Purification and characterisation of a lactococcal aminoacylase

The amd1-encoded aminoacylase from Lactococcus lactis MG1363 was cloned and overexpressed in Escherichia coli and purified. The assumed dimeric enzyme has a subunit molecular mass of about 42 kDa and contains 2.0±0.1 g-atoms of zinc and cobalt, in equimolar amounts, per subunit of Amd1. The enzyme was characterised with respect to substrate specificity, pH, temperature and metal dependence. Amd1 exhibited a broad activity range towards N-acetylated-L-amino acids with a strong preference towards those containing neutral aliphatic and aromatic side chains. It hydrolysed N-acetyl-L-alanine most efficiently, and exhibited temperature and pH optima of 30°C and 7.0, respectively. The activity of Amd1 towards N-acetyl-L-alanine was enhanced by the divalent cation Co²⁺, while Cd²⁺ inhibited activity. Interestingly, Amd1 was shown to catalyse the hydrolysis of several dipeptides at pH 7.0, although with reduced V_max values as compared to hydrolysis of N-acetylated-L-amino acids. This characteristic has also biological significance since Amd1 was able to complement a growth deficiency in a L. lactis triple peptidase mutant.