Effect of calcium ions on the irreversible denaturation of a recombinant Bacillus halmapalus alpha-amylase: a calorimetric investigation.
The effect of temperature and calcium ions on the denaturation of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been studied using calorimetry. It was found that thermal inactivation of BHA is irreversible and that calcium ions have a significant effect on stability. Thus an apparent denaturation temperature ( T (d)) of 83 degrees C in the presence of excess calcium ions was observed, whereas T (d) decreased to 48 degrees C when calcium was removed. The difference in thermal stability with and without calcium ions has been used to develop an isothermal titration calorimetric (ITC) procedure that allows simultaneous determination of kinetic parameters and enthalpy changes of the denaturation of calcium-depleted BHA. An activation energy E (A) of 101 kJ/mol was found for the denaturation of calcium-depleted BHA. The results support a kinetic denaturation mechanism where the calcium-depleted amylase denatures irreversibly at low temperature and if calcium ions are in excess, the amylase denatures irreversibly at high temperatures. The two denaturation reactions are coupled with the calcium-binding equilibrium between calcium-bound and -depleted amylase. A combination of the kinetic denaturation results and calcium-binding constants, determined by isothermal titration calorimetry, has been used to estimate kinetic stability, expressed in terms of the half-life of BHA as a function of temperature and free-calcium-ion concentration. Thus it is estimated that the apparent E (A) can be increased to approx. 123 kJ/mol by increasing the free-calcium concentration.
Nielsen
Anders D
AD
Department of Life Sciences and Chemistry, Roskilde University, PO Box 260, DK-4000 Roskilde, Denmark.
Fuglsang
Claus C
CC
Westh
Peter
P
D001407:Bacillus / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D002151:Calorimetry; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008024:Ligands; D011489:Protein Denaturation / Q000187:drug effects; D011994:Recombinant Proteins; D013816:Thermodynamics; D000516:alpha-Amylases / Q000378:metabolism*
D008024:Ligands; D011994:Recombinant Proteins; D000516:alpha-Amylases; D002118:Calcium