Aleksandra Marsavelski, University of Zagreb

Ancestral Sequence Reconstruction as a Strategy for Engineering Thermostable Plastic-Degrading Enzymes

Plastic pollution presents a growing environmental and health crisis, driving the need for sustainable degradation strategies for synthetic polymers like polylactic acid (PLA). This study, supported by the WiRE Women in Research (WiRe) fellowship, focuses on engineering a novel PLA-degrading enzyme, MGY, identified from a metagenomic database. Despite its promising activity, the native MGY enzyme exhibited low solubility, poor expression yield, and limited thermostability, restricting its practical application. To address these limitations, we employed ancestral sequence reconstruction to generate three ancestral MGY variants. All variants demonstrated significant PLA-degrading activity, with one variant also exhibiting activity against polycaprolactone (PCL). The ancestral variants showed substantial improvements in solubility, expression levels, and thermal stability, with differential scanning calorimetry (DSC) revealing a remarkable melting temperature (Tm) of 84 °C—the highest reported for any PLA-degrading enzyme to date. Our findings provide critical insights into the structural determinants of enzyme stability and polymer degradation, underscoring the potential of ancestral sequence reconstruction as a powerful strategy for engineering robust biocatalysts. This work paves the way for developing efficient enzyme-based systems for plastic recycling and environmental remediation, contributing to industrial biotechnology and sustainability.