Thermodynamic Stability of a Cold-Active -Amylase from the Antarctic Bacterium Alteromonas haloplanctis
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- 作者：Georges Feller ; Dominique d'Amico ; and Charles Gerday
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：April 6, 1999
- 年：1999
- 卷：38
- 期：14
- 页码：4613 - 4619
- 全文大小：96K
- 年卷期：v.38,no.14(April 6, 1999)
- ISSN：1520-4995
文摘
The thermal stability of the cold-active 
-amylase (AHA) secreted by the Antarctic bacteriumAlteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differentialscanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain proteinexhibiting a reversible two-state unfolding, as demonstrated by the recovery of 
Hcal values after consecutivecalorimetric transitions, a Hcal/Heff ratio close to unity, and the independence of unfolding thermodynamicparameters of scan rates. By contrast, the mesophilic -amylases investigated here (from porcine pancreas,human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfoldirreversibly according to a non-two-state mechanism. Unlike mesophilic -amylases, the melting point ofAHA is independent of calcium and chloride binding while the allosteric and structural functions of theseions are conserved. The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7
C, a Hcal of 238 kcal mol-1, and a Cp of 8.47 kcal mol-1 K-1. These values were used to calculatethe Gibbs free energy of unfolding over a wide range of temperatures. This stability curve shows that (a)the specific Gmax of AHA [22 cal (mol of residue)-1] is 4 times lower than that of mesophilic -amylases,(b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperatureof cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinantheat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued thatthe cold-active -amylase has evolved toward the lowest possible conformational stability of its nativestate.
-amylase (AHA) secreted by the Antarctic bacteriumAlteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differentialscanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain proteinexhibiting a reversible two-state unfolding, as demonstrated by the recovery of Hcal values after consecutivecalorimetric transitions, a Hcal/Heff ratio close to unity, and the independence of unfolding thermodynamicparameters of scan rates. By contrast, the mesophilic -amylases investigated here (from porcine pancreas,human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfoldirreversibly according to a non-two-state mechanism. Unlike mesophilic -amylases, the melting point ofAHA is independent of calcium and chloride binding while the allosteric and structural functions of theseions are conserved. The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7C, a Hcal of 238 kcal mol-1, and a Cp of 8.47 kcal mol-1 K-1. These values were used to calculatethe Gibbs free energy of unfolding over a wide range of temperatures. This stability curve shows that (a)the specific Gmax of AHA [22 cal (mol of residue)-1] is 4 times lower than that of mesophilic -amylases,(b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperatureof cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinantheat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued thatthe cold-active -amylase has evolved toward the lowest possible conformational stability of its nativestate.