Biologia, Bratislava, 57/Suppl. 11: 213-219, 2002.

ISSN 0006-3088 (Biologia).

 

Review

Structural determinants of cold adaptation and stability in a psychrophilic a

a-amylase.

 

Salvino D’Amico, Charles Gerday & Georges Feller*

 

Laboratory of Biochemistry, Institute of Chemistry B6, University of Liege, B-4000 Liege, Belgium; tel.: ++ 32 4 366 33 43, fax: ++ 32 4 366 33 64, e-mail: gfeller@ulg.ac.be

* corresponding author

Received: October 4, 2001 / Accepted: February 12, 2002

 

AbstractBiologia, Bratislava, 57/Suppl. 11: xxx-xxx, 2002; ISSN 0006-3088 (Biologia). ISSN 1335-6399 (Biologia. Section Cellular and Molecular Biology).

 

Key words: extremophiles, protein engineering, microcalorimetry.

_____________________

*Corresponding author

 

The heat-labile aa-amylase from an Antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition, as shown by differential scanning calorimetry. Mutants of this enzyme were produced, carrying intended additional weak interactions of a type found in thermostable aa-amylases. It is shown that single amino  acid side  chain substitutions can significantly modify the melting point Tm, the calorimetric enthalpy DHcal, the cooperativiity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters kcat and Km. Although all mutations were located far from the active site, their overall trend is to decrease both kcat and Km , probably by making the molecule more rigid, but this protects rigidifying the molecule and to protect mutants against thermal inactivation.

 

Key words: extremophiles, protein engineering, microcalorimetry.