Penicillin Acylase in the Industrial Production of β-Lactam Antibiotics

(1987). Possible Physiological Roles of Carboxylic Ester Hydrolases. Drug Metabolism Reviews: Vol. 18, No. 4, pp. 379-439.

Possible Physiological Roles of Carboxylic Ester Hydrolases

Biotransformations catalyzed by multimeric enzymes: stabilization of tetrameric ampicillin acylase permits the optimization of ampicillin synthesis under dissociation conditions.

In the course of more than 60-year history, penicillin G acylase (PGA) gained a unique position among enzymes used by pharmaceutical industry for production of β-lactam antibiotics. Kinetically controlled enzymatic syntheses of cephalosporins of novel generations in which PGA catalyzes coupling of activated acyl donor with nucleophile belong among the latest large-scale applications. Contrary to rather specific roles of other enzymes involved in β-lactam biocatalyses, the PGA seems to have the greatest potential. On the laboratory scale, other applications with industrial potential were described, e.g., directed evolution of the enzyme to meet specific demands of industrial processes or its modification into the enzyme catalyzing reactions with novel substrates. The fact that β-lactams represent the most important group of antibiotics comprising 65 % of the world antibiotic market explains such a tremendous and continuous interest in this enzyme. Indeed, the annual consumption of PGA has recently been estimated to range from 10 to 30 million tons. The application potential of the enzyme goes beyond the β-lactam biocatalysis due to its enantioselectivity and promiscuity: the PGA can be used for the production of achiral and chiral compounds convenient for the preparation of synthons and active pharmaceutical ingrediences, respectively. These biocatalyses, however, still wait for large-scale application.

Current state and perspectives of penicillin G acylase-based biocatalyses

Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method.

A partially purified preparation of an α-amino acid ester hydrolase was obtained from Acetobacter turbidans A.T.C.C. 9325, which catalyses synthesis of 7-(d-α-amino-α-phenylacetamido)-3-cephem-3-methyl-4- carboxylic acid (cephalexin) from methyl d-α-aminophenylacetate and 7-amino-3-deacetoxycephalosporanic acid. The enzyme preparation catalysed both cephalosprin synthesis from 7-amino-3-deacetoxycephalosporanic acid and suitable amino acid esters (e.g. methyl d-α-aminophenylacetate, l-cysteine methyl ester, glycine ethyl ester, d-alanine methyl ester, methyl dl-α-aminoiso-butyrate, l-serine methyl ester, d-leucine methyl ester, l-methionine methyl ester) and the hydrolysis of such esters. The substrate specificity of the enzyme preparation for the hydrolysis closely paralleled the acyl-donor specificity for cephalosporin synthesis, even to the reaction rates. Only α-amino acid derivatives could act as acyl donors. The hydrogen atom on the α-carbon atom was not always required by acyl donors. The hydrolysis rate was markedly diminished by adding 7-amino-3-deacetoxycephalosporanic acid to reaction mixtures, but no effect on the total reaction rate (the hydrolysis rate plus synthesis rate) was observed with various concentrations of 7-amino-3-deacetoxycephalosporanic acid. Both the hydrolytic and the synthetic activities of the enzyme preparation were inhibited by high concentrations of some acyl donors (e.g. methyl d-α-aminophenylacetate, ethyl d-α-aminophenylacetate). The enzyme preparation hydrolysed α-amino acid esters much more easily than α-amino acid derivatives with an acid–amide bond.

Substrate specificity of an α-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325

BACTERIOLYTIC ENZYME IS PRODUCED BY CULTIVATING A BACTERIOLYTIC ENQYME-PRODUCING MICROORGANISM BELONGING TO THE GENUS ACHROMOBACTER IN A GROWTH MEDIUM AND RECOVERING THE ACCUMLATING BACTERIOLYTIC ENZYME THEREFROM. THE BACTERIOLYTIC ENZYME AND PREPARATIONS CONTAINING IT ARE SPECIFICALLY USEFUL IN DETERGENT COMPOSITIONS, ORAL COMPOSITIONS FOR USE IN PREVENTING DENTAL CAVITIES AND EYEWASH COMPOSITION.

Takeshi Takahashi

Papers

Substrate specificity of an α-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325

Bacteriolytic enzyme and process for the production thereof