Purification and properties of penicillin acylase of Bovista plumbea

Abstract

1. 1.|A penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) formed constitutively in the basidiomycete Bovista plumbea was purified 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. 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. K m for the best substrate phenoxymethylpenicillin was 1.67 mM. The maximum of activity occurred at 52°C and pH 7.5. The activation energy calculated by Arrhenius' graphic method was 16.45 kJ/mol. 3. 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. 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.