Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli

A trehalose synthase (TSase) gene from a hyperacidophilic, thermophilic archaea, Picrophilus torridus, was synthesized using overlap extension PCR and transformed into Escherichia coli for expression. The purified recombinant P. torridus TSase (PTTS) showed an optimum pH and temperature of 6.0 and 45°C, respectively, and the enzyme maintained high activity at pH 5.0 and 60°C. Kinetic analysis showed that the enzyme has a 2.5-fold higher catalytic efficiency (k_cat/K_M) for maltose than for trehalose, indicating maltose as the preferred substrate. The maximum conversion rate of maltose into trehalose by the enzyme was independent of the substrate concentration, tended to increase at lower temperatures, and reached ≈71% at 20°C. Enzyme activity was inhibited by Hg²⁺, Al³⁺, and SDS. Five amino acid residues that are important for α-amylase family enzyme catalysis were shown to be conserved in PTTS (Asp²⁰³, Glu ²⁴⁵, Asp³¹¹, His¹⁰⁶, and His³¹⁰) and required for its activity, suggesting this enzyme might employ a similar hydrolysis mechanism.