Temperature-induced denaturation of beta-glycosidase from the archaeon Sulfolobus solfataricus.
The beta-glycosidase isolated from the extreme thermophilic archaeon Sulfolobus solfataricus, grown at 87 degrees C, is a tetrameric protein with a molecular mass of 240 kDa. This enzyme is barely active at 30 degrees C and has optimal activity, over 95 degrees C, at pH 6.5. Its thermal stability was investigated at pH 10.1 and 10.6 by means of functional studies, circular dichroism and differential scanning calorimetry. There was no evidence of thermal activation of the enzyme and the temperature-induced denaturation was irreversible and not well represented by the two-state transition model. A more complex process occurred, involving the dissociation and unfolding of subunits, and subsequent nonspecific association and/or aggregation. Denaturation temperature was around 85 degrees C, depending on protein concentration. The denaturation enthalpy change was between 7,500 and 9,800 kJ.mol-1, depending on the pH. The collapse of the native structure around 85 degrees C was confirmed by circular dichroism measurements and time-dependent activity studies. Finally, preliminary investigations were performed on the recombinant enzyme expressed in Escherichia coli.
Institute of Protein Biochemistry and Enzymology, National Research Council, Naples, Italy.
S
S
D'Auria
M
M
Rossi
G
G
Barone
F
F
Catanzano
P
P
Del Vecchio
G
G
Graziano
R
R
Nucci
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases