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A thermostable L-aminoacylase from Thermococcus litoralis: cloning, overexpression, characterization, and applications in biotransformations
Toogood, H.S.; Hollingsworth, E.J.; Brown, R.C.; Taylor, I.N.; Taylor, S.J.C.; McCague, R.; Littlechild, J.A. (2002). A thermostable L-aminoacylase from Thermococcus litoralis: cloning, overexpression, characterization, and applications in biotransformations. Extremophiles 6(2): 111-122. hdl.handle.net/10.1007/s007920100230
In: Extremophiles. Springer: Tokyo. ISSN 1431-0651, more
Peer reviewed article  

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Keyword
    Marine

Authors  Top 
  • Toogood, H.S.
  • Hollingsworth, E.J.
  • Brown, R.C.
  • Taylor, I.N.
  • Taylor, S.J.C.
  • McCague, R.
  • Littlechild, J.A., more

Abstract
    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°C in Tris-HCl at pH 8.0. The recombinant enzyme is thermostable, with a half-life of 25 h at 70°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.

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