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 degrees C and 7.0, respectively. The activity of Amd1 towards N-acetyl- l-alanine was enhanced by the divalent cation Co(2+), while Cd(2+ )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.
Curley
Peter
P
Department of Microbiology, National University of Ireland, Western Road, Cork, Ireland.
van der Does
Chris
C
Driessen
Arnold J M
AJ
Kok
Jan
J
van Sinderen
D
D
D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008670:Metals / Q000378:metabolism; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D013696:Temperature
D004269:DNA, Bacterial; D008670:Metals; D010447:Peptide Hydrolases; D000581:Amidohydrolases; C019672:aminoacylase I