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Optimizing the composition of a synthetic cellulosome complex for the hydrolysis of softwood pulp: identification of the enzymatic core functions and biochemical complex characterization.

ABSTRACT: Background:The development of efficient cellulase blends is a key factor for cost-effectively valorizing biomass in a new bio-economy. Today, the enzymatic hydrolysis of plant-derived polysaccharides is mainly accomplished with fungal cellulases, whereas potentially equally effective cellulose-degrading systems from bacteria have not been developed. Particularly, a thermostable multi-enzyme cellulase complex, the cellulosome from the anaerobic cellulolytic bacterium Clostridium thermocellum is promising of being applied as cellulolytic nano-machinery for the production of fermentable sugars from cellulosic biomass. Results:In this study, 60 cellulosomal components were recombinantly produced in E. coli and systematically permuted in synthetic complexes to study the function-activity relationship of all available enzymes on Kraft pulp from pine wood as the substrate. Starting from a basic exo/endoglucanase complex, we were able to identify additional functional classes such as mannanase and xylanase for optimal activity on the substrate. Based on these results, we predicted a synthetic cellulosome complex consisting of seven single components (including the scaffoldin protein and a ?-glucosidase) and characterized it biochemically. We obtained a highly thermostable complex with optimal activity around 60-65 °C and an optimal pH in agreement with the optimum of the native cellulosome (pH 5.8). Remarkably, a fully synthetic complex containing 47 single cellulosomal components showed comparable activity with a commercially available fungal enzyme cocktail on the softwood pulp substrate. Conclusions:Our results show that synthetic bacterial multi-enzyme complexes based on the cellulosome of C. thermocellum can be applied as a versatile platform for the quick adaptation and efficient degradation of a substrate of interest.

SUBMITTER: Leis B

PROVIDER: S-EPMC6083626 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Optimizing the composition of a synthetic cellulosome complex for the hydrolysis of softwood pulp: identification of the enzymatic core functions and biochemical complex characterization.

Leis Benedikt B Held Claudia C Andreeßen Björn B Liebl Wolfgang W Graubner Sigrid S Schulte Louis-Philipp LP Schwarz Wolfgang H WH Zverlov Vladimir V VV

Biotechnology for biofuels 20180809

<h4>Background</h4>The development of efficient cellulase blends is a key factor for cost-effectively valorizing biomass in a new bio-economy. Today, the enzymatic hydrolysis of plant-derived polysaccharides is mainly accomplished with fungal cellulases, whereas potentially equally effective cellulose-degrading systems from bacteria have not been developed. Particularly, a thermostable multi-enzyme cellulase complex, the cellulosome from the anaerobic cellulolytic bacterium <i>Clostridium thermo ...[more]

PMID: 30116297

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Project description:BackgroundSoftwood is a promising feedstock for lignocellulosic biorefineries, but as it contains galactoglucomannan efficient mannan-degrading enzymes are required to unlock its full potential.ResultsBoosting of the saccharification of pretreated softwood (Canadian lodgepole pine) was investigated for 10 fungal endo-β(1→4)-mannanases (endomannanases) from GH5 and GH26, including 6 novel GH26 enzymes. The endomannanases from Trichoderma reesei (TresMan5A) and Podospora anserina (PansMan26) were investigated with and without their carbohydrate-binding module (CBM). The pH optimum and initial rates of enzyme catalysed hydrolysis were determined on pure β-mannans, including acetylated and deacetylated spruce galactoglucomannan. Melting temperature (Tm) and stability of the endomannanases during prolonged incubations were also assessed. The highest initial rates on the pure mannans were attained by GH26 endomannanases. Acetylation tended to decrease the enzymatic rates to different extents depending on the enzyme. Despite exhibiting low rates on the pure mannan substrates, TresMan5A with CBM1 catalysed highest release among the endomannanases of both mannose and glucose during softwood saccharification. The presence of the CBM1 as well as the catalytic capability of the TresMan5A core module itself seemed to allow fast and more profound degradation of portions of the mannan that led to better cellulose degradation. In contrast, the presence of the CBM35 did not change the performance of PansMan26 in softwood saccharification.ConclusionsThis study identified TresMan5A as the best endomannanase for increasing cellulase catalysed glucose release from softwood. Except for the superior performance of TresMan5A, the fungal GH5 and GH26 endomannanases generally performed on par on the lignocellulosic matrix. The work also illustrated the importance of using genuine lignocellulosic substrates rather than simple model substrates when selecting enzymes for industrial biomass applications.

Dataset Information

Optimizing the composition of a synthetic cellulosome complex for the hydrolysis of softwood pulp: identification of the enzymatic core functions and biochemical complex characterization.

Publications

Optimizing the composition of a synthetic cellulosome complex for the hydrolysis of softwood pulp: identification of the enzymatic core functions and biochemical complex characterization.

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