Thermodynamics of family 1 cellulose-binding modules from T. reesei Cel7A and Cel6A

Cellulose binding modules (CBMs) are discrete protein modules existed in most cellulolytic enzymes which function in improving the binding and facilitating the activity of the catalytic domain on the insoluble cellulose substrates. In this work fungus CBMs from Trichoderma reesei cellobiohydrolase Cel7A and Cel6A were heterologously expressed and purified from Escherichia coli, the binding properties between these CBMs and cellulose substrate were studied as well. Since the Cel7A and Cel6A are preferably binding to the crystalline region of cellulose other than the amorphous region, cellulose nanowhiskers (CNWs), which are the crystalline portion of cellulose fibers extracted through a sulfuric acid / hydrochloride acid hydrolysis process from cotton, as well as Avicel (microcrystalline cellulose), are used as representative model cellulose to better understand the binding interaction between the CBMs and the crystalline cellulose. Isothermal titration calorimetry (ITC) was used to get a thermodynamic profile of the binding between each CBM and CNWs by titrating CBM into the suspension of CNWs, from which the binding constant, stoichiometry, enthalpy change and entropy change were obtained. The ITC results showed the binding constant of CBM_Cel7A to CNWs is - 10⁵ M^-1, while - 10⁶ M^-1 for CBM_Cel6A, suggesting a higher binding affinity of CBM_Cel6A to CNWs. Additionally, the binding reactions were driven by a favorable enthalpy change, offset a little bit by an unfavorable entropy change.