Characterization of a Recombinant Trehalose Synthase from Arthrobacter chlorophenolicus and its Unique Kinetics Indicating a Substrate Cooperativity.

Trehalose is a non-reducing disaccharide with beneficial physiological properties and commercial potential. Trehalose synthase (EC 5.4.99.16) catalyzes the reversible conversion between maltose and trehalose. A recombinant trehalose synthase from Arthrobacter chlorophenolicus SK 33.001 (ACTS) was cloned, expressed, and characterized. The recombinant enzyme encoded a protein of 598 amino acids with a molecular mass of 66 kDa. Gel filtration showed that ACTS is a tetramer in sodium phosphate buffer. The enzyme was metal ion independent and exhibited maximal activity in sodium phosphate buffer (pH 7.5) at 30 °C. The kinetic investigations resulted in a KM value of 120.5 ± 4.5 mM for maltose and a KM value of 343.1 ± 13.8 mM for trehalose. The catalytic efficiency (Vmax/KM) for maltose and trehalose were 0.2 and 0.15 U mg−1 mM−1, respectively. In addition, a cooperative substrate binding was found displayed by the determined Hill coefficients (nH) of 2.8 for maltose and 2.1 for trehalose as a substrate, respectively. The final trehalose yield of various maltose concentrations (50–1000 mM) was constant between 58 and 59%, implying that substrate concentration had no inhibitory influence on ACTS activity.