Purification and characterization of L-aminoacylase from Pseudomonas maltophila B1

The constitutive L-aminoacylase, which is used for optical resolution of DL-α-aminosuberic acid (DL-Asu), has been purified and characterized from Pseudomonas maltophila B1. The crude enzyme showed a specific activity of 0.062 units/mg for N-acetyl(Ac)-L-Asu. This value is very high compared with those from Aspergillus melleus, porcine kidney, and Bacillus stearothermophilus. Molecular masses of 108 kDa for the native enzyme and 50 kDa for the subunit were determined, indicating a dimer. The enzyme activity was optimal at pH 8.0 and at 55°C. The enzyme hydrolyzed N-acyl derivatives of various neutral L-amino acids and acidic L-amino acids, L-glutamate and L-Asu. The enzyme also had dipeptidase activity. The K_m values for N-Ac-L-alanine and N-Ac-DL-Asu were determined at 2.32 and 12.7 mM, respectively. The apoenzyme was activated using Zn²⁺, Ca²⁺, and Co²⁺. Glyoxylate, DL-lactate, phenylboronic acid (PBA), butaneboronic acid (BBA), diethylpyrocarbonate (DEP), and phenylglyoxal (PGO) inhibited enzyme activity.