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

Area of Science:

• Biochemistry
• Materials Science
• Biotechnology

Background:

• Cellulose binding modules (CBMs) are crucial for understanding cellulose degradation and utilization.
• Different CBMs exhibit varying affinities for cellulose based on its structural properties.
• Family 1 CBMs, specifically CBMCel7A and CBMCel6A, are key components in cellulase systems.

Purpose of the Study:

• To investigate the binding properties of Family 1 CBMs (CBMCel7A and CBMCel6A) to different cellulose substrates.
• To elucidate the influence of cellulose crystallinity on CBM binding interactions.
• To determine the thermodynamic parameters governing CBM-cellulose binding.

Main Methods:

• Heterologous expression and purification of CBMCel7A and CBMCel6A from Escherichia coli.
• Utilized cellulose nanowhiskers (CNWs, crystalline), Avicel PH101 (partially crystalline), and PASC (amorphous) as model substrates.
• Employed isothermal titration calorimetry (ITC) and adsorption-isotherm experiments.
• Determined accessible surface area using N2 adsorption and static light scattering (SLS).
• Developed a novel method for determining cellulose molarity based on available surface area.

Main Results:

• CBMCel6A demonstrated a higher binding constant (Ka ≈ 10^6 M^-1) to crystalline CNWs compared to CBMCel7A (Ka ≈ 10^5 M^-1).
• Both CBMs exhibited lower binding affinities to partially crystalline Avicel and weak binding to amorphous PASC, indicating a correlation with cellulose crystallinity.
• Binding reactions were primarily driven by favorable enthalpy changes, counteracted by unfavorable entropy changes.
• CBMCel6A showed preferential binding to the reducing end of cellulose chains, a specificity not observed for CBMCel7A.
• Cello-oligosaccharides with fewer than two glucose units did not bind, while longer oligosaccharides showed improved binding.

Conclusions:

• The binding affinity of CBMCel7A and CBMCel6A to cellulose is significantly influenced by the substrate's crystallinity.
• CBMCel6A possesses a higher affinity for crystalline cellulose and exhibits end-binding specificity.
• Understanding these binding characteristics is vital for optimizing enzymatic cellulose hydrolysis and biofuel production.