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Ethanol inducible expression of a mesophilic cellulase avoids adverse effects on plant development.

Klose H, Günl M, Usadel B, Fischer R, Commandeur U - Biotechnol Biofuels (2013)

Bottom Line: However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development.These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, Aachen, 52074, Germany. commandeur@molbiotech.rwth-aachen.de.

ABSTRACT

Background: Plant-produced biomass-degrading enzymes are promising tools for the processing of lignocellulose to fermentable sugars. A major limitation of in planta production is that high-level expression of such enzymes could potentially affect the structure and integrity of the plant cell wall and negatively influence plant growth and development.

Results: Here, we evaluate the impact on tobacco plant development of constitutive versus alcohol-inducible expression of the endoglucanase TrCel5A from the mesophilic fungus Trichoderma reesei. Using this system, we are able to demonstrate that constitutive expression of the enzyme, controlled by the doubled Cauliflower Mosaic Virus promoter, leads to lower cellulose content of the plant combined with severe effects on plant growth. However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.

Conclusion: We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development. These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

No MeSH data available.


Related in: MedlinePlus

Enzymatic activity of TrCel5A at different pH and temperature values using the azoCMC assay. Activity of TrCel5A was determined for pH values between 3.0 and 7.0 (A) and temperature between 20–70°C (B). The maximum activity measured for each system (at pH 4.8 and 55°C, respectively) was set to 100%. For panel A, the buffer systems are as indicated in the figure and temperature was set at 55°C. For panel B 50 mM Na-Acetate, pH 4.8 buffer was used throughout.
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Figure 3: Enzymatic activity of TrCel5A at different pH and temperature values using the azoCMC assay. Activity of TrCel5A was determined for pH values between 3.0 and 7.0 (A) and temperature between 20–70°C (B). The maximum activity measured for each system (at pH 4.8 and 55°C, respectively) was set to 100%. For panel A, the buffer systems are as indicated in the figure and temperature was set at 55°C. For panel B 50 mM Na-Acetate, pH 4.8 buffer was used throughout.

Mentions: The biochemical properties of purified TrCel5A were tested on azoCMC, within the pH range 3.0–7.0 and the temperature range 20–70°C (Figure 3). The optimal conditions were pH 5.4 and 55°C, at which 50% of the enzyme remained active for 90 min. There was no significant activity against highly crystalline substrates, such as avicel, however we detected significant activity against heterogeneous substrates including lichenan and barley-β-glucan (Table 1).


Ethanol inducible expression of a mesophilic cellulase avoids adverse effects on plant development.

Klose H, Günl M, Usadel B, Fischer R, Commandeur U - Biotechnol Biofuels (2013)

Enzymatic activity of TrCel5A at different pH and temperature values using the azoCMC assay. Activity of TrCel5A was determined for pH values between 3.0 and 7.0 (A) and temperature between 20–70°C (B). The maximum activity measured for each system (at pH 4.8 and 55°C, respectively) was set to 100%. For panel A, the buffer systems are as indicated in the figure and temperature was set at 55°C. For panel B 50 mM Na-Acetate, pH 4.8 buffer was used throughout.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643885&req=5

Figure 3: Enzymatic activity of TrCel5A at different pH and temperature values using the azoCMC assay. Activity of TrCel5A was determined for pH values between 3.0 and 7.0 (A) and temperature between 20–70°C (B). The maximum activity measured for each system (at pH 4.8 and 55°C, respectively) was set to 100%. For panel A, the buffer systems are as indicated in the figure and temperature was set at 55°C. For panel B 50 mM Na-Acetate, pH 4.8 buffer was used throughout.
Mentions: The biochemical properties of purified TrCel5A were tested on azoCMC, within the pH range 3.0–7.0 and the temperature range 20–70°C (Figure 3). The optimal conditions were pH 5.4 and 55°C, at which 50% of the enzyme remained active for 90 min. There was no significant activity against highly crystalline substrates, such as avicel, however we detected significant activity against heterogeneous substrates including lichenan and barley-β-glucan (Table 1).

Bottom Line: However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development.These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, Aachen, 52074, Germany. commandeur@molbiotech.rwth-aachen.de.

ABSTRACT

Background: Plant-produced biomass-degrading enzymes are promising tools for the processing of lignocellulose to fermentable sugars. A major limitation of in planta production is that high-level expression of such enzymes could potentially affect the structure and integrity of the plant cell wall and negatively influence plant growth and development.

Results: Here, we evaluate the impact on tobacco plant development of constitutive versus alcohol-inducible expression of the endoglucanase TrCel5A from the mesophilic fungus Trichoderma reesei. Using this system, we are able to demonstrate that constitutive expression of the enzyme, controlled by the doubled Cauliflower Mosaic Virus promoter, leads to lower cellulose content of the plant combined with severe effects on plant growth. However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.

Conclusion: We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development. These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

No MeSH data available.


Related in: MedlinePlus