Now, Isabelle André, Alain Marty and co-workers report a masterbatch-based melt extrusion process to create an ‘enzymated’ PLA film that contains embedded PLA depolymerase (PLAase) that undergoes complete degradation under home-composting conditions within roughly 20 weeks. Specifically, a new PLAase named PAM was isolated from Actinomadura keratinilytica T16-1, a thermophilic bacterium, and expressed in Escherichia coli before undergoing purification. The obtained PAM exhibited superior PLA depolymerization performance compared with proteinase K (a reference for PLA depolymerase activity). Furthermore, molecular dynamics simulations and site-saturation mutagenesis were applied to optimize the PAM enzyme further. The improved PAMFLI variant demonstrated pronounced improvements in specific activity compared with PAM under home-composting conditions (pH 7.5–9.0 and 28–45 °C).
In addition, to enable the masterbatch-based melt extrusion process at 70 °C, a more thermostable PLAase is essential. The researchers successfully transferred the catalytic properties of PAMFLI to protein T (named ‘ProteinT’), an extracellular serine proteinase, using a structure-based engineering approach due to their similar three-dimensional organization. The ProteinTFLTIER multivariant emerged as a highly thermostable PLAase with a melting temperature (Tm) of approximately 79 °C, surpassing that of PAMFLI by about 22 °C. Notably, ProteinTFLTIER displayed an 80-fold increase in specific activity compared with ProteinT and similar activity levels to PAMFLI.
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