Cloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization

Ozkan M., Yilmaz E., Lynd L., Ozcengiz G.

CANADIAN JOURNAL OF MICROBIOLOGY, vol.50, no.10, pp.845-851, 2004 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 50 Issue: 10
  • Publication Date: 2004
  • Doi Number: 10.1139/w04-071
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.845-851
  • Keywords: L-lactate dehydrogenase purification, thermophilic bacteria, THERMUS-CALDOPHILUS GK24, NUCLEOTIDE-SEQUENCE, ALIGNMENT, BINDING
  • Middle East Technical University Affiliated: Yes


The structural gene for L-lactate dehydrogenase (LDH) (EC. from Clostridium thermocellum 27405 was cloned in Escherichia coli by screening the Lambda Zap 11 phage library of C. thermocellum genomic DNA. In one positive clone, an open reading frame of 948 base pairs corresponded to C. thermocellum ldh gene encoding for the predicted 315-residue protein. The ldh gene was successfully expressed in E. coli FMJ39 (ldh mutant) under the lac promoter. The recombinant enzyme was partially purified from E. coli cell extracts and its kinetic properties were determined. Clostridium thermocellum LDH was shown to catalyze a highly reversible reaction and to be an allosteric enzyme that is activated by fructose-1,6-diphosphate (FDP). For pyruvate, partially purified LDH had K-m and V-max values of 7.3 mmol/L and 87 mumol/min, respectively, and in the presence of FDP, a 24-fold decrease in K-m and a 5.7-fold increase in V-max were recorded. The enzyme exhibited no marked catalytic activity for lactate in the absence of FDP, whereas K-m and V-max values were 59.5 mmol/L and 52 mumol/min, respectively, in its presence. The enzyme did not lose activity when incubated at 65degreesC for 5 min.