The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A

Harry Boer (Corresponding Author), Anu Koivula

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Abstract

The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its three‐dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild‐type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild‐type enzyme show an optimal thermostability between pH 3.5–5.6 (Tm, 62 ± 2°C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerström, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19–30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild‐type enzyme. In addition, an interesting time‐dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range.
Original languageEnglish
Pages (from-to)841-848
Number of pages8
JournalEuropean Journal of Biochemistry
Volume270
Issue number5
DOIs
Publication statusPublished - 2003
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Thermodynamic stability
Hot Temperature
Enzymes
Fluorescence
Cellulases
Cellulase
Substrates
Fungi
Tryptophan
Proteins
Engineers
Point Mutation

Keywords

  • cellulase
  • circular dichroism
  • fluorescence
  • pH optimum
  • thermostability

Cite this

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title = "The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A",
abstract = "The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its three‐dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild‐type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild‐type enzyme show an optimal thermostability between pH 3.5–5.6 (Tm, 62 ± 2°C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerstr{\"o}m, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19–30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild‐type enzyme. In addition, an interesting time‐dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range.",
keywords = "cellulase, circular dichroism, fluorescence, pH optimum, thermostability",
author = "Harry Boer and Anu Koivula",
year = "2003",
doi = "10.1046/j.1432-1033.2003.03431.x",
language = "English",
volume = "270",
pages = "841--848",
journal = "FEBS Journal",
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T1 - The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A

AU - Boer, Harry

AU - Koivula, Anu

PY - 2003

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N2 - The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its three‐dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild‐type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild‐type enzyme show an optimal thermostability between pH 3.5–5.6 (Tm, 62 ± 2°C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerström, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19–30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild‐type enzyme. In addition, an interesting time‐dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range.

AB - The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its three‐dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild‐type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild‐type enzyme show an optimal thermostability between pH 3.5–5.6 (Tm, 62 ± 2°C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerström, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19–30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild‐type enzyme. In addition, an interesting time‐dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range.

KW - cellulase

KW - circular dichroism

KW - fluorescence

KW - pH optimum

KW - thermostability

U2 - 10.1046/j.1432-1033.2003.03431.x

DO - 10.1046/j.1432-1033.2003.03431.x

M3 - Article

VL - 270

SP - 841

EP - 848

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 5

ER -