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dc.contributor.authorLiu, Ziyuan-
dc.descriptionPhD Thesisen_US
dc.description.abstractGrass stains containing chlorophyll are the most difficult stains to remove from cotton fabrics in laundry processes. The grass cell walls are highly complex networks of polysaccharides interlocking with each other, which makes it recalcitrant to enzyme degradation. In nature, glycoside hydrolases that target plant cell walls often contain noncatalytic carbohydrate binding modules (CBMs), which promote enzymes access to insoluble substrates and thus potentiate catalysis. This project aims to use xylan-specific CBMs to target enzymes in washing powder onto grass stains to improve the removal of these stains. The binding capacity of CBMs to grass stains arrayed on nitrocellulose was initially assessed using immunological methodologies that focused on the detection of the His6 tag of the CBMs. The data showed that CBM15 and CBM2b1,2 bound to grass weakly, in the presence of Marvel. When the surfactant Sodium Lauryl Ethoxy Sulphate (SLES) was added, both CBMs showed strong binding to grass stains arrayed on the membranes in the presence of Marvel. Different methods to detect the two CBMs binding to grass stains displayed on cotton were explored in the absence of Marvel. The most appropriate method was direct labelling of CBM2b and CBM15 using fluorescent dye (Alexa 488) followed by confocal and fluorescent microscopy. The confocal data showed that both CBMs bound to grass stains and cotton, in the absence of SLES. The confocal data also showed that SLES strongly increased CBM2b1,2 targeting to grass stains. To quantify the CBM adsorption to grasses and cotton, standard fluorescence microscopy was used to provide a quick and efficient image formation system. Statistical analysis showed that neither CBMs significantly bound to grass stains, in the absence of SLES. There was significant increased targeting of both CBMs to grass stains in the presence of SLES. They both showed significant binding to non-stained cotton, either with or without SLES. CBM2b1,2 and CBM15 were fused to the xylanases C. japonicus Xyn10A and N. patriciarum Xyn11A respectively; the CBM-enzymes were purified and assessed in detergent assays. Statistical analysis showed that CBM2b1,2-Xyn10A efficiently released grass stains the most and showed a significant difference to the control with no enzymes, in surfactant formulation containing a high percentage of SLES (high SLES blend). Xyn10A, Xyn11A, CBM15- Xyn10A, CBM15-Xyn11A and CBM2b1,2-Xyn11A were not significantly different to the control, but they showed higher averages of the amount of grass dye released than the control, in high SLES blend. This thesis also reports the biochemical properties and crystal structure of vCBM60, a homolog of CBM60 in C. japonicus, which uniquely displays broad ligand specificity, targeting xylans, galactans and cellulose. The crystal structure of vCBM60 displays a β- sandwich with the ligand binding site comprising a broad cleft formed by the loops connecting the two β-sheets. Ligand recognition at site 1 is, exclusively, through hydrophobic interactions, while binding at site 2 is conferred by polar interactions between a protein-bound calcium and the O2 and O3 of the sugar. The observation that ligand recognition at site 2 requires only a β-linked sugar that contains equatorial hydroxyls at C2 and C3, explains the broad ligand specificity displayed by vCBM60. Another CBM from family 35 in C. thermocellum has been studied in this project. Site directed mutagenesis of ten residues around its binding site were carried out. The binding of wild type and mutants of CtCBM35 to galactomannan was determined by Isothermal Titration Calorimetry (ITC). The data showed that all ten mutants abolished binding to galactomannan. Within a wider context for the CBM35 family, where the ligand binding sites of galactose-specific CtCBM35 and uronate-specific CBM35s are similar structures, but display divergent ligand specificity, the signature residues in their ligand binding sites are compared and discussed in this study.en_US
dc.publisherNewcastle Universityen_US
dc.titleDeveloping novel enzymes for the laundry industryen_US
Appears in Collections:Institute for Cell and Molecular Biosciences

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