Binding specificity and thermodynamics of cellulose-binding modules from trichoderma reesei Cel7A and Cel6A

In this work, Family 1 cellulose binding modules CBM_Cel7A and CBM_Cel6A were heterologously expressed and purified from Escherichia coli, and the binding properties between these CBMs and cellulose substrates were studied. Cellulose nanowhiskers (CNWs, the crystalline portion of cellulose), microcrystalline cellulose Avicel PH101 (partially crystalline cellulose), and phosphoric acid swollen cellulose (PASC, amorphous cellulose) were used as representative models for cellulose to better understand the binding interactions between the CBMs and different regions of native cellulose. Isothermal titration calorimetry (ITC) was combined with adsorption-isotherm experiment to analyze the thermodynamics of CBM binding to various cellulose substrates. N₂ adsorption and static light scattering (SLS) data were used to estimate the accessible surface area of cellulose which was then used for ITC data analysis. A new method of determining the cellulose molarity based on the available surface area for CBM binding was developed, which allows different cellulose substrates to be compared for binding experiments. The ITC results showed that the binding constant (K_a) to crystalline CNWs was ∼10⁵ M^-1 for CBM_Cel7A, while ∼10 ⁶ M^-1 for CBM_Cel6A, suggesting a higher binding affinity of CBM_Cel6A to CNWs. For Avicel, lower binding constants for both CBMs were observed, and weak bindings to PASC were characterized, suggesting that the binding between CBM_Cel7A,Cel6A and cellulose to some extent relates to the crystallinity of cellulose. Additionally, the binding reactions were driven by a favorable enthalpy change, offset partially by an unfavorable entropy change. It is suggested that CBM_Cel6A preferentially binds to the reducing end of cellulose chain, while CBM _Cel7A does not show such end binding specificities. Cello-oligosaccharides less than two glucose units did not bind with CBMs, and improved binding affinities were observed for cello-oligosaccharides with longer glucose units.