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

Abstract 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 -CD lipid 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, TΔS 0 =17.72 kJ mol −1 ; for 130 literature [ α - and β -CD] values: α =0.92, TΔS 0 =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 CD guest 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. 13 C NMR and ITC experiments indicated that HP- β -CD lipid 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 -CD 16-OH-P complex was formed nor background ΔH * observed. At pH 9 no substantial binding was evident when synthetic cutin ( ΔQ=−240±61 μ J , ΔQ control =−231±31 μ J ) 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 -CD synthetic cutin system tends to argue for the latter interpretation.