For the overproduction of 5-aminolevulinic acid (ALA) from recombinant Escherichia coli, the inhibition of ALA dehydratase on both small scale by using an eppendorf tube, and on a large scale by using a fermenter, the in vitro glycation and the inactivation of enzymes on the ALA dehydratase under several experimental conditions were investigated. The presence of 0.5-10 mM of D-glucose caused a concentration-dependent inhibition of recombinant E. coli ALA dehydratase activity. The ALA dehydratase levels were dependent on the pH of the medium, with the maximal activities occurring at 8.0. The inhibition constant, K-i, of intracellular ALA dehydratase by D-glucose and levulinic acid (LA) were 1.02 and 0.32 mM, respectively. The addition Of 10 MM Of D-glucose drastically inhibited the ALA dehydratase activity (85% inhibition), in turn, the highest level of extracellular ALA production (3.8 g/l) was achieved. Based upon those results, we concluded that D-glucose decreased the ALA dehydratase activity both by the competitive inhibition with substrate and by the inactivation of enzyme protein, and that the inactivation of ALA dehydratase by D-glucose may require glycation metabolism Of D-glucose at least in part.