Hydrolysis of penicillins and related compounds by the cell-bound penicillin acylase of Escherichia coli.
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-alpha-hydroxybenzyl-, 2-furylmethyl-, 2-thienylmethyl-, d-alpha-aminobenzyl-, n-propoxymethyl- and isobutoxymethyl-penicillin. Phenylpenicillin and dl-alpha-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 degrees the optimum pH was 8.2. The optimum temperature was 60 degrees 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 degrees and pH8.2, the K(m) for benzylpenicillin was found to be about 30mm and the K(m) for 2-furylmethylpenicillin, about 10mm. The V(max.) values were 0.6 and 0.24mumole/min./mg. of bacterial cells respectively.
D000577:Amides / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism*; D000667:Ampicillin / Q000378:metabolism; D002511:Cephalosporins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D004952:Esters / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008712:Methicillin / Q000378:metabolism; D010400:Penicillin G / Q000378:metabolism; D010404:Penicillin V / Q000031:analogs & derivatives / Q000378:metabolism; D010406:Penicillins / Q000378:metabolism*; D013696:Temperature
D000577:Amides; D002511:Cephalosporins; D004952:Esters; D010406:Penicillins; D000667:Ampicillin; D000581:Amidohydrolases; D010400:Penicillin G; D008712:Methicillin; D010404:Penicillin V