Cloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization.
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 Km and Vmax values of 7.3 mmol/L and 87 micromol/min, respectively, and in the presence of FDP, a 24-fold decrease in Km and a 5.7-fold increase in Vmax were recorded. The enzyme exhibited no marked catalytic activity for lactate in the absence of FDP, whereas Km and Vmax values were 59.5 mmol/L and 52 micromol/min, respectively, in its presence. The enzyme did not lose activity when incubated at 65 degrees C for 5 min.
Ozkan
Melek
M
Biology Department, Middle East Technical University, Ankara 06531, Turkey.
Yilmaz
Ebru I
EI
Lynd
Lee R
LR
Ozcengiz
Gülay
G
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli; D005635:Fructosediphosphates / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D019289:Pyruvic Acid / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D020536:Enzyme Activators; D005635:Fructosediphosphates; D011993:Recombinant Fusion Proteins; D019344:Lactic Acid; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase; C029063:fructose-1,6-diphosphate