Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis

The thermal stability of the cold-active alpha-amylase (AHA) secreted by th e Antarctic bacterium Alteromonas haloplanctis has been investigated by int rinsic fluorescence, circular dichroism, and differential scanning calorime try. It was found that this heat-labile enzyme is the largest known multido main protein exhibiting a reversible two-state unfolding, as demonstrated b y the recovery of Delta H-cal values after consecutive calorimetric transit ions, a Delta H-cal/Delta H-eff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesop hilic alpha-amylases investigated here (from porcine pancreas, human saliva ry glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus lic heniformis) unfold irreversibly according to a non-two-state mechanism, Unl ike mesophilic alpha-amylases, the melting point of AHA is independent of c alcium and chloride binding while the allosteric and structural functions o f these ions are conserved. The thermostability of AHA at optimal condition s is characterized by a T-m of 43.7 degrees C, a Delta H-cal of 238 kcal mo l(-1), and a Delta C-p of 8.47 kcal mol(-1) K-1. These values were used to calculate the Gibbs free energy of unfolding over a wide range of temperatu res. This stability curve shows that (a) the specific Delta G(max) of AHA [ 22 cal (mol of residue)(-1)] is 4 times lower than that of mesophilic alpha -amylases, (b) group hydration plays a crucial role in the enzyme flexibili ty at low temperatures, (c) the temperature of cold unfolding closely corre sponds to the lower limit of bacterial growth, and (d) the recombinant heat -labile enzyme can be expressed in mesophilic hosts at moderate temperature s. It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state.