Improving thermostability for biodegradation via discovery and protein engineering

Overview

This research addresses a critical bottleneck in enzymatic plastic recycling: most known polymer-degrading enzymes are mesophilic, meaning they operate only between 20–45°C, which is well below the optimal temperature range (~58°C) for efficient hydrolysis of biodegradable plastics such as polylactic acid (PLA) and polyhydroxyalkanoates (PHA). The project pursues a dual strategy of (1) bioinformatic genome mining to identify novel depolymerases from the α/β hydrolase enzyme family based on sequence and structural similarity to known PLA/PHA-degrading enzymes, and (2) computational protein engineering to enhance the thermostability of these candidates. The ultimate goal is to produce engineered enzymes that perform at higher temperatures, accelerating polymer breakdown into monomers or smaller subunits that can be recovered and reused through chemical upcycling.

Technical specifications

Discovery approach:

• Bioinformatic screening of genomic databases for α/β hydrolase family homologs with sequence and structural similarity to known PLA and PHA depolymerases
• Gene synthesis and expression in E. coli, followed by purification via affinity chromatography
• Activity assays adapted to test native function on polymers and copolymers

Engineering approach:

• Computational design tools including AlphaFold2, Rosetta Modeling Suite, PROSS (Protein Repair One-Stop Shop), HotSpot Wizard 3.0, FireProt, and MutCompute
• Structure-function analysis to understand enzyme-substrate interactions before engineering
• Iterative in silico modeling combined with in vivo testing of variants
• Integration of thermal challenge assays to evaluate thermostability improvements
• Analytical methods to monitor polymer degradation and monomer formation

Prior studies using similar combinatorial strategies have achieved melting temperature improvements of up to 30°C in related hydrolases, demonstrating feasibility of the approach.

Technology readiness level

The project is at an early-to-mid stage of development. The discovery and engineering workflows draw on validated computational tools (PROSS, Rosetta, AlphaFold2) that have demonstrated success in improving thermostability of related enzymes such as PETase, cutinase, and lipases. Future validation will proceed through gene synthesis, expression, purification, activity screening, and iterative engineering cycles. The approach is currently focused on laboratory validation; scaling to industrial bioprocessing conditions has not yet been addressed.