Purification and characterisation of a lactococcal aminoacylase

Abstract

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.

Purification and Characterization of a Novel Aminoacylase fromStreptomyces mobaraensis

“…A number of studies have reported data on the substrate specificity of aminoacylases from various sources. [1][2][3][4][5][6][7][8][9][10][11][12][13] Table 5 shows the substrate specificity of some aminoacylases. As shown in Table 5, aminoacylase from S. mobaraensis shows wider substrate specificity than those from the other sources.…”

Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

“…Methionine, alanine and phenylalanine are produced using aminoacylase on a commercial scale [4][5][6][7]. Aminoacylase is an essential enzyme found in almost all organisms, and many of these enzymes are classified into the M20 family of metallopeptidases [4,[8][9][10][11]. In hyperthermophiles, several enzymes showing aminoacylase activity have been identified [5,12,13], but, to our knowledge, few reports have dealt with the amino acid residues involved in metal binding.…”

Aminoacylase 3 binds to and cleaves the N‐terminus of the hepatitis C virus core protein

“…Dipeptidase and carboxypeptidase activity of several microbial aminoacylases have been reported [21–24]. Cleavage of the neighbor amino acids in the C-terminus of HCVCP-short suggests that AA3 also possesses carboxypeptidase activity.…”

“…Our study provides the first evidence for the presence of endopeptidase activity in a mammalian aminoacylase. Dipeptidase and carboxypeptidase activity of several microbial aminoacylases have been reported [21][22][23][24]. Cleavage of the neighbor amino acids in the C-terminus of HCVCP-short suggests that AA3 also possesses carboxypeptidase activity.…”