Hydrolysis of penicillins and related compounds by the cell-bound penicillin acylase of Escherichia coli

Abstract

1. A method is given for the preparation of penicillin acylase by using Escherichia coli N.C.I.B. 8743 and a strain selected for higher yield. The enzyme is associated with the bacterial cells and removes the side chains of penicillins to give 6-amino-penicillanic acid and a carboxylic acid. 2. The rates of penicillin deacylation indicated that p-hydroxybenzylpenicillin was the best substrate, followed in diminishing order by benzyl-, dl-α-hydroxybenzyl-, 2-furylmethyl-, 2-thienylmethyl-, d-α-aminobenzyl-, n-propoxymethyl- and isobutoxymethyl-penicillin. Phenylpenicillin and dl-α-carboxybenzylpenicillin were not substrates and phenoxymethyl-penicillin was very poor. 3. Amides and esters of the above penicillins were also substrates for the deacylation reaction, as were cephalosporins with a thienylmethyl side chain. 4. For the deacylation of 2-furylmethylpenicillin at 21° the optimum pH was 8·2. The optimum temperature was 60° at pH7. 5. By using selection A of N.C.I.B. 8743 and determining reaction velocities by assaying yields of 6-amino-penicillanic acid in a 10min. reaction at 50° and pH8·2, the Km for benzylpenicillin was found to be about 30mm and the Km for 2-furylmethylpenicillin, about 10mm. The Vmax. values were 0·6 and 0·24μmole/min./mg. of bacterial cells respectively.