Enzymatic degradation tests are carried out on polymeric films obtained by hot pressing the polymer powder between two Teflon plates, containing a spacer, for a minute under an appropriate pressure at 25-30°C above the melting temperature. Filmed samples are incubated at 37°C in vials containing 1.5 ml of 0.1 M potassium phosphate buffer (pH=7.4), in the presence of enzyme (Lipase or Proteinase). The films are removed from the enzymatic solution after 20 h, washed with distilled water several times, and dried under vacuum in the presence of P2O5 at room temperature, to constant weight.
This procedure is repeated dipping the partly degraded film into a fresh enzymatic solution for several 20 h incubation intervals. The degree of biodegradation is evaluated as weight loss normalised by initial sample surface area.
High molar mass PBSu, PBAd, PBSe and 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 assayed to enzymatic hydrolysis by Lipase from Mucor miehei or from Rhizopus arrhizus. The relative normalised weight loss rates were compared. The results point out that the copolyesters have reduced crystallinity with respect to the homopolyesters, and that the rate of enzymatic degradation is increased when the sample crystallinity decreases. Furthermore, it was observed that structural changes in the repeat unit of the copolyesters influence the rate of enzymatic degradation, independent of sample crystallinity. Instead, molar mass changes in the polymer investigated do not affect the rate of enzymatic degradation.
The enzymatic hydrolysis of homo and co-polyesters 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.
Related papers:
1. Giorgio Montaudo, Paola Rizzarelli
Synthesis and enzymatic degradation of aliphatic copolyesters
Polymer Degradation and Stability 70 (2000) 305-314
2. Paola Rizzarelli, Concetto Puglisi, Giorgio Montaudo
Soil burial and enzymatic degradation in solution of aliphatic co-polyesters
Polymer Degradation and Stability 85 (2004) 855-863
3. Paola Rizzarelli, Giuseppe Impallomeni, Giorgio Montaudo
Evidence for Selective Hydrolysis of Aliphatic Copolyesters Induced by Lipase Catalysis
Biomacromolecules 5 (2004) 433-444
Related posters:
1. Synthesis, characterisation and enzymatic degradation of aliphatic copolyesters
2. Role of structure and crystallinity of synthetic aliphatic copolyesters on their enzymatic degradation
3. HPLC/ESI-MS studies on enzymatic hydrolysis of synthetic aliphatic polyesters
The biodegradability is also tested by soil burial degradation. Tests are carried out at 30°C, under moisture controlled conditions in a series of darkened vessels
containing a multi-layer substrate (Scheme n.2: Soil Burial Test).
Polymer films are sandwiched between two layers of a mixture of milled perlite and soil, moistened with distilled water. The bottom and top layers are filled with perlite moistened with distilled water. Perlite is added to increase aeration of the soil and the amount of water retained. A flow of moistened air is supplied from the bottom of each vessel. Polymeric films are removed after intervals of 5, 10, 15, 30 and 45 days, brushed softly, washed with distilled water several times and dried under vacuum in the presence of P2O5 at room temperature, to constant weight. The degree of biodegradation is evaluated as the weight loss divided by the initial sample weight or surface area.
The biodegradability of high molar mass aliphatic polyesters and poly(ester amide)s was investigated under controlled soil burial conditions. The relative normalised weight loss rates were compared. The influence of crystallinity, molar mass, chemical structure and melting temperature upon biodegradation was studied. The weight loss of commercial poly(3-hydroxy butyrate), P(HB), of poly(3-hydroxy butyrate-co-3-hydroxy valerate) 76/24, P(HB-co-HV) 76/24, and of two commercial Bionolle samples, was also examined.
Related papers:
1. Paola Rizzarelli, Concetto Puglisi, Giorgio Montaudo
Soil burial and enzymatic degradation in solution of aliphatic co-polyesters
Polymer Degradation and Stability 85 (2004) 855-863
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1. Biodegradation of aliphatic polyesters by soil burial tests and enzymatic degradation in solution
Aliphatic polyesters have acquired significant interest as environmentally friendly thermoplastics for a wide range of applications and understanding their degradation behaviour has relevance both for processing and end uses. We have investigated the thermal, photo and thermo-oxidative degradation processes occurring in synthetic and commercial PBSu samples. The new compounds were analyzed by SEC, NMR spectroscopy, and MALDI-TOF MS.
Degradation produced a sensible reduction of the molar mass of the polyesters, promoting the formation of PBSu oligomers with different end groups. Applications of MALDI-TOF MS to the study of polymer degradation are quite recent, and involve the collection of MALDI spectra at different temperatures or light exposure to observe the structural changes induced by heat or light under an oxidizing atmosphere or nitrogen flow. The polymer sample can be directly analyzed and the recorded MALDI spectrum arises from a mixture of original and degraded chains. The identification of the structures and end groups attached to the oligomers produced is of remarkable importance, since the end groups may reveal the particular mechanism that has been active in the degradation processes.
According to the structure of the oxidation products identified, three photo-oxidation processes have been unambiguously ascertained to occur in PBSu. Beside the hydrogen abstraction and subsequent hydroperoxide formation, a process active in several other polymeric materials, two other cleavage processes appear to be operating in PBSu, i.e., the chain cleavage reaction Norrish I and the hydroxyl end groups oxidation (Scheme n.3: Overall Photo-oxidation Processes in PBSu).
Remarkably, these three processes have been revealed to be time-resolved, each showing a different induction period before appearance.
Related papers:
1. Sabrina Carroccio, Paola Rizzarelli, Concetto Puglisi, Giorgio Montaudo
MALDI Investigation of Photooxidation in Aliphatic Polyesters: Poly(butylene succinate)
Macromolecules 2004, 37, 6576-6586
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1. MALDI-TOF investigation of photo-degradation mechanisms in aliphatic polyesters
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