Characterization of a hyperthermostable glycogen phosphorylase from Aquifex aeolicus expressed in Escherichia coli

The glycogen phosphorylase gene (glgP) of Aquifex aeolicus (Aae), a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli and the characteristics of the expressed enzyme were examined. The recombinant enzyme was purified to homogeneity by heat-treatment at 70 °C for 15 min to denature the contaminating E. coli proteins, followed by Ni–NTA agarose column chromatography to selectively trap the His-tagged enzyme. The purified enzyme gave a single band on SDS–PAGE with a molecular mass of approximately 80 kDa. The enzyme displayed optimal activity at pH 6.5 and was stable in the pH range from 4.0 to 10.0. The temperature at which optimal enzyme activity was observed was 100 °C and the enzyme retained 66% of its original activity after heating at 100 °C for 30 min. Kinetic studies using the purified enzyme demonstrated that the smallest primer molecule accepted for catalysis in the synthetic direction was maltotriose (G3) and that the smallest effective substrate for the reverse process, phosphorolysis, was maltotetraose (G4). The Km and kcat values were determined for various oligosaccharides (G3–G7) in both synthetic and phosphorolytic reactions and, remarkably, a maximum degree of specificity was observed toward substrates in the phosphorolytic direction.