The l‐lactate dehydrogenase gene of the hyperthermophilic bacterium Thermotoga maritima cloned by complementation in Escherichia coli

Ralf OSTENDORP, Wolfgang LIEBL, Hartmut SCHURIG, Rainer JAENICKE

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Abstract

The gene for a l(+)‐lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was cloned by complementation of an Escherichia coli pfl. ldh mutant. The gene is part of a 4.5 kb SauIIIA fragment obtained by partial digestion of the Thermotoga genome. The DNA fragment was physically mapped and the putative Shine‐Dalgarno sequence within the non‐coding region determined. The gene contains 960 bp, including the stop codon, corresponding to 319 amino acids/subunit of the homotetrameric enzyme. Part of the amino acid sequence was confirmed by Edman degradation of peptides obtained from nanomolar quantities of the purified enzyme by tryptic digestion. A comparison of the amino acid sequenc̀e with those of known prokaryotic l‐lactate dehydrogenases reveals a high similarity, especially with the enzyme from thermophilic sources, where up to 48% identity is found. The gene was expressed as an active enzyme in a heterologous host.

Original languageEnglish
Pages (from-to)709-715
Number of pages7
JournalEuropean Journal of Biochemistry
Volume216
Issue number3
DOIs
StatePublished - Sep 1993
Externally publishedYes

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