Dataset Information

Elucidation of Changes in Cellulose Ultrastructure and Accessibility in Hardwood Fractionation Processes with Carbohydrate Binding Modules.

ABSTRACT: We have recently presented a sequential treatment method, in which steam explosion (STEX) was followed by hydrotropic extraction (HEX), to selectively fractionate cellulose, hemicellulose, and lignin in hardwood into separate process streams. However, above a treatment severity threshold, the structural alterations in the cellulose-enriched fraction appeared to restrict the enzymatic hydrolyzability and delignification efficiency. To better understand the ultrastructural changes in the cellulose, hardwood chips were treated by single (STEX or HEX) and combined treatments (STEX and HEX), and the cellulose accessibility quantified with carbohydrate-binding modules (CBMs) that bind preferentially to crystalline (CBM2a) and paracrystalline cellulose (CBM17). Fluorescent-tagged versions of the CBMs were used to map the spatial distribution of cellulose substructures with confocal laser scanning microscopy. With increasing severities, STEX increased the apparent crystallinity (CBM2a/CBM17-ratio) and overall accessibility (CBM2aH6 + CBM17) of the cellulose, whereas HEX demonstrated the opposite trend. The respective effects could also be discerned in the combined treatments where increasing severities further resulted in higher hemicellulose dissolution and, although initially beneficial, in stagnating accessibility and hydrolyzability. This study suggests that balancing the severities in the two treatments is required to maximize the fractionation and simultaneously achieve a reactive and accessible cellulose that is readily hydrolyzable.

SUBMITTER: Novy V

PROVIDER: S-EPMC7202243 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Elucidation of Changes in Cellulose Ultrastructure and Accessibility in Hardwood Fractionation Processes with Carbohydrate Binding Modules.

Novy Vera V Nielsen Fredrik F Olsson Johanna J Aïssa Kevin K Saddler Jack N JN Wallberg Ola O Galbe Mats M

ACS sustainable chemistry & engineering 20200331 17

We have recently presented a sequential treatment method, in which steam explosion (STEX) was followed by hydrotropic extraction (HEX), to selectively fractionate cellulose, hemicellulose, and lignin in hardwood into separate process streams. However, above a treatment severity threshold, the structural alterations in the cellulose-enriched fraction appeared to restrict the enzymatic hydrolyzability and delignification efficiency. To better understand the ultrastructural changes in the cellulose ...[more]

PMID: 32391215

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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.

Project description:Background:Effective enzymatic degradation of crystalline polysaccharides requires a synergistic cocktail of hydrolytic enzymes tailored to the wide-ranging degree of substrate crystallinity. To accomplish this type of targeted carbohydrate recognition, nature produces multi-modular enzymes, having at least one catalytic domain appended to one or more carbohydrate binding modules (CBMs). The Type B CBM categorization encompasses several families (i.e., protein folds) of CBMs that are generally thought to selectively bind oligomeric polysaccharides; however, a subset of cellulose-specific CBM families (17 and 28) appear to bind non-crystalline cellulose more tightly than oligomers and in a manner that discriminates between surface topology. Results:To provide insight into this unexplained phenomenon, we investigated the molecular-level origins of oligomeric and non-crystalline carbohydrate recognition in cellulose-specific Type B CBMs using molecular dynamics (MD) simulation and free energy calculations. Examining two CBMs from three different families (4, 17, and 28), we describe how protein-ligand dynamics contribute to observed variations in binding affinity of oligomers within the same CBM family. Comparisons across the three CBM families identified factors leading to modified functionality prohibiting competitive binding, despite similarity in sequence and specificity. Using free energy perturbation with Hamiltonian replica exchange MD, we also examined the hypothesis that the open topology of the binding grooves in families 17 and 28 necessitates tight binding of an oligomer, while the more confined family 4 binding groove does not require the same degree of tight binding. Finally, we elucidated the mechanisms of non-crystalline carbohydrate recognition by modeling CBMs complexed with a partially decrystallized cellulose substrate. Molecular simulation provided structural and dynamic data for direct comparison to oligomeric modes of carbohydrate recognition, and umbrella sampling MD was used to determine ligand binding free energy. Comparing both protein-carbohydrate interactions and ligand binding free energies, which were in good agreement with experimental values, we confirmed the hypothesis that family 17 and 28 CBMs bind non-crystalline cellulose and oligomers with different affinities (i.e., high and low). Conclusions:Our study provides an unprecedented level of insight into the complex solid and soluble carbohydrate substrate recognition mechanisms of Type B CBMs, the findings of which hold considerable promise for enhancing lignocellulosic biomass conversion technology and development of plant cell wall probes.

Dataset Information

Elucidation of Changes in Cellulose Ultrastructure and Accessibility in Hardwood Fractionation Processes with Carbohydrate Binding Modules.

Publications

Elucidation of Changes in Cellulose Ultrastructure and Accessibility in Hardwood Fractionation Processes with Carbohydrate Binding Modules.

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