Abstract: To express and purify the key genes of trehalose synthesis and degradation, trehalose-6-phosphate synthase(TPS) and trehalase(Tre), in vitro in Escherichia coli, and study the enzymatic characteristics of TPS, soluble trehalase(Tre1), and membrane-bound trehalase(Tre2) of the Hyphantria cunea. The prokaryotic expression plasmid vectors of HcTPS, HcTre1, and HcTre2 were successfully constructed. Subsequently, these recombinant proteins were heterologously expressed in Escherichia coli BL21(DE3) using the pET-28a(+) vector. SDS-PAGE was used to identify the recombinant proteins which were then purified via Ni-affinity chromatography. The enzymatic characteristics of the purified recombinant proteins were determined, including the effects of temperature and pH on enzyme activity. The findings indicated that the soluble recombinant proteins of TPS and Tre1 were expressed in Escherichia coli, while Tre2 was expressed as inclusion body. The recombinant proteins were purified via Ni-affinity chromatography, and the molecular weights of the purified proteins were 92.62, 63.34 kDa and 72.79 kDa for TPS, Tre1 and Tre2, respectively; the optimal temperature for recombinant TPS, Tre1 and Tre2 was 25, 35 ℃ and 55 ℃, respectively, with maximum enzyme activities of(50.97 ± 2.06),(80.59 ± 3.49)U/mg and(41.42 ± 1.23)U/mg at these temperature. The optimal pH values for recombinant TPS, Tre1 and Tre2 were 7.0, 6.0 and 7.0, respectively, with maximum enzyme activities of(58.50 ± 1.42),(85.60 ± 3.45)U/mg and(44.17 ± 1.23)U/mg at these pH levels. The prokaryotic expression plasmid of pET-28a(+)-TPS, pET-28a(+)-Tre1 and pET-28a(+)-Tre2 were successfully constructed, the optimal temperature and pH of recombinant proteins related to trehalose synthesis and degradation in H. cunea were determined.