NAD-dependent Thermotoga maritima glycerol dehydrogenase (TmGlyDH) converts glycerol into dihydroxyacetone (DHA), a valuable synthetic precursor and sunless tanning agent. In this work, recombinant TmGlyDH was characterized to determine if it can be used to catalyze DHA production. The pH optima for glycerol oxidation and DHA reduction at 50 A degrees C were 7.9 and 6.0, respectively. Under the conditions tested, TmGlyDH had a linear Arrhenius plot up to 80 A degrees C. TmGlyDH was more thermostable than other glycerol dehydrogenases, remaining over 50 % active after 7 h at 50 A degrees C. TmGlyDH was active on racemic 1,2-propanediol and produced (R)-1,2-propanediol from hydroxyacetone with an enantiomeric excess above 99 %, suggesting that TmGlyDH can also be used for chiral synthesis. (R)-1,2-propanediol production from hydroxyacetone was demonstrated for the first time in a one-enzyme cycling reaction using glycerol as the second substrate. Negative cooperativity was observed with glycerol and DHA, but not with the cofactor. Apparent kinetic parameters for glycerol, DHA, and NAD(H) were determined over a broad pH range. TmGlyDH showed little activity with N-6-carboxymethyl-NAD(+) (N-6-CM-NAD), an NAD(+) analog modified for easy immobilization to amino groups, but the double mutation V44A/K157G increased catalytic efficiency with N-6-CM-NAD(+) ten-fold. Finally, we showed for the first time that a GlyDH is active with immobilized N-6-CM-NAD(+), suggesting that N-6-CM-NAD(+) can be immobilized on an electrode to allow TmGlyDH activity in a system that reoxidizes the cofactor electrocatalytically.