UBC Theses and Dissertations
Characterisation of the cellulose binding domain of endo-β-1,4-glucanase A from Cellulomonas fimi Din, Neena
Endoglucanase A (CenA) from the bacterium Cellulomonasfimi comprises a catalytic domain and a non-hydrolytic cellulose binding domain (CBD) which can function independently. The DNA fragment encoding CBD[sub CenA] was cloned and expressed in E. coli and the polypeptide produced was characterised in an attempt to examine the mechanisms by which CBDs adsorb to their substrate and to determine the role of CBDs in the process of cellulose hydrolysis. CBD[sub CenA] and related CBDs from other bacterial glycanases have a characteristic motif of four highly conserved tryptophan residues. Each of two of these residues (W14 and W68) was mutated in CBD[sub CenA] to give the mutant polypeptides CBD [sub CenA]W14A and CBD[sub CenA]W68A. The binding affinities of CBD[sub CenA]W14A and CBD[sub CenA]W68A for crystalline cellulose were reduced 50 and 30 fold respectively compared with CBD[sub CenA]. Data obtained from CD, ¹H NMR and tryptophan fluorescence spectroscopy indicated that the mutant polypeptides have a similar conformation to CBDp. Tryptophan fluorescence data also suggested that W14 and W68 are exposed on the surface of CBD[sub CenA] and hence positioned to interact with residues on the cellulose surface upon adsorption of the polypeptide. Tryptophan fluorescence spectroscopy was used to examine the interaction of CBD CenA with the soluble glucans cellohexaose, carboxymethylcellulose and hydroxyethylcellulose and the insoluble substrate, bacterial microcrystalline cellulose (BMCC). No interaction of CBD[sub CenA] with the soluble glucans was detected, however fluorescence quenching of CBD[sub CenA] was observed upon addition of BMCC. The isolated binding domain of CenA released small particles from cotton and disrupted the structure of cellulose fibres. This is the first demonstration of the dispersive effects of a CBD and implies art active role for some CBDs in cellulose hydrolysis. The related CBD[sub Cex] showed similar properties to CBD[sub CenA] in disrupting the structure of cellulose fibres; however the mutant polypeptides CBD[sub CenA]W14A and CBD[sub CenA]W68A did not release small particles from cotton. A co-operative interaction between the isolated binding and catalytic domains of CenA in the release of soluble and insoluble sugar from cotton fibres was observed. The two domains interacted synergistically in the hydrolysis of cotton fibres. This intramolecular synergism is distinct from the previously described intermolecular synergism observed between individual cellulases.