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Gene expression and characterization of thermostable glutamate decarboxylase from Pyrococcus furiosus
- Lee, Eon-Seok, Kim, Han-Woo, Kim, Dong-Eun, Kim, Yeon-Hee, Nam, Soo-Wan, Kim, Byung-Woo, Jeon, Sung-Jong
- Biotechnology and bioprocess engineering 2013 v.18 no.2 pp. 375-381
- Escherichia coli, Pyrococcus furiosus, bacteria, calcium chloride, gene expression, genes, glutamate decarboxylase, glutamic acid, half life, heat inactivation, molecular weight, pH, polyacrylamide gel electrophoresis, potassium chloride, sodium chloride, temperature, thermal stability
- Gene encoding for a putative glutamate decarboxylase (GAD: EC 126.96.36.199) from the hyperthermophilic archaeon Pyrococcus furiosus was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (PF1159) from P. furiosus showed some identity with other group II decarboxylases from an archaea and bacteria. The GAD from P. furiosus (PfGAD) was expressed in Escherichia coli, and the recombinant protein has a molecular mass of 41 kDa, determined by SDS-PAGE. The optimum temperature and pH for GAD activity were 75°C and 6.0, respectively. The half-life of heat inactivation was approximately 60 min at 90°C. The GAD activity was found to be dependent on various salts, such as CaCl₂, NaCl, KCl, and NaBr, with an optimum concentration of 400 mM, but not (NH₄)₂SO₄. PfGAD demonstrated activity against various substrates, such as L-glutamate, L-aspartate, and L-tyrosine. The results of the kinetics experiment indicated that L-aspartate was a better substrate of PfGAD than L-glutamate and Ltyrosine.