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Applied and Environmental Microbiology, February 2004, p. 937-942, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.937-942.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Highly Stable L-Lysine 6-Dehydrogenase from the Thermophile Geobacillus stearothermophilus Isolated from a Japanese Hot Spring: Characterization, Gene Cloning and Sequencing, and Expression

Mojgan Heydari, Toshihisa Ohshima,* Naoki Nunoura-Kominato, and Haruhiko Sakuraba

Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan

Received 8 August 2003/ Accepted 13 November 2003

L-Lysine dehydrogenase, which catalyzes the oxidative deamination of L-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be {Delta}1-piperideine-6-carboxylate, indicating that the enzyme is L-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70°C, respectively. No activity was lost at temperatures up to 65°C in the presence of 5 mM L-lysine. The enzyme was relatively selective for L-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for L-lysine, NAD, and NADP at 50°C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.

* Corresponding author. Mailing address: Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1, Minamijosanjimacho, Tokushima 770-8506, Japan. Phone: 81-88-656-7518. Fax: 81-88-656-9071. E-mail: ohshima{at}bio.tokushima-u.ac.jp.

Applied and Environmental Microbiology, February 2004, p. 937-942, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.937-942.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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