Synthesis of 1,3-propanediol (1,3-PD) from glycerol through the biotransformation process requires two steps, catalyzed by glycerol dehydratase (GDHt) and 1,3-PD oxidoreductase. GDHt is the rate-limiting enzyme in this process. All recombinant microorganisms for production of 1,3-PD so far utilized the natural genes that may not have been optimized. Two positions, which are 19.3A and 29.6A away from the active site in GDHt from Klebsiella pneumoniae, were subjected to saturation-mutagenesis and 38 mutants were characterized. The catalytic activity of a mutant in beta-subunit (beta-Q42F, 29.6A from the active site) was 8.3-fold higher than the wild type, and the enzyme efficiency of other two mutants beta-Q42L and beta-Q42S for substrate glycerol was 336-fold and 80-fold higher than that for 1,2-propanediol. This investigation supplied further evidence that distant mutations could be a good source of diversity and therefore, made a contribution to the toolbox of industrial enzyme improvement.