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Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP ( k/ K = 2618 mM s, k = 249 s, K = 0.10 ± 0.01 mM) as cofactor, although NAD ( k/ K = 138 mM s, k = 604 s, K = 4.37 ± 0.56 mM) could also be accepted. The K values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP and NAD as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8-9.0 for NADP and at pH 7.0-8.6 for NAD while the optimal temperature was 80 °C for NADP and 70 °C for NAD. This was the first observation that the NADP-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase. [ABSTRACT FROM AUTHOR]