Cloning, expression and functional characterization of a novel trehalose synthase from marine Pseudomonas sp. P8005.

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose. A novel treS gene with a length of 3,369 bp, encoding a protein of 1,122 amino acid residues with a predicted molecular mass of 126 kDa, was cloned from a marine Pseudomonas sp. P8005 (CCTCC: M2010298) and expressed in Escherichia coli. The amino acid sequence identities between this novel TreS and other reported TreS is relatively low. The purified recombinant TreS showed an optimum pH and temperature of 7.2 and 37 °C, respectively. The enzyme displayed a high conversion rate (70 %) of maltose to trehalose during equilibrium and had a higher catalytic efficiency (k cat/K m) for maltose than for trehalose, suggesting its application in the production of trehalose. In addition to maltose and trehalose, this enzyme can also act on sucrose, although this activity is relatively low. Mutagenesis studies demonstrated that enzymatic activity was reduced dramatically by individually substitution with alanine for D78, Y81, H121, D219, E261, H331 or D332, which implied that these residues might be important in P8005-TreS. Experiments using isotope-labeled substrates showed that [(2)H2]trehalose combined with unlabeled trehalose was converted to [(2)H2]maltose and maltose, but without any production of [(2)H]maltose or [(2)H]trehalose and with no incorporation of exogenous [(2)H7]glucose into the disaccharides during the conversion catalyzed by this enzyme. This finding indicated the involvement of an intramolecular mechanism in P8005-TreS catalyzing the reversible interconversion of maltose and trehalose.