are more and more in demand in modern pharmaceutics), as well as for the protection of hydroxy and amino groups in peptide and fine organic synthesis.


  1. Van Langen, L.M. Penicillin acylase: Properties, applications and prospects in the biocatalytic production of fine chemicals. 2001.
  2. Tishkov, V.I, Savin, S.S., Yasnaya, A.S. Protein engineering of penicillin acylase. Acta Naturae, 2010, 2(3):47-61.
  3. D.B. Janssen. Biocatalyst engineering for the production of semi-synthetic antibiotics. University of Groningen.

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