Abstract
The cellulosomes produced by Clostridium cellulovorans are organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain in C. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30-to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces of C. cellulovorans using its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that in C. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.
| Original language | English |
|---|---|
| Pages (from-to) | 5942-5948 |
| Number of pages | 7 |
| Journal | Applied and Environmental Microbiology |
| Volume | 79 |
| Issue number | 19 |
| DOIs | |
| Publication status | Published - 2013 Sep 20 |
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ASJC Scopus subject areas
- Applied Microbiology and Biotechnology
- Food Science
- Biotechnology
- Ecology
Cite this
Unique contribution of the cell wall-binding endoglucanase G to the cellulolytic complex in Clostridium cellulovorans. / Jeon, Sang Duck; Lee, Ji Eun; Kim, Su Jung; Park, Sung Hyun; Choi, Gi Wook; Han, Sung Ok.
In: Applied and Environmental Microbiology, Vol. 79, No. 19, 20.09.2013, p. 5942-5948.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Unique contribution of the cell wall-binding endoglucanase G to the cellulolytic complex in Clostridium cellulovorans
AU - Jeon, Sang Duck
AU - Lee, Ji Eun
AU - Kim, Su Jung
AU - Park, Sung Hyun
AU - Choi, Gi Wook
AU - Han, Sung Ok
PY - 2013/9/20
Y1 - 2013/9/20
N2 - The cellulosomes produced by Clostridium cellulovorans are organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain in C. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30-to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces of C. cellulovorans using its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that in C. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.
AB - The cellulosomes produced by Clostridium cellulovorans are organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain in C. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30-to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces of C. cellulovorans using its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that in C. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.
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UR - http://www.scopus.com/inward/citedby.url?scp=84884181164&partnerID=8YFLogxK
U2 - 10.1128/AEM.01400-13
DO - 10.1128/AEM.01400-13
M3 - Article
C2 - 23872560
AN - SCOPUS:84884181164
VL - 79
SP - 5942
EP - 5948
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
SN - 0099-2240
IS - 19
ER -