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The synergistic action of accessory enzymes enhances the hydrolytic potential of a "cellulase mixture" but is highly substrate specific.

Hu J, Arantes V, Pribowo A, Saddler JN - Biotechnol Biofuels (2013)

Bottom Line: Currently, the amount of protein/enzyme required to achieve effective cellulose hydrolysis is still too high.The GH10 EX was more effective on steam pretreated agriculture residues and hardwood substrates whereas GH5 XG addition was more effective on softwood substrates.However, the extent of improved hydrolysis was highly substrate dependent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Forestry Products Biotechnology/Bioenergy Group, Wood Science Department, University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada. jack.saddler@ubc.ca.

ABSTRACT

Background: Currently, the amount of protein/enzyme required to achieve effective cellulose hydrolysis is still too high. One way to reduce the amount of protein/enzyme required is to formulate a more efficient enzyme cocktail by adding so-called accessory enzymes such as xylanase, lytic polysaccharide monooxygenase (AA9, formerly known as GH61), etc., to the cellulase mixture. Previous work has shown the strong synergism that can occur between cellulase and xylanase mixtures during the hydrolysis of steam pretreated corn stover, requiring lower protein loading to achieve effective hydrolysis. However, relatively high loadings of xylanases were required. When family 10 and 11 endo-xylanases and family 5 xyloglucanase were supplemented to a commercial cellulase mixture varying degrees of improved hydrolysis over a range of pretreated, lignocellulosic substrates were observed.

Results: The potential synergistic interactions between cellulase monocomponents and hemicellulases from family 10 and 11 endo-xylanases (GH10 EX and GH11 EX) and family 5 xyloglucanase (GH5 XG), during hydrolysis of various steam pretreated lignocellulosic substrates, were assessed. It was apparent that the hydrolytic activity of cellulase monocomponents was enhanced by the addition of accessory enzymes although the "boosting" effect was highly substrate specific. The GH10 EX and GH5 XG both exhibited broad substrate specificity and showed strong synergistic interaction with the cellulases when added individually. The GH10 EX was more effective on steam pretreated agriculture residues and hardwood substrates whereas GH5 XG addition was more effective on softwood substrates. The synergistic interaction between GH10 EX and GH5 XG when added together further enhanced the hydrolytic activity of the cellulase enzymes over a range of pretreated lignocellulosic substrates. GH10 EX addition could also stimulate further cellulose hydrolysis when added to the hydrolysis reactions when the rate of hydrolysis had levelled off.

Conclusions: Endo-xylanases and xyloglucanases interacted synergistically with cellulases to improve the hydrolysis of a range of pretreated lignocellulosic substrates. However, the extent of improved hydrolysis was highly substrate dependent. It appears that those accessory enzymes, such as GH10 EX and GH5 XG, with broader substrate specificities promoted the greatest improvements in the hydrolytic performance of the cellulase mixture on all of the pretreated biomass substrates.

No MeSH data available.


Related in: MedlinePlus

Relative improvement in cellulose hydrolysis by supplementation of binary mixtures of accessory enzymes (GH11EX/GH10EX, GH11EX/GH5XG and GH10EX/GH5XG) to Cel7A during the hydrolysis of various pretreated lignocellulosic substrates (SPCS, SPLP and SPP200) at 72 h. Substrate control: dissolving pulp (DP). Enzyme control: GH10EX and GH10EX/Cel5A.
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Figure 3: Relative improvement in cellulose hydrolysis by supplementation of binary mixtures of accessory enzymes (GH11EX/GH10EX, GH11EX/GH5XG and GH10EX/GH5XG) to Cel7A during the hydrolysis of various pretreated lignocellulosic substrates (SPCS, SPLP and SPP200) at 72 h. Substrate control: dissolving pulp (DP). Enzyme control: GH10EX and GH10EX/Cel5A.

Mentions: To assess the possible synergistic interaction between the accessory enzymes and Cel7A, binary mixtures of GH10 EX/GH11 EX, GH10 EX/GH5 XG, and GH11 EX/GH5 XG were formulated and compared with the hydrolysis obtained when Cel7A was supplemented with GH10 EX alone (Figure 3). With the exception of the binary mixture GH11 EX/GH5 XG, all of the other binary mixtures resulted in a similar or an increase in hydrolytic activity as compared to when Cel7A was supplemented with GH10 EX alone. The greatest improvement was observed with binary mixture GH10 EX/GH5 XG, which enhanced the glucose yields obtained from the SPCS, SPP200, DP and SPLP substrates by 120, 60, 60 and 20% respectively (Figure 3). As GH5 XG had previously exhibited detectable CMCase activity (Table 2), we next substituted GH5 XG in the binary mixture GH10 EX/GH5 XG with a purified endo-glucanase (Cel5A) to assess the interaction of GH10 EX with a “true” endoglucanase enzyme. Although the addition of the GH10 EX/Cel5A mixture resulted in the improved hydrolytic activity of the Cel7A on the SPLP and SPP200 substrates as compared to the addition of GH 10 EX alone, these improvements were similar (on SPLP) or lower (on DP, SPCS and SPP200) to those observed with the GH10 EX/GH5 XG mixture. This suggested that the detected endo-glucanase activity of GH5 XG did not explain the strong synergistic interaction observed with the binary mixture GH10 EX/GH5 XG.


The synergistic action of accessory enzymes enhances the hydrolytic potential of a "cellulase mixture" but is highly substrate specific.

Hu J, Arantes V, Pribowo A, Saddler JN - Biotechnol Biofuels (2013)

Relative improvement in cellulose hydrolysis by supplementation of binary mixtures of accessory enzymes (GH11EX/GH10EX, GH11EX/GH5XG and GH10EX/GH5XG) to Cel7A during the hydrolysis of various pretreated lignocellulosic substrates (SPCS, SPLP and SPP200) at 72 h. Substrate control: dissolving pulp (DP). Enzyme control: GH10EX and GH10EX/Cel5A.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750293&req=5

Figure 3: Relative improvement in cellulose hydrolysis by supplementation of binary mixtures of accessory enzymes (GH11EX/GH10EX, GH11EX/GH5XG and GH10EX/GH5XG) to Cel7A during the hydrolysis of various pretreated lignocellulosic substrates (SPCS, SPLP and SPP200) at 72 h. Substrate control: dissolving pulp (DP). Enzyme control: GH10EX and GH10EX/Cel5A.
Mentions: To assess the possible synergistic interaction between the accessory enzymes and Cel7A, binary mixtures of GH10 EX/GH11 EX, GH10 EX/GH5 XG, and GH11 EX/GH5 XG were formulated and compared with the hydrolysis obtained when Cel7A was supplemented with GH10 EX alone (Figure 3). With the exception of the binary mixture GH11 EX/GH5 XG, all of the other binary mixtures resulted in a similar or an increase in hydrolytic activity as compared to when Cel7A was supplemented with GH10 EX alone. The greatest improvement was observed with binary mixture GH10 EX/GH5 XG, which enhanced the glucose yields obtained from the SPCS, SPP200, DP and SPLP substrates by 120, 60, 60 and 20% respectively (Figure 3). As GH5 XG had previously exhibited detectable CMCase activity (Table 2), we next substituted GH5 XG in the binary mixture GH10 EX/GH5 XG with a purified endo-glucanase (Cel5A) to assess the interaction of GH10 EX with a “true” endoglucanase enzyme. Although the addition of the GH10 EX/Cel5A mixture resulted in the improved hydrolytic activity of the Cel7A on the SPLP and SPP200 substrates as compared to the addition of GH 10 EX alone, these improvements were similar (on SPLP) or lower (on DP, SPCS and SPP200) to those observed with the GH10 EX/GH5 XG mixture. This suggested that the detected endo-glucanase activity of GH5 XG did not explain the strong synergistic interaction observed with the binary mixture GH10 EX/GH5 XG.

Bottom Line: Currently, the amount of protein/enzyme required to achieve effective cellulose hydrolysis is still too high.The GH10 EX was more effective on steam pretreated agriculture residues and hardwood substrates whereas GH5 XG addition was more effective on softwood substrates.However, the extent of improved hydrolysis was highly substrate dependent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Forestry Products Biotechnology/Bioenergy Group, Wood Science Department, University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada. jack.saddler@ubc.ca.

ABSTRACT

Background: Currently, the amount of protein/enzyme required to achieve effective cellulose hydrolysis is still too high. One way to reduce the amount of protein/enzyme required is to formulate a more efficient enzyme cocktail by adding so-called accessory enzymes such as xylanase, lytic polysaccharide monooxygenase (AA9, formerly known as GH61), etc., to the cellulase mixture. Previous work has shown the strong synergism that can occur between cellulase and xylanase mixtures during the hydrolysis of steam pretreated corn stover, requiring lower protein loading to achieve effective hydrolysis. However, relatively high loadings of xylanases were required. When family 10 and 11 endo-xylanases and family 5 xyloglucanase were supplemented to a commercial cellulase mixture varying degrees of improved hydrolysis over a range of pretreated, lignocellulosic substrates were observed.

Results: The potential synergistic interactions between cellulase monocomponents and hemicellulases from family 10 and 11 endo-xylanases (GH10 EX and GH11 EX) and family 5 xyloglucanase (GH5 XG), during hydrolysis of various steam pretreated lignocellulosic substrates, were assessed. It was apparent that the hydrolytic activity of cellulase monocomponents was enhanced by the addition of accessory enzymes although the "boosting" effect was highly substrate specific. The GH10 EX and GH5 XG both exhibited broad substrate specificity and showed strong synergistic interaction with the cellulases when added individually. The GH10 EX was more effective on steam pretreated agriculture residues and hardwood substrates whereas GH5 XG addition was more effective on softwood substrates. The synergistic interaction between GH10 EX and GH5 XG when added together further enhanced the hydrolytic activity of the cellulase enzymes over a range of pretreated lignocellulosic substrates. GH10 EX addition could also stimulate further cellulose hydrolysis when added to the hydrolysis reactions when the rate of hydrolysis had levelled off.

Conclusions: Endo-xylanases and xyloglucanases interacted synergistically with cellulases to improve the hydrolysis of a range of pretreated lignocellulosic substrates. However, the extent of improved hydrolysis was highly substrate dependent. It appears that those accessory enzymes, such as GH10 EX and GH5 XG, with broader substrate specificities promoted the greatest improvements in the hydrolytic performance of the cellulase mixture on all of the pretreated biomass substrates.

No MeSH data available.


Related in: MedlinePlus