Isolation and Spectral Characterization of Plant-Cuticle Polyesters

An efficient protocol combining enzymatic and chemical methods has been developed for the isolation of lime fruit cutin and potato suberin in pure form and with excellent yields. Starting with the procedures specified by commercial assay kits for model substrates, the breakdown of cellulose, pectin, and hemicellulose cell-wall constituents was optimized with respect to enzyme concentration, substrate concentration, reaction time, and temperature. The optimized procedures were then applied to the plant polyesters cutin and suberin, with the isolation process monitored in parallel by gravimetric methods and with cross-polarization magic-angle spinning ¹³C NMR. For lime cutin, the optimized protocol avoids spectral interferences from cell-wall carbohydrates, waxes, and exogenous oxalate, confirming that esters of both primary and secondary alcohols are present in the biopolymer. For suberized potato periderm, the optimized protocol removes more than 95% of the unsuberized cell walls and waxes, making it possible to generate an NMR difference spectrum of suberin that includes resonances from all major functional groups of this aromatic-aliphatic polyester.