HPLC/ESI-MS INVESTIGATION OF ENZYMATIC HYDROLYSIS IN SYNTHETIC ALIPHATIC COPOLYESTERS

High molar mass random poly(butylene succinate-co-butylene sebacate), P(BSu-co-BSe), and poly(butylene succinate-co-butylene adipate), P(BSu-co-BAd), with different composition, were synthesized and subjected to enzymatic hydrolysis by Lipase from Mucor miehei or from Rhizopus arrhizus. The enzymatic hydrolysis of P(BSu-co-BSe)s and P(BSu-co-BAd)s films produced a mixture of water-soluble monomers and co-oligomers that were separated and identified by on-line High Performance Liquid Chromatography/ElectroSpray Ionization Mass Spectrometry (HPLC/ESI-MS). Optimization of the HPLC analysis allowed the separation of isobar co-oligomers, differing only for the co-monomers sequence. Oligomers with the same monomer composition and molar mass but different sequence were identified by HPLC/ESI-MS-MS on-line analysis. The results obtained show a preferential hydrolytic cleavage induced by the lipases used. In particular, these enzymes prefer cleaving sebacic ester bonds in P(BSu-co-BSe) copolymers, whereas succinic ester bonds appear to be hydrolyzed faster than adipic ester bonds in P(BSu-co-BAd) copolyesters. 1H-NMR analysis further substantiates these findings. The primary products generated by lipase hydrolysis of polyester films underwent further degradation at longer reaction times. The HPLC/ESI-MS analysis of these mixtures at various times provided the first evidence that lipase catalysis is active also in water solution, a hydrophobic effect induced by the aliphatic units of these polyesters.