It was reported that glycerol and 1,2-propanediol was converted to 1,3-propanediol and 1-propanol in Lactobacillus diolivorans(DSM14421)under the anaerobic condition，respectively. Its putative genes encoding diol dehydratase, a key enzyme in the pathway, were sequenced. However, their reactivating factor (gldG and gldH) was not completely sequenced. Based on several glycerol dehydratase and diol dehydratase reactivating factors sequence alignment, it was amplified from L. diolivorans with degenerated primers. Then it was inserted into expression vector pSE380. A recombinant plasmid pSE-gldGH was constructed and transformed into Escherichia coli BL21. Recombinant gldG and gldH protein were co-purified by metal chelating affinity chromatography and gel filtration from cell-free extracts of E. coli overexpressing the gldGH genes. They existed a putative reactivating factor, with an apparent molecular mass of 325 000. The protein complex consisted of equimolar amounts of the two subunits with M_r of 68 000 (α) and 13 000 (β), encoded by the gldG and gldH genes, respectively. Therefore, its subunit structure was most likely α₄β₄, different from the common structure α₂β₂ of the other diol or glycerol dehydratase reactivating factors. In the presence of free adenosylcobalamin, ATP, and Mg²⁺, the factor reactivated diol dehyadratase from L. diolivorans, which had been inactivated to be enzyme-cyanocobalamin complex.