Purification and Characterization of Thermostable Trehalose Phosphorylase from Thermoanaerobium brockii.

Abstract

A thermostable trehalose phosphorylase (EC 2.4.1.64) from a thermophilic anaerobe, Thermoanaero bium brockii ATCC 35047, was purified to an electrophoretically homogeneous state by successive column chromatographies on DEAE-Toyopearl 6505, Butyl-Toyopearl 650 M and Ultrogel AcA 44 . The molecular weight of the enzyme was estimated to be 190, 000 Da by gel filtration, and 88, 000 Da by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity in the range of pH 7.0 to 7.5 for phosphorolysis, and pH 6.0 to 7.0 for synthesis . The optimum temperature of the enzyme was 70°C in both directions. The enzyme was stable from pH 6.0 to 9.0, and up to 60°C . This result showed that our trehalose phosphorylase was the most thermostable in those reported to date . Activity was inhibited by Cue, Hg2+, Mg2+, Mn2+, Pb2+, and Zn2+ The Kms for trehalose, Pi, D-glucose and R-D-glucose 1-phosphate were 0.97, 0.57, 2.4 and 0.75 mM, respectively.