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|Title:||Structure and function of novel cellulosic, hemicellulosic and pectic glycoside hydrolases|
|Abstract:||Cellulose is a major component of the plant cell wall and is the most abundant organic molecule in the biosphere. Efficient degradation of this polysaccharide is required if the plant cell wall is to be used as a viable source of renewable biofuels. Bacteria express an arsenal of different cellulases that catalyse the degradation of cellulose. The reason why many different cellulases are expressed rather than one highly active cellulase is unclear, but probably lies in the structural diversity displayed by cellulose, which is much greater than its invariant chemical composition suggests. Part of this work describes a novel cellulase from the plant cell wall degrading bacterium Clostridium thermocellum. This cellulase, CtCel119, is the first of this class of enzyme to display the α8 helical fold, is the founding member of a new GH family, performs catalysis through a possible “Grothuss style mechanism” and shares features typical of lytic transglycoslases. Structural data also seem to suggest that the enzyme may attack a novel structure in crystalline cellulose, which could contribute to understanding why bacteria such as C. thermocellum employ a diverse variety of cellulases. A study on glycoside hydrolase family (GH) 26, which consists mainly of endo-β-1,4 mannanases, was also conducted. The work presented in this thesis focused on two novel members of the family. One component of this section provided a thorough kinetic analysis of mutants of active site residues of a GH26 endo-β-1,4-1,3-glucanase. This identified crucial interactions at the -2 subsite, which contribute to the stabilisation of the 4H3 transition state. The other component of this section was the identification and characterisation of an exo-acting mannanase, CjMan26C, also termed a mannobiohydrolase. CjMan26C is the only mannanase characterised to date to release mannobiose. The mannobiohydrolase displayed an extremely high catalytic efficiency of 3 x 109 min-1 M-1 against mannotetraose. The crystal structure revealed a -1 sugar in a 1S5 pre transition state, providing further support for a B2,5 transition state in GH26. The exo activity was conferred by a four amino acid insertion in loop 3 at the -2 subsite. Mutation of D130, to Gly or Ala, in loop 3 was enough to partially convert the enzyme to an endo-mode of action, while removal of D130 plus two flanking resides caused a full conversion to an endo-mode of action. The gene expansion observed in family GH43 enzymes was also investigated. Eleven genes encoding GH43 enzymes were cloned, expressed and investigated for catalytic activity. Three arabinofuranosidases were characterised, two exo α-1,5-L-arabinofuranosidases and a novel sugar beet arabinan specific α-1,2-L-arabinofuranosidase that could attack both single and double substitutions named CjAbf43B. The crystal structure of CjAbf43B was solved in complex with ligand. The structure revealed a curved binding cleft, around a deep active site pocket, that was specific for the curved nature of sugar beet arabinan backbone. The curved binding cleft also had a groove into which α-1,3-L-arabinofuranosides could be accommodated, explaining how the enzyme has plasticity for single and double substitutions. A β-1,4 xylosidase was also characterised, while four enzymes were identified that displayed „trace‟ activity against xylans. Three of these appeared to display endo-activity, while the fourth enzyme displayed very weak arabinfuranosidase activity against xylans.|
|Appears in Collections:||Institute for Cell and Molecular Biosciences|
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|Cartmell 11.pdf||Thesis||7.31 MB||Adobe PDF||View/Open|
|dspacelicence.pdf||Licence||43.82 kB||Adobe PDF||View/Open|
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