Cloning, Expression and Characterization of a Trehalose Synthase Gene From Rhodococcus opacus

Abstract

Trehalose is a unique disaccharide capable of protecting proteins against environmental stress. A novel trehalose synthase (TreS) gene from Rhodococcus opacus was cloned and expressed in Escherichia coli Top10 and BL21 (DE3) pLysS, respectively. The recombinant TreS showed a molecular mass of 79 kDa. Thin layer chromatography (TLC) result suggested that this enzyme had the ability to catalyze the mutual conversion of maltose and trehalose. Moreover, high-performance liquid chromatography (HPLC) result suggested that glucose appeared as a byproduct with a conversion rate of 12 %. The purified recombinant enzyme had an optimum temperature of 25 °C and pH optimum around 7.0. Kinetic analysis revealed that the K _m for trehalose was around 98 mM, which was a little higher than that of maltose. The preferred substrate of TreS was maltose according to the analysis of k _cat/K _m. Both 1 and 10 mM of Hg²⁺, Cu²⁺ and Al³⁺ could inhibit the TreS activity, while only 1 mM of Ca²⁺ and Mn²⁺ could increase its activity. Five amino acid residues, Asp²⁴⁴, Glu²⁸⁶, Asp³⁵⁴, His¹⁴⁷ and His³⁵³, were shown to be conserved in R. opacus TreS, which were also important for α-amylase family enzyme catalysis.