Thermodynamic Stability of a Cold-Active α-Amylase from the Antarctic Bacterium Alteromonas haloplanctis

Abstract

The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of DeltaHcal values after consecutive calorimetric transitions, a DeltaHcal/DeltaHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic alpha-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism. Unlike mesophilic alpha-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7 degrees C, a DeltaHcal of 238 kcal mol-1, and a DeltaCp 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 temperatures. This stability curve shows that (a) the specific DeltaGmax 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 flexibility at low temperatures, (c) the temperature of cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinant heat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state.

Structural, Kinetic, and Calorimetric Characterization of the Cold-active Phosphoglycerate Kinase from the AntarcticPseudomonas sp. TACII18

“…Such an organization of the psychrophilic enzyme into thermodynamic units of different stability has profound implications for our current view of protein adaptation to cold. From the limited number of cold-adapted enzymes studied so far, it was concluded that they are characterized by a low conformational stability, that even multidomain proteins display a reduced stability of all calorimetric units and that they have evolved toward the lowest available stability of the native state (40,43). The results obtained here for the Antarctic PGK demonstrate that this evolution can affect only one particular domain of the molecule.…”

Isolation and characterization of a cold-active amylase from marine Wangia sp. C52

“…The effect of temperature on the amylase activity revealed that the enzyme was optimally active at 30°C ( Figure 5). This optimal temperature was much lower than those of cold-active -amylases from Alteromonas haloplanctis (43.7°C) (Feller et al, 1999) and E. foetide (50°C) (Ueda et al, 2008). When the temperature exceeded 30°C, the enzyme activity decreased rapidly.…”

Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples

“…Earlier isolated cold-adapted Pseudoalteromonas haloplanktis strains were studied as interesting hydrolytic enzyme producers, such as xylanase (EC 3.2.1.8) (Collins et al, 2005), cellulase (EC 3.2.1.4) (Garsoux et al, 2004), amylase (EC 3.2.1.1) (Feller et al, 1999) and esterase (EC 3.1.1.1) (Aurilia et al, 2007). Moreover, several genomes from isolated psychrophylic microorganisms have been completely sequenced (Rabus et al, 2004;Médigue et al, 2005;Methé et al, 2005;Duchaud et al, 2007), offering the possibility of discovering new phenotypes and of unraveling new cold-adapted metabolic pathways.…”