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The Thermal Stability of the Fusarium solani pisi Cutinase as a Function of pH.

Petersen SB, Fojan P, Petersen EI, Petersen MT - J. Biomed. Biotechnol. (2001)

Bottom Line: The ratio between the calorimetric enthalpy (DeltaH(cal)) and the van't Hoff enthalpy (DeltaH(v)) obtained, is far from unity, indicating that cutinase does not exhibit a simple two state unfolding behaviour.The role of pH on the electrostatic contribution to the thermal stability was assessed using TITRA.We propose a molecular interpretation for the pH-variation in enzymatic activity.

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ABSTRACT
We have investigated the thermal stability of the Fusarium solani pisi cutinase as a function of pH, in the range from pH 2-12. Its highest enzymatic activity coincides with the pH-range at which it displays its highest thermal stability. The unfolding of the enzyme as a function of pH was investigated by microcalorimetry. The ratio between the calorimetric enthalpy (DeltaH(cal)) and the van't Hoff enthalpy (DeltaH(v)) obtained, is far from unity, indicating that cutinase does not exhibit a simple two state unfolding behaviour. The role of pH on the electrostatic contribution to the thermal stability was assessed using TITRA. We propose a molecular interpretation for the pH-variation in enzymatic activity.

No MeSH data available.


Reversibility of cutinase at pH 4.0. Cutinase has been heated and after cooling to 5 degrees different wait times have been introduced to account for the time necessary to refold the enzyme, before reheating the protein solution. The % refolding values have been obtained as relative values based on the values obtained from the first scan.
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Figure 2: Reversibility of cutinase at pH 4.0. Cutinase has been heated and after cooling to 5 degrees different wait times have been introduced to account for the time necessary to refold the enzyme, before reheating the protein solution. The % refolding values have been obtained as relative values based on the values obtained from the first scan.

Mentions: The thermograms also revealed that cutinase showed irreversible unfolding beyond pH 6.0. In the acidic region between pH 3.0 to pH 6.0 a denaturation peak has been detected upon reheating. At pH 4.0 the refolding capability of cutinase has been investigated and resulted in an 80% refolding of the native structure (Figure 2). The time necessary to regain 80% of the native protein was 3 hours at 5degrees. Whereas in the basic region the refolding pathway seems to be blocked, since no transition can be detected upon reheating.


The Thermal Stability of the Fusarium solani pisi Cutinase as a Function of pH.

Petersen SB, Fojan P, Petersen EI, Petersen MT - J. Biomed. Biotechnol. (2001)

Reversibility of cutinase at pH 4.0. Cutinase has been heated and after cooling to 5 degrees different wait times have been introduced to account for the time necessary to refold the enzyme, before reheating the protein solution. The % refolding values have been obtained as relative values based on the values obtained from the first scan.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC113781&req=5

Figure 2: Reversibility of cutinase at pH 4.0. Cutinase has been heated and after cooling to 5 degrees different wait times have been introduced to account for the time necessary to refold the enzyme, before reheating the protein solution. The % refolding values have been obtained as relative values based on the values obtained from the first scan.
Mentions: The thermograms also revealed that cutinase showed irreversible unfolding beyond pH 6.0. In the acidic region between pH 3.0 to pH 6.0 a denaturation peak has been detected upon reheating. At pH 4.0 the refolding capability of cutinase has been investigated and resulted in an 80% refolding of the native structure (Figure 2). The time necessary to regain 80% of the native protein was 3 hours at 5degrees. Whereas in the basic region the refolding pathway seems to be blocked, since no transition can be detected upon reheating.

Bottom Line: The ratio between the calorimetric enthalpy (DeltaH(cal)) and the van't Hoff enthalpy (DeltaH(v)) obtained, is far from unity, indicating that cutinase does not exhibit a simple two state unfolding behaviour.The role of pH on the electrostatic contribution to the thermal stability was assessed using TITRA.We propose a molecular interpretation for the pH-variation in enzymatic activity.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
We have investigated the thermal stability of the Fusarium solani pisi cutinase as a function of pH, in the range from pH 2-12. Its highest enzymatic activity coincides with the pH-range at which it displays its highest thermal stability. The unfolding of the enzyme as a function of pH was investigated by microcalorimetry. The ratio between the calorimetric enthalpy (DeltaH(cal)) and the van't Hoff enthalpy (DeltaH(v)) obtained, is far from unity, indicating that cutinase does not exhibit a simple two state unfolding behaviour. The role of pH on the electrostatic contribution to the thermal stability was assessed using TITRA. We propose a molecular interpretation for the pH-variation in enzymatic activity.

No MeSH data available.