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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

Azo-CMC assay for endocellulase activity. Each 500 μl reaction was carried out for 30 min at 50°C in 50 mM sodium acetate buffer (pH 5.5) containing 0.5% Azo-CMC (w/v) and values were calculated for 1 mg TSP of cell or leaf extract for better comparison. The reaction was stopped by addition of 1.25 ml precipitation solution. After centrifugation (10 min, 1000 × g, room temperature), the absorbance of the supernatant at 590 nm was measured photometrically. All measurements were performed in triplicate. Error bars show standard deviation.
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Figure 4: Azo-CMC assay for endocellulase activity. Each 500 μl reaction was carried out for 30 min at 50°C in 50 mM sodium acetate buffer (pH 5.5) containing 0.5% Azo-CMC (w/v) and values were calculated for 1 mg TSP of cell or leaf extract for better comparison. The reaction was stopped by addition of 1.25 ml precipitation solution. After centrifugation (10 min, 1000 × g, room temperature), the absorbance of the supernatant at 590 nm was measured photometrically. All measurements were performed in triplicate. Error bars show standard deviation.

Mentions: Endocellulases E1, E2, and E5 were tested for endocellulase activity using the model substrate azo-carboxymethyl-cellulose (Azo-CMC). The results for each expression system were adjusted to 1 mg of TSP and were compared in Figure 4. Although E2 clearly showed activity against the test substrate when produced in E. coli, we observed almost no detectable activity for E1 and E5 in this expression system. Plant extracts containing the three endocellulases were active against Azo-CMC, and all extracts were more active than their recombinant bacterial counterparts when adjusted to equal TSP content.


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)

Azo-CMC assay for endocellulase activity. Each 500 μl reaction was carried out for 30 min at 50°C in 50 mM sodium acetate buffer (pH 5.5) containing 0.5% Azo-CMC (w/v) and values were calculated for 1 mg TSP of cell or leaf extract for better comparison. The reaction was stopped by addition of 1.25 ml precipitation solution. After centrifugation (10 min, 1000 × g, room temperature), the absorbance of the supernatant at 590 nm was measured photometrically. 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 4: Azo-CMC assay for endocellulase activity. Each 500 μl reaction was carried out for 30 min at 50°C in 50 mM sodium acetate buffer (pH 5.5) containing 0.5% Azo-CMC (w/v) and values were calculated for 1 mg TSP of cell or leaf extract for better comparison. The reaction was stopped by addition of 1.25 ml precipitation solution. After centrifugation (10 min, 1000 × g, room temperature), the absorbance of the supernatant at 590 nm was measured photometrically. All measurements were performed in triplicate. Error bars show standard deviation.
Mentions: Endocellulases E1, E2, and E5 were tested for endocellulase activity using the model substrate azo-carboxymethyl-cellulose (Azo-CMC). The results for each expression system were adjusted to 1 mg of TSP and were compared in Figure 4. Although E2 clearly showed activity against the test substrate when produced in E. coli, we observed almost no detectable activity for E1 and E5 in this expression system. Plant extracts containing the three endocellulases were active against Azo-CMC, and all extracts were more active than their recombinant bacterial counterparts when adjusted to equal TSP content.

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