Development of enzyme-embedded biopolymer blends with improved self-biodegradation

The global production of biodegradable plastics is rapidly increasing; however, in agricultural mulch film applications, degradation in the field can be highly dependent on environmental factors. Enzyme inclusion into biopolymer blends represents a promising strategy to promote biodegradation. The cutinase-like enzyme from Cryptococcus sp. S-2 (CLE) is a promising biocatalyst for this purpose, owing to its ability to hydrolyse aliphatic polyesters under mild conditions. Therefore, this thesis aimed to investigate CLE-based formulations and their inclusion into biodegradable polymer systems, to preserve enzymatic activity during melt extrusion of biopolymer blends. CLE was produced by an engineered Saccharomyces cerevisiae Ethanol Red® strain, and the cell-free supernatant was tested in solid and liquid formulations. Enzyme thermostability under polymer-processing-relevant conditions was investigated measuring hydrolytic activity using a spectrophotometric turbidity assay on emulsified PLA and PCL substrates. Based on previous literature, a masterbatch approach was developed to incorporate the enzyme into biodegradable polymer blends by melt extrusion. The degradation behaviour of the resulting enzyme-embedded films was evaluated through hydrolysis tests, monitoring monomer release and weight loss. Powder formulations showed higher thermal stability than liquid formulations and were selected for inclusion in the final blend. The masterbatch approach enabled the production of enzyme-containing blends with good mechanical properties and self-hydrolysis in controlled laboratory conditions. Overall, this work demonstrated the feasibility of incorporating CLE into biodegradable polymer systems, providing a proof of concept for the development of enzyme-embedded bioplastics with improved self-biodegradation.