Binding of Thermobifida fusca CD_Ce15A, CD_Ce16B and CD_Ce148A to easily hydrolysable and recalcitrant cellulose fractions on BMCC

The binding of the catalytic domain of Thermobifida fusca endoglucanase (EC 3.2.1.4) CD_Ce15A and exoglucanases (EC 3.2.1.91) CD_Ce16B and CD_Ce148A to BMCC was studied. At 5°C, the binding of these CDs to BMCC is rapid with maximum binding occurring just after CD loading. An observed desorption of the bound CDs with time was attributed to the loss of binding sites due to hydrolysis of the easily hydrolysable BMCC. This conclusion was supported by prehydrolysis experiments where the easily hydrolysable BMCC fraction was removed, and binding to the recalcitrant fraction was observed. More of the CDs were bound to the recalcitrant fraction. This was especially true for the exoglucanases, CD_Ce16B and CD_Ce148A, where 81-76% of the total CD binding was to the recalcitrant BMCC fraction. The binding to the easily hydrolysable fraction for the endo CD_Ce15A was 1.5-4 times higher than that of the exo CD_Ce16B and CD_Ce148A at the same enzyme concentration. Although CD_Ce15A saturated the easily hydrolysable fraction of BMCC first, the ratio of the bound to the recalcitrant substrate was constant for CD_Ce16B and CD_Ce148A as 81 and 76%, respectively. As the reaction temperature was increased to 50°C, CD_Ce15A and CD_Ce148A exhibited a rapid increase in the rate and extent of binding, while CD_Ce16B exhibited a decrease in the extent of binding.