Sucrose phosphorylase (SPase) is capable of specifically catalysing transglucosylation reactions and can be employed in the enzymatic synthesis of α-D-glycosides. In the present study, a putative Thermobacillus SPase gene (TSPase) was synthesised with optimised codons and overexpressed in Escherichia coli. The 1467 bp gene encodes a 488-amino acid protein with a calculated molecular mass of 55.8 kDa. The specific activity of the recombinant TSPase (rTSPase) was 6.42 U/mg for sucrose, and the optimum temperature and pH were 65 °C and pH 7.0. The T1/2 value of the rTSPase was 212 h at 50 °C and 98 h at 60 °C. A stimulating effect on the activity of the rTSPase was observed in the presence of 5 mM Co2+. The rTSPase showed increased stability against DMSO as organic co-solvent at 50 °C. The Km and kcat of the rTSPase with sucrose were determined as 6.24 mM and 5.73 s-1 respectively. The rTSPase produced 2-O-α-D-Glucopyranosyl-L-ascorbic acid (AA-2 G) from ascorbic acid in both crude extract and whole-cell forms. A maximum yield of 19.7% (39.94 ± 0.17 g/L) was achieved after incubation of ascorbic acid sodium salt and sucrose (1:2) with 19.76 U/mL of the rTSPase at pH 7.0 and 50 °C for 24 h.,Copyright © 2019 Elsevier B.V. All rights reserved.