Purification, characterization and induction of L‐phenylalanine ammonia‐lyase in Phaseolus vulgaris

The enzyme L‐phenylalanine ammonia‐lyase was purified from leaves of Phaseolus vulgaris by Sephacryl S‐200 gel filtration and Sepharose‐4‐B–succinyl‐aminoethyl‐L‐phenylalanine affinity chromatography. L‐Phenylalanine ammonia‐lyase was specifically eluted from the affinity matrix with its substrate L‐phenylalanine at 20–25°C. The purified enzyme was shown to be homogeneous by gel electrophoresis both in presence and absence of SDS. Its M r , determined by gel filtration and non‐denaturing gel electrophoresis, was 320000 ± 9000 and 330000 ± 4000 respectively. After SDS electrophoresis only one band of M r 83000 ± 4000 was detected, indicating that the enzyme is an oligomer containing four subunits. The pH optimum of enzyme activity was 8.8–9.2. Ampholyte isoelectrofocusing in polyacrylamide demonstrated the presence of a single charged species at pH 4.2. The homogeneous enzyme catalyzed the deamination of L‐phenylalanine to trans ‐cinnamate but did not catalyze the transamination of L‐phenylalanine to L‐phenylpyruvate. The enzyme showed K m 1.25 mM for L‐phenylalanine. Antibodies to homogeneous L‐phenylalanine ammonia‐lyase recognised specific epitopes on L‐phenylalanine aminotransferase as demonstrated by immunoaffinity purification and immunoblotting. The induction of L‐phenylalanine ammonia‐lyase activity during phaseollin biosynthesis in the Phaseolus vulgaris – Colletotrichum lindemuthianum interaction was regulated by an increase in enzyme concentration resulting from an increase in de novo synthesis of L‐phenylalanine ammonia‐lyase protein.