Purification and biochemical characterization of recombinant alanine dehydrogenase from Thermus caldophilus GK24

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Title
Purification and biochemical characterization of recombinant alanine dehydrogenase from Thermus caldophilus GK24
Author(s)
Jung Don Bae; Y J Cho; Doo Il Kim; Dae Sil Lee; H J Shin
Bibliographic Citation
Journal of Microbiology and Biotechnology, vol. 13, no. 4, pp. 628-631
Publication Year
2003
Abstract
The recombinant alanine dehydrogenase (ADH) from E. coli containing Thermus caldophilus ADH was purified to homogeneity from a cell-free extract. The enzyme was purified 38-fold with a yield of 68% from the starting cell-free extract. The purified enzyme gave a single band in polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 45 kDa. The pH optimum was 8.0 for reductive amination of pyruvate and 12.0 for oxidative deamination of L-alanine. The enzyme was stable up to 70°C. The activity of the enzyme was inhibited by 1 mM Zn2+, 20% hexane, and 20% CHCl3. However, 10 mM Mg2+ and 40% propanol had no effect on the enzyme activity. The Michaelis constants (Km) for the substrates were 50 μM for NADH, 0.2 mM for pyruvate, 39.4 mM for NH4+, 2.6 mM for L-alanine, and 1.8 mM for NAD+.
Keyword
alanine dehydrogenasecharacterizationenzyme purificationthermus caldophilus GK24alaninethermus caldophilusthermus
ISSN
1017-7825
Publisher
Korea Soc-Assoc-Inst
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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