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Characterization of Recombinant Glyoxylate Reductase from Thermophile Thermus thermophilus HB27
Author(s) -
Ogino Hiroyasu,
Nakayama Hitoshi,
China Hideyasu,
Kawata Takuya,
Doukyu Noriyuki,
Yasuda Masahiro
Publication year - 2008
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp0702469
Subject(s) - thermus thermophilus , thermostability , thermophile , glyoxylate cycle , enzyme kinetics , thermus , biochemistry , enzyme , reductase , escherichia coli , chemistry , biology , stereochemistry , active site , gene
A glyoxylate reductase gene from the thermophilic bacterium Thermus thermophilus HB27 (TthGR) was cloned and expressed in Escherichia coli cells. The recombinant enzyme was highly purified to homogeneity and characterized. The purified TthGR showed thermostability up to 70 °C. In contrast, the maximum reaction condition was relatively mild (45 °C and pH 6.7). Although the k cat values against co‐enzyme NADH and NADPH were similar, the K m value against co‐enzyme NADH was ∼18 times higher than that against NADPH. TthGR prefers NADPH rather than NADH as an electron donor. These results indicate that a phosphate group of a co‐enzyme affects the binding affinity rather than the reaction efficiency, and TthGR demands appropriate amount of phosphate for a high activity. Furthermore, it was found that the half‐lives of TthGR in the presence of 25% dimethyl sulfoxide and diethylene glycol were significantly longer than that in the absence of an organic solvent.