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The use of fluorescent protein-tagged carbohydrate-binding modules to evaluate the influence of drying on cellulose accessibility and enzymatic hydrolysis.

ABSTRACT: The influence of drying on cellulose accessibility and enzymatic hydrolysis was assessed. Dissolving pulp was differentially dried by freeze-, air- and oven-drying at 50 °C and subsequently hydrolyzed using the commercial CTec 3 cellulase preparation. It was apparent that drying reduced the ease of enzymatic hydrolysis of all of the substrates with a pronounced reduction (48%) exhibited by the oven-dried pulp. To assess if the ease of hydrolysis was due to enzyme accessibility to the substrate, microscopy (SEM), FTIR spectroscopy, water retention value (WRV), fiber aspect ratio analysis, Simons' stain and the selective binding of Fluorescent Protein-tagged Carbohydrate Binding Modules (FP-CBMs): CBM3a (crystalline cellulose) and CBM17 (amorphous cellulose) in combination with confocal laser scanning microscopy (CLSM) were used. The combined methods indicated that, if the gross characteristics of the substrate limited enzyme accessibility, the cellulases, as represented by the FP-CBMs, could not in turn access the finer structural components of the cellulosic substrates.

SUBMITTER: Mboowa D

PROVIDER: S-EPMC9055586 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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The use of fluorescent protein-tagged carbohydrate-binding modules to evaluate the influence of drying on cellulose accessibility and enzymatic hydrolysis.

Mboowa Drake D Khatri Vinay V Saddler Jack N JN

RSC advances 20200721 45

The influence of drying on cellulose accessibility and enzymatic hydrolysis was assessed. Dissolving pulp was differentially dried by freeze-, air- and oven-drying at 50 °C and subsequently hydrolyzed using the commercial CTec 3 cellulase preparation. It was apparent that drying reduced the ease of enzymatic hydrolysis of all of the substrates with a pronounced reduction (48%) exhibited by the oven-dried pulp. To assess if the ease of hydrolysis was due to enzyme accessibility to the substrate, ...[more]

PMID: 35515757

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Project description:Background:Lignocellulosic biomass will progressively become the main source of carbon for a number of products as the Earth's oil reservoirs disappear. Technology for conversion of wood fiber into bioproducts (wood biorefining) continues to flourish, and access to reliable methods for monitoring modification of such fibers is becoming an important issue. Recently, we developed a simple, rapid approach for detecting four different types of polymer on the surface of wood fibers. Named fluorescent-tagged carbohydrate-binding module (FTCM), this method is based on the fluorescence signal from carbohydrate-binding modules-based probes designed to recognize specific polymers such as crystalline cellulose, amorphous cellulose, xylan, and mannan. Results:Here we used FTCM to characterize pulps made from softwood and hardwood that were prepared using Kraft or chemical-thermo-mechanical pulping. Comparison of chemical analysis (NREL protocol) and FTCM revealed that FTCM results were consistent with chemical analysis of the hemicellulose composition of both hardwood and softwood samples. Kraft pulping increased the difference between softwood and hardwood surface mannans, and increased xylan exposure. This suggests that Kraft pulping leads to exposure of xylan after removal of both lignin and mannan. Impact of enzyme cocktails from Trichoderma reesei (Celluclast 1.5L) and from Aspergillus sp. (Carezyme 1000L) was investigated by analysis of hydrolyzed sugars and by FTCM. Both enzymes preparations released cellobiose and glucose from pulps, with the cocktail from Trichoderma being the most efficient. Enzymatic treatments were not as effective at converting chemical-thermomechanical pulps to simple sugars, regardless of wood type. FTCM revealed that amorphous cellulose was the primary target of either enzyme preparation, which resulted in a higher proportion of crystalline cellulose on the surface after enzymatic treatment. FTCM confirmed that enzymes from Aspergillus had little impact on exposed hemicelluloses, but that enzymes from the more aggressive Trichoderma cocktail reduced hemicelluloses at the surface. Conclusions:Overall, this study indicates that treatment with enzymes from Trichoderma is appropriate for generating crystalline cellulose at fiber surface. Applications such as nanocellulose or composites requiring chemical resistance would benefit from this enzymatic treatment. The milder enzyme mixture from Aspergillus allowed for removal of amorphous cellulose while preserving hemicelluloses at fiber surface, which makes this treatment appropriate for new paper products where surface chemical responsiveness is required.

Project description:BackgroundDuring cellulosic ethanol production, cellulose hydrolysis is achieved by synergistic action of cellulase enzyme complex consisting of multiple enzymes with different mode of actions. Enzymatic hydrolysis of cellulose is one of the bottlenecks in the commercialization of the process due to low hydrolysis rates and high cost of enzymes. A robust hydrolysis model that can predict hydrolysis profile under various scenarios can act as an important forecasting tool to improve the hydrolysis process. However, multiple factors affecting hydrolysis: cellulose structure and complex enzyme-substrate interactions during hydrolysis make it diffucult to develop mathematical kinetic models that can simulate hydrolysis in presence of multiple enzymes with high fidelity. In this study, a comprehensive hydrolysis model based on stochastic molecular modeling approch in which each hydrolysis event is translated into a discrete event is presented. The model captures the structural features of cellulose, enzyme properties (mode of actions, synergism, inhibition), and most importantly dynamic morphological changes in the substrate that directly affect the enzyme-substrate interactions during hydrolysis.ResultsCellulose was modeled as a group of microfibrils consisting of elementary fibrils bundles, where each elementary fibril was represented as a three dimensional matrix of glucose molecules. Hydrolysis of cellulose was simulated based on Monte Carlo simulation technique. Cellulose hydrolysis results predicted by model simulations agree well with the experimental data from literature. Coefficients of determination for model predictions and experimental values were in the range of 0.75 to 0.96 for Avicel hydrolysis by CBH I action. Model was able to simulate the synergistic action of multiple enzymes during hydrolysis. The model simulations captured the important experimental observations: effect of structural properties, enzyme inhibition and enzyme loadings on the hydrolysis and degree of synergism among enzymes.ConclusionsThe model was effective in capturing the dynamic behavior of cellulose hydrolysis during action of individual as well as multiple cellulases. Simulations were in qualitative and quantitative agreement with experimental data. Several experimentally observed phenomena were simulated without the need for any additional assumptions or parameter changes and confirmed the validity of using the stochastic molecular modeling approach to quantitatively and qualitatively describe the cellulose hydrolysis.

Project description:Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

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The use of fluorescent protein-tagged carbohydrate-binding modules to evaluate the influence of drying on cellulose accessibility and enzymatic hydrolysis.

Publications

The use of fluorescent protein-tagged carbohydrate-binding modules to evaluate the influence of drying on cellulose accessibility and enzymatic hydrolysis.

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