A thermostable L-aminoacylase from Thermococcus litoralis: cloning, overexpression, characterization, and applications in biotransformations.
A thermostable L-aminoacylase from Thermococcus litoralis was cloned, sequenced, and overexpressed in Escherichia coli. The enzyme is a homotetramer of 43 kDa monomers and has an 82% sequence identity to an aminoacylase from Pyrococcus horikoshii and 45% sequence identity to a carboxypeptidase from Sulfolobus solfataricus. It contains one cysteine residue that is highly conserved among aminoacylases. Cell-free extracts of the recombinant enzyme were characterized and were found to have optimal activity at 85 degrees C in Tris-HCl at pH 8.0. The recombinant enzyme is thermostable, with a half-life of 25 h at 70 degrees C. Aminoacylase inhibitors, such as mono-tert-butyl malonate, had only a slight effect on activity. The enzyme was partially inhibited by EDTA and p-hydroxymercuribenzoate, suggesting that the cysteine residue and a metal ion are important, but not essential, for activity. Addition of Zn2+ and Co2+ to the apoenzyme increased the enzyme activity, whereas Sn4+ and Cu2+ almost completely abolished enzyme activity. The enzyme was most specific for substrates containing N-benzoyl- or N-chloroacetyl-amino acids. preferring substrates containing hydrophobic, uncharged, or weakly charged amino acids such as phenylalanine, methionine, and cysteine.
Toogood
Helen S
HS
School of Chemistry, University of Exeter, UK.
Hollingsworth
Edward J
EJ
Brown
Rob C
RC
Taylor
Ian N
IN
Taylor
Stephen J C
SJ
McCague
Ray
R
Littlechild
Jennifer A
JA
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001711:Biotransformation; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019766:Genes, Archaeal; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012997:Solvents; D013379:Substrate Specificity; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics*
D008670:Metals; D011994:Recombinant Proteins; D012997:Solvents; D000581:Amidohydrolases; C019672:aminoacylase I