Polyester(ase) Activity of Bacterial Esterases on Aliphatic and Aromatic Polyesters

Abstract

The overuse and accumulation of plastics in the environment are concerning and urgent challenges that need to be solved. While recycling can offer a partial solution, emerging biodegradation approaches involving microorganisms capable of breaking down complex polymers, offer a significant alternative. Microbial enzymes, in particular, have shown efficiency depolymerising synthetic polymers. However, each polymer requires specific treatments according to their chemical structure. In this study, various polyethylene terephthalate (PET) hydrolases were tested for their ability to degrade aliphatic and aromatic synthetic polyesters, including PET, AkestraTM 90, Poly(ε-caprolactone) (PCL) and Polylactic acid (PLA). Four autotransporter-domain containing esterase and five extracellular PET hydrolases were expressed and analysed. The autotransporter-domain containing esterase enzymes exhibited no degradation activity while the PET hydrolases demonstrated effective depolymerization. Among these, IsPETase and Trx-IsPETase enzymes derived from Piscinibacter sakaiensis had the greatest activity against PET. Additionally, HiCut, a commercial cutinase from Humicola insolens exhibited extensive degradation in PCL and was the only one enzyme capable of depolymerizing AkestraTM 90. None of the enzymes reported degradation of PLA. Moreover, a comparative analysis of a thermostable PET hydrolase, PET2, fused with a histidine-tag at either the N- or C-terminus revealed significant enhanced expression, 5.8-fold higher, when the His-tag was positioned at the N-terminal. Both PET2 constructs reported comparable stability and effective depolymerization activity against PET and PCL.

Popular Abstract

Biodegradation of plastics using microbial proteins.

The overuse, accumulation, and pollution of plastics have become one of the most important environmental concerns of our time. From oceans filled with floating plastic islands to nano- and microplastics found animals, drinking water and even human tissues, plastic pollution represents an enormous global thread. Petroleum-based plastics, such as polyethylene terephthalate (PET), used in bottle packing or textiles, have created an international necessity for recycling systems. To address this crisis, researchers are studying microbial enzymes capable of degrading these plastics into the original building blocks of their synthetic polymers.

This thesis explores the use of microbial enzymes that can break down the plastic chains into simpler molecules, which can be reused by industries to generate new plastics, closing the loop and recycling the materials that have been already consumed, in a more sustainable circular economy. The focus is on esterases, a specific type of enzyme that can hydrolyze plastic polymers such as PET, widely used for food packaging and everyday clothing.

To test their potential, the enzymes must first be expressed and purified before they can react with the plastics and degrade them. Some enzymes used were autotransporter-domain containing esterase, which are located in the bacterial outer membrane, and they theoretically can degrade the plastics from that position. However, these enzymes couldn’t be effectively purified and their activity degrading the plastics used was very limited.

Given the purification challenges, alternative enzymes were used, ones that are secreted outside the bacterial cell to degrade extracellularly the polyesters, enabling an easier expression and purification. Among these enzymes IsPETase is a well-characterized enzyme, known for its properties and ability to degrade polyesters, and its engineered variant Trx-IsPETase, with previously characterized activity on PET. Moreover, a commercial enzyme, HiCut, was also included to validate the biodegradation reactions on PET and AkestraTM 90, a complex plastic known to be resistant to depolymerization because of its intricate chemical structure. Lastly, two PET2 variants were subjected to the degradation and stability analyses. All these last proteins were successfully expressed and purified, allowing the reactions.

Which enzyme had the highest activity?
Biodegradation reactions were carried out with four different types of polyesters: PET, AkestraTM 90, Polycaprolactone (PCL), and Polylactic acid (PLA). For PET, the enzymes with the highest activity were Thx-IsPETase, followed by IsPETase and then the PET2 variants, with near half the activity of the first two. AkestraTM 90 could only be degraded by HiCut. For PCL, plastic commonly used for biomedical applications and 3D printing, HiCut and the PET2 variants could fully degrade it within 72 hours. However, none of the enzymes tested in this project were able to degrade PLA, which is used in food containers, textiles and medical implants.

Master’s Degree Project in Molecular Biology, Microbiology and Biotechnology. 60 credits 2025.
Department of Biology, Lund University.

Advisor: Javier Linares-Pastén.
Advisors Department of Chemistry: Applied Microbiology & Biotechnology.

• author: Huertas Díaz, Carlos
• supervisor: Javier Linares-Pastén
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20242
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9214502
• date added to LUP: 2025-10-24 14:17:55
• date last changed: 2025-10-24 14:17:55