中国生物工程杂志

1,3-propanediol (1,3-PD) is an important material for chemical industry, therefore, there is much interest in the production of 1,3-PD. The genes dhaF and dhaG encoding glycerol dehydratase reactivase were amplified by using the genomic DNA of Citrobacter freundii. Two segment genes about 1.8Kb and 0.4Kb were obtained and inserted into pMD-18T. There had not more than 86% similarity between the cloning genes and other corresponding genes; then dhaF and dhaG were inserted into pSE380 and co-expressed in E.coli. The interesting recombinant reactivase was about 30 per in the whole crude protein and was purified homogeneous. α and β subunits of reactivase had apparent molecular masses about 63 and 12 kDa by SDS-PAGE, nondenaturing PAGE analysis the molecular mass was about 150kDa. Therefore, its subunit structure was most likely α2β2. In the presence of free adenosylcobalamin, ATP, and Mg2+, the factor reactivated glycerol dehydratase from C. freundii, which had been inactivated. This research is help to make clear the mechanism of reactivase from C. freundii and improve the manufacture of 1，3-PD by the biological pathway.