Cellulose nanostructure and binding characteristics

The objective of this work was to characterize the surface area and porosity of cellulose nanowhiskers (CNWs) from different origins (plant cotton/bacterium Gluconacetobacter xylinus) and different acid treatments (H 2SO4/HCl) by N2 adsorption; as well as to compare surface area and porosity of bacterial cellulose synthesized by static and agitated cultures.;Phage display technology was used to screen peptides that selectively bind to crystalline cellulose nanowhiskers (CNWs) from a library of a number of different polypeptides. These peptides were selected on immobilized CNWs by biopanning, by which all possible heptapeptides were screened based on the binding affinity to the crystalline CNWs. A consensus peptide of WHWTYYW was identified to efficiently bind to CNWs. Binding affinities of isolated phage particles were quantitatively analyzed by single-phage biopanning assay and enzyme-linked immunesorbent assay (ELISA). This consensus peptide was synthesized and exhibited a binding affinity of ∼105 M -1 towards CNWs as measured by isothermal titration calorimetry (ITC), in good agreement with ELISA results. In addition, the affinities of this peptide to cello-oligosaccharides were also measured with UV spectroscopy and fluorescence quenching experiments. The molecular details of the interaction of this heptapeptide with cellohexaose were determined by nuclear magnetic resonance (NMR). The interaction with crystalline cellulose was investigated by molecular modeling (MM) studies. Both NMR and MM experiments suggest that this heptapeptide has adopted a bended structure for binding and Y5 can form CH/pi stacking and hydrogen bond with glucose ring of cellulose.;Family I cellulose binding modules CBMCel7A and CBMCel6A were heterologously expressed and purified from Escherichia coli, and the binding properties between these CBMs and cellulose substrates were studied. Cellulose nanowhiskers (CNWs), the crystalline portions of cellulose, microcrystalline cellulose Avicel PH101 (partially crystalline cellulose), and phosphoric acid swollen cellulose (PASC, amorphous cellulose), were used as representative models for cellulose to better understand the binding interactions between the CBMs and different regions of native cellulose. Isothermal titration calorimetry (ITC) was combined with adsorption-isotherm experiment to analyze the thermodynamics of CBMs binding to various cellulose substrates. N2 adsorption and static light scattering (SLS) data were used to estimate the accessible surface area of cellulose which was then used for ITC data analysis. A new method of determining the cellulose molarity based on the available surface area for CBM binding was developed, which allows different cellulose substrates to be compared for binding experiments. The ITC results showed that the binding constant (Ka) to crystalline CNWs are ∼105 M-1 for CBMCel7A , while ∼106 M-1 for CBM Cel6A, suggesting a higher binding affinity of CBMCel6A to CNWs.;Natural cellulose binding modules (CBMs) bind to cellulose via hydrogen bonding and CH-pi stacking interactions between aromatic amino acids (especially tyrosines) and cellulose glucose rings. To identify the CBM binding motif and explore the binding mechanism at the molecular level, tyrosine-containing peptides mimicking CBM key motifs while having simpler structures than natural CBMs were synthesized and the binding properties to cellulose were characterized. In particular, the involvement of tyrosines in cellulose binding was studied. Information obtained by Fourier transform infra-red spectroscopy (FTIR), isothermal titration calorimetry (ITC), and quantum mechanical calculations provides evidence that these CBM-mimic peptides had a capacity of binding cellulose. Modeling results reveal that the peptide with more than one tyrosine is favorable for binding towards cellulose and that the binding is enhanced with decreased distance between the two hydrophobic residues (tyrosine). Additionally, the random coiling nature of the peptide backbone provides enough room for intermolecular hydrogen bonds with the cellulose surface along with the weak hydrophobic interaction. (Abstract shortened by UMI.)...