Five different sources of trehalose synthase genes and four plasmids have been used to construct recombinant plasmids for the highest trehalose bioconversion in Escherichia coli Rosetta (DE3). The results showed that plasmids with different promoters and copy numbers played an important role in the expression of trehalose synthase genes in E. coli. The trehalose synthase from Thermus thermophilus in the plasmid pET-22b was selected for the subsequent studies for its high activity in trehalose production, with 1.996 U/mg protein. We also coexpressed molecular chaperones sigma32, GroEL, GroES, DnaK and DnaJ with trehalose synthase gene tttreS and this greatly increased the solubility of the trehalose synthase protein. The enzymatic reaction conditions for the trehalose bioconversion was optimized, and the optimal temperature, pH, reaction time and substrate concentration were determined as 50 degrees C, 9.0, 10 h and 10% maltose solution, respectively. Following the addition of Ca2+ and reductant DTT and Vc, the activity of trehalose synthase was enhanced up to 19% by Ca2+ and 41% by DTT. Crown Copyright (C) 2015 Published by Elsevier Ltd. All rights reserved.