Purification and properties of penicillin acylase of Bovista plumbea.

1. A penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) formed constitutively in the basidiomycete Bovista plumbea was purifed 220-fold by a combination of two gel filtration runs, ion-exchange chromatography on DEAE-cellulose, ultrafiltration and final chromatography on hydroxyapatite. Recovery was 40%. 2. The enzyme was clearly distinguished from penicillin acylases previously characterized: the molecular weight of the purified enzyme was evaluated by gel filtration to be 88 000. Km for the best substrate phenoxymethylpenicillin was 1.67 mM. The maximum of activity occurred at 52 degrees C and pH 7.5. The activation energy calculated by Arrhenius' graphic method was 16.45 kJ/mol. 3. Neither 8-hydroxyquinoline nor EDTA, iodoacetic acid, or the products of enzymatic cleavage, 6-aminopenicillanic acid or phenoxyacetic acid, showed any characteristic inhibition effect. 4. The substrate spectrum of the enzyme was elucidated. Phenoxymethylpenicillin was the best substrate. N-Acylamino acids, dipeptides, and tripeptides were not hydrolyzed; affinity occurred only towards penicillins lacking a nitrogen atom in the side chain acid. Penicillins with aryloxy residues possessing hydrophilic groups are favoured above aryl residues and short side chains above bulky ones.