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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.


Original DSC scans of native cutinase at different pH values. The protein concentrations were 0.7 mg/ml for all experiments. The thermograms were all baseline corrected. The buffers were chosen as described inmaterials and methods.
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Figure 1: Original DSC scans of native cutinase at different pH values. The protein concentrations were 0.7 mg/ml for all experiments. The thermograms were all baseline corrected. The buffers were chosen as described inmaterials and methods.

Mentions: The thermal unfolding of cutinase was investigated as a function of pH in different buffers. In the pH range from 2 to 4 NaH2PO4, was used in the range of 4 to 5.5 NaOAc buffer, in the range of 6 to 7.5 Sodium citrate buffer, and above 7.5 TRIS buffer was used at a buffer concentration of 20 mM for the calorimetric investigations. The pH of the buffer was adjusted with NaOH. Except for the TRIS buffer, having the highest ionization enthalpy, buffers displaying a low buffer ionization enthalpy over the temperature range applied for the calorimeter scans have been selected and therefore provide the best pH stability throughout the experiment. The excess heat capacities varysignificantly with pH (Table 1). At pH 3.0 a value of14 kcal/mol ± 8 kcal/mol was obtained, and at the pH optimum for cutinase (pH 8.5), these values rose to about 46 kcal/mol ± kcal/mol. Asseen in Table 1Tm displays a broad maximum in the range between pH 6.0 and 9.0. Original scans of cutinase at a scanrate of 90°C/h are given in Figure 1. The pH at which cutinase reveals its highest enzymatic activity (8.5) towards tributyrin [11], coincides with high thermal stability. Outside this pH range, Tm is shifting towards lower temperatures.


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)

Original DSC scans of native cutinase at different pH values. The protein concentrations were 0.7 mg/ml for all experiments. The thermograms were all baseline corrected. The buffers were chosen as described inmaterials and methods.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Original DSC scans of native cutinase at different pH values. The protein concentrations were 0.7 mg/ml for all experiments. The thermograms were all baseline corrected. The buffers were chosen as described inmaterials and methods.
Mentions: The thermal unfolding of cutinase was investigated as a function of pH in different buffers. In the pH range from 2 to 4 NaH2PO4, was used in the range of 4 to 5.5 NaOAc buffer, in the range of 6 to 7.5 Sodium citrate buffer, and above 7.5 TRIS buffer was used at a buffer concentration of 20 mM for the calorimetric investigations. The pH of the buffer was adjusted with NaOH. Except for the TRIS buffer, having the highest ionization enthalpy, buffers displaying a low buffer ionization enthalpy over the temperature range applied for the calorimeter scans have been selected and therefore provide the best pH stability throughout the experiment. The excess heat capacities varysignificantly with pH (Table 1). At pH 3.0 a value of14 kcal/mol ± 8 kcal/mol was obtained, and at the pH optimum for cutinase (pH 8.5), these values rose to about 46 kcal/mol ± kcal/mol. Asseen in Table 1Tm displays a broad maximum in the range between pH 6.0 and 9.0. Original scans of cutinase at a scanrate of 90°C/h are given in Figure 1. The pH at which cutinase reveals its highest enzymatic activity (8.5) towards tributyrin [11], coincides with high thermal stability. Outside this pH range, Tm is shifting towards lower temperatures.

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.