Cyclomaltooligosaccharide binding and solubilization of hydroxyfatty acid matrices in aqueous solution: calorimetric titration and 13C NMR investigations of molecular recognition

Cyclomaltooligosaccharides (cyclodextrins, CDs) increase cutinase activity with both naturally occurring and synthetic cuticular substrates. Little is known about the interactions of CDs with cutin or cutin-like substrates such as 16-hydroxypalmitate (16-OH-P). We report herein investigations into the thermochemistry of β-CD, hydroxypropyl-β-CD (HP-β-CD) or α-CD interactions with palmitic acid (P), 16-OH-P and polyesters (synthetic cutin) derived therefrom under conditions coincident with maximal cutinase activity (pH 9, glycine/NaOH buffer) at 25 °C using isothermal titration calorimetry (ITC). The thermodynamic parameters for HP-β-CDradical dotlipid inclusion complex formation and subsequent solubilization, which were studied in heterogeneous phase suspensions, displayed enthalpy–entropy compensation typical of processes driven by solvation phenomena (α=T∂ΔS/ΔH=1.03, TΔS0=17.72 kJ mol−1; for 130 literature [α- and β-CD] values: α=0.92, TΔS0=15.11 kJ mol−1). In the 16-OH-P (Na+) experiments ΔH and ΔS (ΔH=42±8 kJ mol−1, ΔS=206±24 J mol−1 K−1) values were large relative to those reported elsewhere for diverse CDradical dotguest complexes (ΔH=−50 to 0 kJ mol−1, ΔS=−170 to 30 J mol−1 K−1) since ΔH resulted from the combined processes of binding and solubilization. 13C NMR and ITC experiments indicated that HP-β-CDradical dotlipid complexes had a 1:1 stoichiometry. A constant background lipid concentration-dependent endothermic process (ΔH*) also observed using both P and 16-OH-P substrates (ΔH* ∼4.8±0.5 kJ mol−1) as HP-β-CD was titrated into the heterogeneous lipid slurry. At a lower pH (6, 100 mM Na+ phosphate buffer) neither a soluble HP-β-CDradical dot16-OH-P complex was formed nor background ΔH* observed. At pH 9 no substantial binding was evident when synthetic cutin (ΔQ=−240±61 μJ, ΔQcontrol=−231±31 μJ) was used as a substrate; a similar result was obtained using β-CD. Titrations using α-CD did, however, display a weak interaction (K=119±53 M−1, ΔH=1.1±0.9 kJ mol−1, ΔS= 43.4±3.7 J mol−1 K−1) with the synthetic cuticular matrix. Thus, either CDs do not bind to the insoluble cutin matrix or they do but with a small ΔH. The fact that HP-β-CD binds the synthetic cutin monomer and weak binding was observed in the α-CDradical dotsynthetic cutin system tends to argue for the latter interpretation.