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Addition of a carbohydrate-binding module enhances cellulase penetration into cellulose substrates.

Reyes-Ortiz V, Heins RA, Cheng G, Kim EY, Vernon BC, Elandt RB, Adams PD, Sale KL, Hadi MZ, Simmons BA, Kent MS, Tullman-Ercek D - Biotechnol Biofuels (2013)

Bottom Line: Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive.Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type.Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film.

View Article: PubMed Central - HTML - PubMed

Affiliation: Deconstruction Division, Joint BioEnergy Institute, Emeryville, CA 94608, US. dtercek@berkeley.edu.

ABSTRACT

Introduction: Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive. To further improve these enzymes for application at industrial conditions, it is critical to gain a better understanding of not only the details of the degradation process, but also the function of accessory modules.

Method: We fused a carbohydrate-binding module (CBM) from family 2a to two thermophilic endoglucanases. We then applied neutron reflectometry to determine the mechanism of the resulting enhancements.

Results: Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type. Importantly, we demonstrate that the wild type enzymes affect primarily the surface properties of an amorphous cellulose film, while the chimeras containing a CBM alter the bulk properties of the amorphous film.

Conclusion: Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film.

No MeSH data available.


Related in: MedlinePlus

Figure 3 Neutron reflectivity data from cellulose films exposed to the chimeric cellulases Cel9A-CBM (a) and Cel5A-CBM (b) at 5 μM and 20°C. The fringe pattern, related to the thicknesses of the various layers, is distinctly different for the two chimeras: the fringes decay uniformly with qz for Cel5A-CBM, whereas for Cel9A-CBM the first three fringes are damped relative to the fringes at higher qz. The latter pattern indicates a distinct layer of enzyme adsorbed at the surface of the film.
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Figure 3: Figure 3 Neutron reflectivity data from cellulose films exposed to the chimeric cellulases Cel9A-CBM (a) and Cel5A-CBM (b) at 5 μM and 20°C. The fringe pattern, related to the thicknesses of the various layers, is distinctly different for the two chimeras: the fringes decay uniformly with qz for Cel5A-CBM, whereas for Cel9A-CBM the first three fringes are damped relative to the fringes at higher qz. The latter pattern indicates a distinct layer of enzyme adsorbed at the surface of the film.

Mentions: For Cel9A-CBM a fringe pattern that is distinctly different from that for Cel5A-CBM resulted, that indicates a layer of protein adsorbed to the film surface (Figure 3 and Additional file 1: Figure S8). The additional layer at the film surface is clearly identified as a layer of protein because the scattering length density (SLD) value for that layer is much lower than that of the swollen cellulose film. This additional layer is not observed for the Cel9A CD alone or for the Cel5A-CBM chimera. Therefore this layer is likely due to strong CBM binding at the surface along with an inherent property of the Cel9A CD that inhibits its penetration (e.g. larger size that slows its entry into the bulk of the film). Thus for Cel9A-CBM the results indicate substantial penetration into the bulk as well as a distinct layer of enzyme remaining at the surface. Film expansion was not observed for Cel9A-CBM as for Cel5A-CBM, but it is possible that for Cel9A-CBM film expansion was compensated by activity at the surface of the film due to the adsorbed layer of enzymes. The cellulose profile shown in Figure 2 for Cel9A-CBM was obtained by truncating the full SLD profile (Additional file 1: Figure S8a) at the interface between the cellulose film and the layer of adsorbed enzyme.


Addition of a carbohydrate-binding module enhances cellulase penetration into cellulose substrates.

Reyes-Ortiz V, Heins RA, Cheng G, Kim EY, Vernon BC, Elandt RB, Adams PD, Sale KL, Hadi MZ, Simmons BA, Kent MS, Tullman-Ercek D - Biotechnol Biofuels (2013)

Figure 3 Neutron reflectivity data from cellulose films exposed to the chimeric cellulases Cel9A-CBM (a) and Cel5A-CBM (b) at 5 μM and 20°C. The fringe pattern, related to the thicknesses of the various layers, is distinctly different for the two chimeras: the fringes decay uniformly with qz for Cel5A-CBM, whereas for Cel9A-CBM the first three fringes are damped relative to the fringes at higher qz. The latter pattern indicates a distinct layer of enzyme adsorbed at the surface of the film.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716932&req=5

Figure 3: Figure 3 Neutron reflectivity data from cellulose films exposed to the chimeric cellulases Cel9A-CBM (a) and Cel5A-CBM (b) at 5 μM and 20°C. The fringe pattern, related to the thicknesses of the various layers, is distinctly different for the two chimeras: the fringes decay uniformly with qz for Cel5A-CBM, whereas for Cel9A-CBM the first three fringes are damped relative to the fringes at higher qz. The latter pattern indicates a distinct layer of enzyme adsorbed at the surface of the film.
Mentions: For Cel9A-CBM a fringe pattern that is distinctly different from that for Cel5A-CBM resulted, that indicates a layer of protein adsorbed to the film surface (Figure 3 and Additional file 1: Figure S8). The additional layer at the film surface is clearly identified as a layer of protein because the scattering length density (SLD) value for that layer is much lower than that of the swollen cellulose film. This additional layer is not observed for the Cel9A CD alone or for the Cel5A-CBM chimera. Therefore this layer is likely due to strong CBM binding at the surface along with an inherent property of the Cel9A CD that inhibits its penetration (e.g. larger size that slows its entry into the bulk of the film). Thus for Cel9A-CBM the results indicate substantial penetration into the bulk as well as a distinct layer of enzyme remaining at the surface. Film expansion was not observed for Cel9A-CBM as for Cel5A-CBM, but it is possible that for Cel9A-CBM film expansion was compensated by activity at the surface of the film due to the adsorbed layer of enzymes. The cellulose profile shown in Figure 2 for Cel9A-CBM was obtained by truncating the full SLD profile (Additional file 1: Figure S8a) at the interface between the cellulose film and the layer of adsorbed enzyme.

Bottom Line: Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive.Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type.Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film.

View Article: PubMed Central - HTML - PubMed

Affiliation: Deconstruction Division, Joint BioEnergy Institute, Emeryville, CA 94608, US. dtercek@berkeley.edu.

ABSTRACT

Introduction: Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive. To further improve these enzymes for application at industrial conditions, it is critical to gain a better understanding of not only the details of the degradation process, but also the function of accessory modules.

Method: We fused a carbohydrate-binding module (CBM) from family 2a to two thermophilic endoglucanases. We then applied neutron reflectometry to determine the mechanism of the resulting enhancements.

Results: Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type. Importantly, we demonstrate that the wild type enzymes affect primarily the surface properties of an amorphous cellulose film, while the chimeras containing a CBM alter the bulk properties of the amorphous film.

Conclusion: Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film.

No MeSH data available.


Related in: MedlinePlus