Novel Polyesterases from Marine Plastisphere Metagenome

Abstract

While it is neither desirable nor viable to halt production of petroplastics, the growing accumulation of mismanaged postconsumer plastic waste highlights the urgent requirement for improved recycling strategies and developing new technologies. Current recycling technologies remain far from fully circular, particularly for mixed or contaminated waste streams. Enzyme-based plastic recycling offers a promising complementary strategy, providing a greener and more flexible solution for processing mixed waste streams with lower energy consumption and potential for upcycled products. However, its widespread application is restricted by the limited availability of diverse, highly active plastic-degrading enzymes.

In this study, marine plastisphere metagenomes derived from seawater incubation experiments were mined to identify novel polyester-degrading enzymes. Twenty-seven putative alpha/beta (α/β) hydrolases were selected based on the presence of the serine-hydrolase catalytic triad and sequence similarity to benchmark PETases, and subjected to phylogenetic, biochemical, and structural analyses. Phylogenetic analysis revealed that candidates span diverse bacterial lineages, predominantly Pseudomonadota, with Group V carboxylesterase candidates forming a well-supported clade adjacent to benchmark PETases. Among the subsequently characterised enzymes, PV31 fell within this Group V clade, while PV34, PV50, PV51, and PV52 grouped with Group IV carboxylesterases. Of the 27 candidates, 10 were successfully purified and screened for carboxylesterase activity using chromogenic para-nitrophenyl (pNP) ester substrates, revealing activity in eight purified proteins. PV26, PV38, PV45, PV47, PV48, and PV55 were not biochemically characterised due to low activity, restricted substrate specificity, or failure to repurify. Five enzymes (PV31, PV34, PV50, PV51, PV52) were selected for detailed biochemical characterisation, including optimisation of pH, temperature, NaCl concentration, and surfactant (Tween 20) concentration, alongside kinetic analysis and oligomeric states. Most of the characterised candidates exhibited activity on medium- to long-chain pNP ester substrates and displayed cold-tolerant, alkaliphilic, and halotolerant behaviour, with enhanced activity in the presence of surfactant. Agar clearance screens and high-performance liquid chromatography (HPLC) product analysis demonstrated polyesterase activity in all five purified candidates across a range of aromatic and aliphatic polyester substrates. PV31 exhibited the broadest substrate range, including unique activity on polyurethane analogue, impranil, while PV34 and PV50 showed the highest activity on 3PET and aPET respectively. Activity varied between candidates, with PV52 showing the lowest overall hydrolysis.

Thus, this study identified five novel, promiscuous polyesterases with the ability to depolymerise polyethylene terephthalate (PET) and other polyesters, as confirmed by HPLC product analysis. These findings expand the known diversity of marine-derived polyesterases and highlight the plastisphere as an underexplored reservoir of novel biocatalysts. Importantly, the results also highlight the limitations of assays with model ester substrates in predicting polyester degradation, emphasising the need for integrated biochemical and analytical validation in enzyme discovery pipelines.

Date of Award	28 Jul 2026
Original language	English
Awarding Institution	Bangor University
Supervisor	Peter Golyshin (Supervisor) & Alexandre Iakounine (Supervisor)