Cloning and expression of theClostridium thermocellumL-lactate dehydrogenase gene inEscherichia coliand enzyme characterization

Abstract

The structural gene for L-lactate dehydrogenase (LDH) (EC.1.1.1.27) from Clostridium thermocellum 27405 was cloned in Escherichia coli by screening the Lambda Zap II 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 Kmand Vmaxvalues of 7.3 mmol/L and 87 µmol/min, respectively, and in the presence of FDP, a 24-fold decrease in Kmand a 5.7-fold increase in Vmaxwere recorded. The enzyme exhibited no marked catalytic activity for lactate in the absence of FDP, whereas Kmand Vmaxvalues were 59.5 mmol/L and 52 µmol/min, respectively, in its presence. The enzyme did not lose activity when incubated at 65 °C for 5 min.Key words: L-lactate dehydrogenase purification, thermophilic bacteria.