Protein Engineering of Enzymes for Plastic Biodegradation

Abstract

Plastics are increasingly prevalent in our daily lives, and the management of plastic waste has developed into a significant environmental concern. Conventional recycling methods fall short of achieving a genuinely circular plastic economy. Enzymatic depolymerisation represents an intriguing opportunity for addressing the limitations of chemical and mechanical plastic recycling. Employing enzymatic hydrolysis to depolymerize post-consumer plastic waste holds the potential to establish a true circular economy. Nevertheless, it is essential to note that natural enzymes, in their current state, are not yet suitable for industrial applications; significant enhancements are required to make them viable for large-scale purposes. The discovery of new plastic-degrading enzymes opens new possibilities for enzyme engineering. A breakthrough occurred in 2016 with the discovery of Ideonella sakaiensis, the first bacteria known to be capable of using PET as its primary carbon source and completely degrading it into its monomers. The initial catalytic step in this process involves IsPETase, an enzyme with great potential for industrial applications. The doctoral thesis aims to improve IsPETase limited thermal stability and enzymatic activity. To achieve this, a combinatory approach of engineering strategies was employed, including ancestral sequence reconstruction (ASR), structure-based rational design, and directed evolution (DE). ASR is a method to understand the evolutionary history of a particular enzyme and is increasingly used for protein engineering due to the beneficial properties of ancestral enzymes. Our study resulted in a better understanding of the path of evolution of IsPETase and resulted in ancestral enzymes with enhanced properties. Simultaneously, a structure-based approach using iterative rounds of engineering and rational design was performed, resulting in the most significant improvement of IsPETase. Finally, to further improve IsPETase this thesis aimed to employ directed evolution. First, this thesis reviewed the successful approaches in improving plastic-degrading enzymes via directed evolution and highlighted the limitations of the research such as other properties including the protein solubility that has not been explored yet. Second, to address this research gap, a directed evolution was employed using a high throughput approach with FACS sorting and a Split-GFP system to screen for active and more soluble variants. Overall, we combined the beneficial mutations from each approach resulting in the Combi-PETase, a variant that has a significantly improved activity, thermal stability of 27 degrees and up to 25-fold in protein yields. Show more