Abstract: the mannitol dehydrogenase(MDH)from P. bacterium 1109 was expressed and purified. Enzymatic properties of the recombinant enzyme and the process conditions to produce mannitol were studied. Results showed that recombinant enzyme was a tetramer with molecular weight of 37 kDa. Amino acid sequence alignment showed that its homology with most MDHs was less than 40%. The optimum pH and temperature of the enzyme were 8.5 and 80℃,respectively. The activity of recombinant MDH could increase to 260% of control group with the presence of metal ion Zn²⁺. In addition,the recombinant MDH could retain more than 85% of residual activity after incubation at 75℃ for 6 h,indicating a higher thermal stability than most MDHs. Substrate specificity studies showed that it had a high specificity for D-fructose. The Michaelis constant(K_m)and catalytic efficiency(k_cat/K_m)of D-fructose catalyzed by recombinant MDH were 20 mmol/L and 7.5 L/(mmol·min),respectively. The recombinant MDH could produce more than 80% mannitol in the reaction system composed of 400 mmol/L of D-fructose substrate. The optimization of the reaction conditions laid the foundation for the subsequent industrial production of mannitol.