The glgP gene encoding alpha-glucan phosphorylase (alpha-GP) from the thermopile Thermus caldophilus GK24 has been identified, cloned, Iq and overexpressed in Escherichia coli and used to synthesize D-glucose-l-phospate (GIP) from an inexpensive starch. The enzyme, purified 6.5-fold, was isolated in 31% yield from the transformed E. coli, and gave a single band. The purified enzyme may exist as a homohexamer with an apparent molecular mass of a 550 kDa molecule, consisting of 90 kDa per subunit. The optimal pH and temperature were 7.0 and 70 degrees C in the a-GP reaction with starch producing G I P. Soluble starch (amylopectin, amylose) turned out to be a better substrate giving a higher yield of GIP than alpha-1,6-branched alpha-1,4-glucans (glycogen, potato starch, etc.). As a result, GIP was obtained in a good yield (47%, w/w) from the reaction containing 5% (w/v) soluble starch in 0.7 M potassium phosphate at pH 7.0. T caldophillis a-GP shows a high tolerance (up to 0.7 M) of potassium phosphate and plays a critical role in shifting the reaction equilibrium in favor of G1P synthesis. The GIP product can be purified simply by ethanol precipitation, after removing the unreacted starch and inorganic phosphate by activated charcoal and magnesium acetate precipitation. It is concluded that T caldophilus a-GP readily utilized in large scale synthesis of GIP. (c) 2005 Elsevier Ltd. All rights reserved.