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A Proposed Mechanism for the Thermal Denaturation of a Recombinant Bacillus Halmapalus Alpha-amylase - the Effect of Calcium IonsThe thermal stability of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This alpha-amylase is homologous to other Bacillus alpha-amylases where previous crystallographic studies have identified the existence of 3 calcium binding sites in the structure. Denaturation of BHA is irreversible with a Tm of approximately 89 C, and DSC thermograms can be described using a one-step irreversible model. A 5 C increase in T(sub m) in the presence of 10 fold excess CaCl2 was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30-40 fold excess calcium chelator (EDTA or EGTA) results in a large destabilization of BHA corresponding to about 40 C lower T(sub m), as determined by both CD and DSC. Ten fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which possibly originate from different populations of BHA:calcium complexes. The observations in the present study have, in combination with structural information of homologous alpha-amylases, provided the basis for the proposal of a simple denaturation mechanism of BHA. The proposed mechanism describes the irreversible thermal denaturation of different BHA:calcium complexes and the calcium binding equilibrium involved. Furthermore, the model accounts for a temperature induced reversible structural change associated with calcium binding.
Document ID
20030062080
Acquisition Source
Marshall Space Flight Center
Document Type
Reprint (Version printed in journal)
Authors
Nielsen, Anders D.
(Roskilde Univ. Denmark)
Pusey, Marc L.
(NASA Marshall Space Flight Center Huntsville, AL, United States)
Fuglsang, Claus C.
(Novo Industri A/S Bagsvaerd, Denmark)
Westh, Peter
(Roskilde Univ. Denmark)
Date Acquired
August 21, 2013
Publication Date
January 1, 2003
Subject Category
Life Sciences (General)
Distribution Limits
Public
Copyright
Other

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