The purification and characterization of an extremely thermostable alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The alpha-amylase from Pyrococcus furiosus, a hyperthermophilic archaebacterium, has been purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 66 kDa. The isoelectric point is 4.3. The enzyme displays optimal activity, with substantial thermal stability, at 100 degrees C, with the onset of activity at approximately 40 degrees C. Unlike mesophilic alpha-amylases there is no dependence on Ca2+ for activity or thermostability. The enzyme displays a broad range of substrate specificity, with the capacity to hydrolyze carbohydrates as simple as maltotriose. No subtrate binding occurs below the temperature threshold of activity, and a decrease in Km accompanies an increase in temperature. Except for a decrease in Asp and an increase in Glu, the amino acid composition does not confirm previously defined trends in thermal adaption. Fourth derivative UV spectroscopy and intrinsic fluorescence measurements detected no temperature-dependent structural reorganization. Hydrogen exchange results indicate that the molecule is rigid, with only a slight increase in conformational flexibility at elevated temperature. Scanning microcalorimetry detected no considerable change in the heat capacity function, at the pH of optimal activity, within the temperature range in which activity is induced. The heat absorption peak due to denaturation, under these conditions, occurred within the temperature range of 90-120 degrees C. When the pH was increased, a change in the shape of the heat absorption peak was observed, which when analyzed thermodynamically shows that the process of heat denaturation is complex, and includes at least three stages, indicating that the protein structure consists of three domains. At temperatures below 90 degrees C no excess heat absorption or change in the CD spectra were observed which could be associated with the cooperative conformational transition of the protein. According to the thermodynamic characteristics of the heat denaturation, the cold denaturation of this protein can be expected only at -3 degrees C. Therefore, the observed inactivation of this enzyme is not caused by the cooperative change of its tertiary structure. It can be associated only with the gradual changes of protein domain interaction.