Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus

Sang Suk Kim, In Geol Choi, Sung Hou Kim, Yeon Gyu Yu

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts L- or D-glutamate to D- or L-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The K(m) and k(cat) values of the overexpressed A. pyrophilus glutamate racemase for the racemization of L-glutamate to the D-form and the conversion of D-glutamate to the L-form were measured as 1.8 ± 0.4 mM and 0.79 ± 0.06s-1 or 0.50 ± 0.07 mM and 0.25 ± 0.01 s-1, respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85°C for 90 min.

Original languageEnglish
Pages (from-to)175-183
Number of pages9
JournalExtremophiles
Volume3
Issue number3
DOIs
Publication statusPublished - 1999 Aug
Externally publishedYes

Keywords

  • Aquifex pyrophilus
  • Glutamate racemase
  • Molecular cloning
  • Overexpression
  • Thermostability

ASJC Scopus subject areas

  • Microbiology
  • Molecular Medicine

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