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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

Western blot and Coomassie stained gel of N. tabacum leaf extracts. Proteins were detected with monoclonal anti-polyhistidine antibodies. His-tagged mCherry was used as a positive control (+) and leaf extract of a plant infiltrated with the empty vector pTRAkc (-) was used as negative control. Lanes for BglC and E1–E6 contain leaf extracts of plants infiltrated with the respective pTRAkc-ERH clones for cellulase expression. Each sample represents 5.625 μg of TSP (total soluble protein).
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Figure 3: Western blot and Coomassie stained gel of N. tabacum leaf extracts. Proteins were detected with monoclonal anti-polyhistidine antibodies. His-tagged mCherry was used as a positive control (+) and leaf extract of a plant infiltrated with the empty vector pTRAkc (-) was used as negative control. Lanes for BglC and E1–E6 contain leaf extracts of plants infiltrated with the respective pTRAkc-ERH clones for cellulase expression. Each sample represents 5.625 μg of TSP (total soluble protein).

Mentions: The leaf extracts (Figure 3) yielded bands for all six cellulases, but no band was visible for β-glucosidase BglC. We observed a single ∼135 kDa band for endocellulase E1 and multiple bands in the range of 48–65 kDa for endocellulase E2, as well as weaker bands (∼28 – 24 kDa) potentially showing evidence of protein degradation. For exocellulase E3 and the processive endocellulase E4, bands were visible at ∼90 kDa and ∼110 kDa, respectively. Weaker E4 bands (∼100 kDa and ca. 80 kDa) represented additional forms of the protein, whereas the additional Band received for E3 (∼65 kDa) might represent a non-glycosylated form of the exocellulase. E5 was represented by a strong band at ∼55 kDa and several weak smaller bands potentially representing degradation fragments. Exocellulase E6 was represented by a ∼120 kDa band and a weaker band at about 100 kDa. The high molecular mass of the main enzyme bands suggested that (partial) N-glycosylation had occurred in the ER, although none of these proteins are glycosylated in their natural host T. fusca. Subsequently, N-glycosylation analysis was performed, using the NetNGlyc 1.0 Server software to predict glycan acceptor sites. As shown in Table 4, all of the enzymes contain 1–8 likely glycosylation sites, suggesting that glycosylated forms are probable to be synthesized in plants. This correlates well with the higher molecular masses of the modified protein variants detected by western blot.


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)

Western blot and Coomassie stained gel of N. tabacum leaf extracts. Proteins were detected with monoclonal anti-polyhistidine antibodies. His-tagged mCherry was used as a positive control (+) and leaf extract of a plant infiltrated with the empty vector pTRAkc (-) was used as negative control. Lanes for BglC and E1–E6 contain leaf extracts of plants infiltrated with the respective pTRAkc-ERH clones for cellulase expression. Each sample represents 5.625 μg of TSP (total soluble protein).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Western blot and Coomassie stained gel of N. tabacum leaf extracts. Proteins were detected with monoclonal anti-polyhistidine antibodies. His-tagged mCherry was used as a positive control (+) and leaf extract of a plant infiltrated with the empty vector pTRAkc (-) was used as negative control. Lanes for BglC and E1–E6 contain leaf extracts of plants infiltrated with the respective pTRAkc-ERH clones for cellulase expression. Each sample represents 5.625 μg of TSP (total soluble protein).
Mentions: The leaf extracts (Figure 3) yielded bands for all six cellulases, but no band was visible for β-glucosidase BglC. We observed a single ∼135 kDa band for endocellulase E1 and multiple bands in the range of 48–65 kDa for endocellulase E2, as well as weaker bands (∼28 – 24 kDa) potentially showing evidence of protein degradation. For exocellulase E3 and the processive endocellulase E4, bands were visible at ∼90 kDa and ∼110 kDa, respectively. Weaker E4 bands (∼100 kDa and ca. 80 kDa) represented additional forms of the protein, whereas the additional Band received for E3 (∼65 kDa) might represent a non-glycosylated form of the exocellulase. E5 was represented by a strong band at ∼55 kDa and several weak smaller bands potentially representing degradation fragments. Exocellulase E6 was represented by a ∼120 kDa band and a weaker band at about 100 kDa. The high molecular mass of the main enzyme bands suggested that (partial) N-glycosylation had occurred in the ER, although none of these proteins are glycosylated in their natural host T. fusca. Subsequently, N-glycosylation analysis was performed, using the NetNGlyc 1.0 Server software to predict glycan acceptor sites. As shown in Table 4, all of the enzymes contain 1–8 likely glycosylation sites, suggesting that glycosylated forms are probable to be synthesized in plants. This correlates well with the higher molecular masses of the modified protein variants detected by western blot.

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