Characterization of recombinant glutamine synthetase from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1

Author:

Adul Rahman R N1,Jongsareejit B1,Fujiwara S1,Imanaka T1

Affiliation:

1. Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.

Abstract

The glnA gene encoding glutamine synthetase was cloned from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1, and its nucleotide sequence was determined. The glnA gene was expressed in Escherichia coli ME8459 (glnA mutant strain), and the protein was purified to homogeneity and shown to be functional in a dodecameric from (637,000 Da), exhibiting both transferase and synthetase activities. However, kinetic studies indicated that the enzyme possessed low biosynthetic activity, suggesting that the reaction was biased towards glutamate production. The optimum temperature for both activities was 60 degrees C, which was lower than the optimal growth temperature of KOD1. Recombinant KOD1 GlnA exhibited different optimum pHs depending on the reaction employed (pH 7.8 for the synthetase reaction and pH 7.2 for the transferase reaction). Of the various nucleoside triphosphates tested, GTP as well as ATP was involved in the synthetase reaction.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

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