Expression and characterization of recombinant trehalose synthase in Bacillus subtilis<\i>

Trehalose synthase (TreS, EC 2.4.1.245) is a potential catalyst for synthesis of trehalose, an important natural disaccharide. In this study, the treS gene of Pseudomonas putida (VTCC 12263) was cloned into pHT01 plasmid at BamHI-XbaI position, expressed in Bacillus subtilis (B. subtilis) 1012, and characterized. The recombinant TreS had molecular weight of 68 kDa when fused with 8xHis tag at the C-terminus. catalyzed conversion of maltose to trehalose in optimal conditions had specific activity of 1.664 U/g. Expression of TreS was highest when B. subtilis 1012 harboring pHT01-treS was cultured in TB medium at 30 o C, induced with 1.0 mM IPTG when OD600 reached 0.8 and harvested after 10 hours of induction. The recombinant TreS purified by Ni-sepharose chromatography had specific activity of 41.700 U/g and formed a single band on Western blot with monoclonal antibody against His-tag. The recombinant TreS had optimal activity at 37 o C in 100 mM pH 7.4 PBS and 300 mM maltose. It was inhibited by NaCl, KCl and MgCl2 (retaining 45% or 75% specific activity in buffer containing 5 mM KCl or 5 mM MgCl2, respectively) and stimulated by imidazol (with specific activity increasing by 30–200%).