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Molecular Genetic Characterization of the Thermostable L-Lactate Dehydrogenase Gene (ldhL) of Thermoanaerobacter ethanolicus JW200 and Biochemical Characterization of the Enzyme

Q. Zhou* and W.-L. Shao

Jiangsu Key Laboratory for Biodiversity and Bio-resources, College of Life Sciences, Nanjing Normal University, Nanjing 210046, China; E-mail: qqzhouqing@hotmail.com

* To whom correspondence should be addressed.

Received October 26, 2009; Revision received November 10, 2009
The structural gene ldhL for a thermostable L-(+)-lactate dehydrogenase was cloned from Thermoanaerobacter ethanolicus JW200. The nucleotide sequence of the ldhL gene was determined and shown to have the capacity to encode a protein of 311 amino acids (33.5 kDa). By 5′-RACE analysis, the ldhL transcription start point was confirmed to be derived from the –10 region closest to the initiation codon. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by nickel-affinity chromatography. It was shown to be allosteric in the presence of fructose-1,6-bisphosphate. The optimum pH and temperature for the enzyme were 5.8 and 60°С in the pyruvate reduction and 7.0 and 70°С in the lactate oxidation reaction, respectively. The kinetic parameters Km,app and kcat,app for pyruvate were 0.18 mM and 520 U/mg, respectively, and in the absence of fructose-1,6-bisphosphate, a 1.2-fold increase in Km,app and a 16-fold decrease in kcat,app were determined. The Km,app and kcat,app values for lactate were 60 mM and 0.58 U/mg, respectively, and they were not affected by fructose-1,6-bisphosphate. The enzyme was greatly inhibited by Zn2+, Ag+, Cu2+, Fe3+, and Pb3+. The extreme thermostability of the enzyme was reflected in its unaltered activity over 5 h at 70°C.
KEY WORDS: L-(+)-lactate dehydrogenase, Thermoanaerobacter ethanolicus JW200, allosteric, thermostable, transcription start point

DOI: 10.1134/S0006297910040188