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Comparison of Thermobifida fusca Cellulases Expressed in Escherichia coli and Nicotiana tabacum Indicates Advantages of the Plant System for the Expression of Bacterial Cellulases.

Klinger J, Fischer R, Commandeur U - Front Plant Sci (2015)

Bottom Line: Only the β-glucosidase showed high activity against 4-MUC.In contrast, all the plant-derived enzymes were active against the respective model substrates.Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biology VII (Molecular Biotechnology), RWTH Aachen University Aachen, Germany.

ABSTRACT
The economic conversion of lignocellulosic biomass to biofuels requires in addition to pretreatment techniques access to large quantities of inexpensive cellulases to be competitive with established first generation processes. A solution to this problem could be achieved by plant based expression of these enzymes. We expressed the complete set of six cellulases and an additional β-glucosidase expressed from Thermobifida fusca in the bacterium Escherichia coli and in tobacco plants (Nicotiana tabacum). This was done to determine whether functional enzyme expression was feasible in these organisms. In extracts of recombinant E. coli cells, five of the proteins were detected by western blot analysis, but exocellulases E3 and E6 were undetectable. In the plant-based expression system we were able to detect all six cellulases but not the β-glucosidase even though activity was detectable. When E. coli was used as the expression system, endocellulase E2 was active, while endocellulases E1 and E5 showed only residual activity. The remaining cellulases appeared completely inactive against the model substrates azo-carboxymethyl-cellulose (Azo-CMC) and 4-methylumbelliferyl-cellobioside (4-MUC). Only the β-glucosidase showed high activity against 4-MUC. In contrast, all the plant-derived enzymes were active against the respective model substrates. Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host.

No MeSH data available.


Related in: MedlinePlus

The 4-MUC assay for enzyme activity. Each 100 μl reaction was carried out for 2 h at 50°C in 50 mM sodium acetate buffer (pH 5.5) containing 1 mM 4-MUC and values were calculated for 1 mg TSP of cell or leaf extract for better comparison. The assay was stopped by adding 100 μL 0.15 M glycine (pH 10) and fluorescence was measured at 465 nm (excitation, 360 nm) against a calibration curve. All measurements were performed in triplicate. Error bars show standard deviation.
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Figure 5: The 4-MUC assay for enzyme activity. Each 100 μl reaction was carried out for 2 h at 50°C in 50 mM sodium acetate buffer (pH 5.5) containing 1 mM 4-MUC and values were calculated for 1 mg TSP of cell or leaf extract for better comparison. The assay was stopped by adding 100 μL 0.15 M glycine (pH 10) and fluorescence was measured at 465 nm (excitation, 360 nm) against a calibration curve. All measurements were performed in triplicate. Error bars show standard deviation.

Mentions: The activity of the remaining enzymes was tested against 4-MUC (Figure 5). The bacterial extracts showed no detectable activity for the remaining cellulases compared to the empty vector control (pRB95). In contrast, the activity of BglC exceeded the detection limit of the assay indicating that the enzyme was highly active when produced in E. coli. All the cellulases appeared to be active compared to the negative control (empty pTRAkc vector) when expressed in tobacco, but the activity of plant-derived BglC was significantly lower than the same enzyme produced in E. coli when adjusted to similar amounts of TSP.


Comparison of Thermobifida fusca Cellulases Expressed in Escherichia coli and Nicotiana tabacum Indicates Advantages of the Plant System for the Expression of Bacterial Cellulases.

Klinger J, Fischer R, Commandeur U - Front Plant Sci (2015)

The 4-MUC assay for enzyme activity. Each 100 μl reaction was carried out for 2 h at 50°C in 50 mM sodium acetate buffer (pH 5.5) containing 1 mM 4-MUC and values were calculated for 1 mg TSP of cell or leaf extract for better comparison. The assay was stopped by adding 100 μL 0.15 M glycine (pH 10) and fluorescence was measured at 465 nm (excitation, 360 nm) against a calibration curve. All measurements were performed in triplicate. Error bars show standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664618&req=5

Figure 5: The 4-MUC assay for enzyme activity. Each 100 μl reaction was carried out for 2 h at 50°C in 50 mM sodium acetate buffer (pH 5.5) containing 1 mM 4-MUC and values were calculated for 1 mg TSP of cell or leaf extract for better comparison. The assay was stopped by adding 100 μL 0.15 M glycine (pH 10) and fluorescence was measured at 465 nm (excitation, 360 nm) against a calibration curve. All measurements were performed in triplicate. Error bars show standard deviation.
Mentions: The activity of the remaining enzymes was tested against 4-MUC (Figure 5). The bacterial extracts showed no detectable activity for the remaining cellulases compared to the empty vector control (pRB95). In contrast, the activity of BglC exceeded the detection limit of the assay indicating that the enzyme was highly active when produced in E. coli. All the cellulases appeared to be active compared to the negative control (empty pTRAkc vector) when expressed in tobacco, but the activity of plant-derived BglC was significantly lower than the same enzyme produced in E. coli when adjusted to similar amounts of TSP.

Bottom Line: Only the β-glucosidase showed high activity against 4-MUC.In contrast, all the plant-derived enzymes were active against the respective model substrates.Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biology VII (Molecular Biotechnology), RWTH Aachen University Aachen, Germany.

ABSTRACT
The economic conversion of lignocellulosic biomass to biofuels requires in addition to pretreatment techniques access to large quantities of inexpensive cellulases to be competitive with established first generation processes. A solution to this problem could be achieved by plant based expression of these enzymes. We expressed the complete set of six cellulases and an additional β-glucosidase expressed from Thermobifida fusca in the bacterium Escherichia coli and in tobacco plants (Nicotiana tabacum). This was done to determine whether functional enzyme expression was feasible in these organisms. In extracts of recombinant E. coli cells, five of the proteins were detected by western blot analysis, but exocellulases E3 and E6 were undetectable. In the plant-based expression system we were able to detect all six cellulases but not the β-glucosidase even though activity was detectable. When E. coli was used as the expression system, endocellulase E2 was active, while endocellulases E1 and E5 showed only residual activity. The remaining cellulases appeared completely inactive against the model substrates azo-carboxymethyl-cellulose (Azo-CMC) and 4-methylumbelliferyl-cellobioside (4-MUC). Only the β-glucosidase showed high activity against 4-MUC. In contrast, all the plant-derived enzymes were active against the respective model substrates. Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host.

No MeSH data available.


Related in: MedlinePlus