Introduction

Environmental Issues

Pollution is one of the largest environmental issues facing our globe today. It is estimated that there is 359 million tons of plastic waste, excluding all other forms of material waste, produced annually around the world. Of this plastic waste, 150–200 million tons ends up sitting in landfills or as pollution in our natural environments. The most common form of plastic waste is poly-ethylene terephthalate (PET), generally used in soft drink containers and plastic water bottles. Only about 10% is recycled. The methods currently in use are not viable on a global scale.

Current Plastic Recycling Methodology

Strides have been made toward recycling PET plastics, but most current methods are not viable globally. Enzymes—especially cutinases and hydrolases—have been promising, but many suffer from low thermal stability. PET needs to be broken down at or above 70°C, which causes most enzymes to denature. Furthermore, many enzymes don’t fully break PET down to its monomers, making complete recycling impossible.

Leaf compost cutinase (LCC), in both its wild-type and mutated forms, is a promising enzyme that can function at higher temperatures and shows potential to help resolve global plastic recycling challenges.

Structure

Overall Topology

LCC is a monomeric serine hydrolase with 258 amino acids and an amphipathic structure. It features a of alpha helices and beta turns, characteristic of the alpha-beta hydrolase family. The in its active site is a hallmark of this family. LCC is 33× more efficient than other studied PET-degrading enzymes.

Alpha-Beta Hydrolase Family

This enzyme contains an alpha-beta hydrolase fold. The fold includes a chymotrypsin-like catalytic triad, hydrophobic binding pocket, and an oxyanion hole. The motif G-X-Nu-X-G allows for a tight nucleophilic loop. Some members also contain HX₄D motifs, providing acyltransferase activity. Functions across this family include proteolysis, signal transduction, and lipid metabolism.

Active Site

Structure

The stabilizes PET through aromatic π-stacking and Van der Waals interactions. No structure of LCC bound to PET exists, so ligand positions have been approximated. [Add table with residues + monomers]

Mechanism

The catalytic triad (insert scene) facilitates proton transfer, activating S165 for nucleophilic attack. It targets the carbonyl carbon of the PET polymer, forming a tetrahedral intermediate. The (Y95 and M166) stabilizes the intermediate. H242 then protonates the leaving group oxygen, cleaving the scissile bond. In a second step, a water molecule—activated by H242 and D210—attacks, forming a second tetrahedral intermediate. H242 protonates the leaving group again, restoring the carbonyl and freeing the product.

Figure Legend

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Mutations

Tournier et al. Mutagenesis

Tournier et al. studied mutations in the active site using a . Out of 11 targeted residues, most mutations decreased activity—except F243I and F243W, which increased it.

ICCG/WCCG Mutants

The ICCG and WCCG variants increased catalytic efficiency and thermal stability. A disulfide bond was introduced by mutating , replacing a metal-binding motif (E208/D238/S283). This raised melting temperature from 84.7°C (WT) to 94.5°C, though activity slightly decreased.

Glycosylation

Three were introduced by Shirke et al. using N-linked modifications. These improved thermal stability and catalytic activity. Patterns used: - 197–199 (N-T-S) - 239–241 (N-A-S) – no individual impact - 266–268 (N-D-T)

Glycosylated LCC was more stable (10°C higher Tm), unfolded more slowly, and retained 85% activity at 80°C (vs. 50% for WT).

Conclusions

Improved Thermal Stability

Thermal stability is essential for PET degradation above 70°C. The disulfide mutation increased the enzyme’s melting point by ~10°C.

Depolymerization Efficiency of Mutant LCCs

ICCG and WCCG reached ~90% depolymerization in 9.3–10.5 hours, compared to 53% in 20 hours for WT.

Implications for Plastic Recycling

These discoveries suggest a path forward for more efficient enzymatic PET recycling—bringing us closer to scalable, environmentally sustainable plastic waste solutions.