The significance of the result lies in what it eliminates. Conventional enzymatic PET recycling depends on thermostable enzymes operating near the glass transition temperature of PET, where polymer chains become flexible enough for the catalyst to access them. That strategy works, but it demands substantial energy input to maintain elevated temperatures, relies on freshwater-based buffer systems, and creates a downstream bottleneck: the monomers released by hydrolysis must then be cooled to 30 to 40 degrees Celsius before bacteria or engineered chassis organisms can assimilate them, adding an energy-intensive cooling step. Running depolymerization in seawater at moderate temperature collapses this entire pipeline into a single, low-energy process, and seawater is free, abundant, and sterile enough in its salinity to support non-sterile bioprocessing.

The team began by screening eight representative PET hydrolases in artificial seawater prepared according to the ASTM D1141-98 standard, at 30 degrees Celsius. Among the candidates were esterases from Bacillus subtilis and Thermobifida fusca, the marine-derived PE-H from Pseudomonas aestusnigri, the polyesterase Cut190, the fungal cutinase FsC, the metagenomic leaf-branch compost cutinase LCC, and IsPETase, the enzyme originally discovered in the bacterium Ideonella sakaiensis in 2016. IsPETase proved the clear winner, outperforming the second-best enzyme by more than 4.5-fold. The catch was fragility: wild-type IsPETase has a half-life of only about one day at 30 degrees Celsius, far too unstable for sustained industrial use.

To fix that, the researchers turned to a semi-rational protein engineering strategy focused on rigidifying flexible sites. They analyzed seven published crystal structures of IsPETase with the program B-FITTER, ranking residues by B-factors to identify the 45 most mobile positions, and added 11 positions where literature had already reported stabilizing mutations. The overlap yielded a final list of 53 target residues. At each site, they built saturation mutagenesis libraries using degenerate NNK codons, which encode all 20 amino acids, and screened the libraries with a clever Petri-dish assay: after a heat shock at 75 degrees Celsius that lyses the host E. coli cells, surviving enzyme converts a chromogenic substrate into a visible blue product, allowing positive mutants to be picked by eye. The screen produced 22 beneficial mutations from 17 positions, raising the enzyme’s T50 inactivation temperature by up to 9.8 degrees in single mutants.

Combining mutations proved the delicate part. Two multi-mutants, M7 and M15, pushed the T50 up by 24.9 and 21.7 degrees respectively, but their hydrolysis activities collapsed to just 29 and 25 percent of the wild-type level, a classic example of the trade-offs and epistatic interactions that plague multiparameter enzyme engineering. Structural analysis suggested that the S207E mutation, sitting adjacent to the catalytic residue D206, was sabotaging activity, so the team removed it to create M6, which restored activity to 92 percent of wild type. Adding the R280A mutation, previously reported to aid substrate binding, lifted activity to 117 percent of wild type, and introducing an engineered disulfide bridge between positions 233 and 282, inspired by stabilizing strategies in other PET hydrolases, boosted the T50 by a further 8.9 degrees without sacrificing activity.

The final variant, M8, is a tour de force of balanced engineering. Its melting temperature rose by 27.3 degrees Celsius over wild type, its catalytic activity increased 1.14-fold, and, unexpectedly, its soluble expression yield in E. coli jumped 14.3-fold. The expression boost traces to the P181V mutation, which appears to promote formation of a more extensive beta-sheet structure that folds more readily in the bacterial cytoplasm. Because enzyme production cost is a major barrier to industrial biocatalysis, a variant that is simultaneously more stable, more active, and far cheaper to manufacture represents a rare triple win. Benchmarked against the leading thermostable enzymes DuraPETase and LCC-ICCG under identical conditions, M8’s overall depolymerization efficiency, combining yield and activity, exceeded them by 32.2-fold and 10.4-fold respectively.

To understand why M8 works so well, the team solved its crystal structure at the Shanghai Synchrotron Radiation Facility and ran extensive molecular dynamics simulations in artificial ocean water. The overall fold was preserved, with a root mean square deviation of only 0.178 angstroms from wild type, but individual mutations each contributed stabilizing interactions: K95N forms a new hydrogen bond with S242, I168R creates a salt bridge with D186, P181V strengthens hydrophobic packing with L167, S214V places a hydrophobic contact near W185, and A248D forms a transient salt bridge with a rotated R100 that appears in nearly 80 percent of simulation frames. The flexible loops spanning residues 202 to 218 and 231 to 240 became markedly more rigid. Meanwhile, the R280A mutation removes a spatial clash between arginine 280 and the terminal phenyl ring of the substrate, and simulations showed the distance between catalytic residues S160 and H237 shortened dramatically in M8, facilitating the proton shuttling that drives catalysis.

The decisive test came in natural seawater collected from the South China Sea, using post-consumer PET powder ground from fruit packaging trays. The team optimized substrate loading from 5 to 15 percent by weight and enzyme concentrations from 500 to 2000 nanomolar, maintaining the pH at 8.2 with periodic sodium hydroxide additions. Over five days of continuous operation, product release remained essentially linear at both 30 and 37 degrees Celsius. At 37 degrees, total soluble products reached 76.8 millimolar, roughly two-thirds of it terephthalic acid, corresponding to a monomer production rate of 15.4 millimolar per day. At 30 degrees the rate was 5.2 millimolar per day. Crucially, 15.4 millimolar per day falls squarely within the 3 to 28.5 millimolar per day uptake rates reported for monomer-assimilating microbes, meaning the enzyme’s output is sufficient to feed a downstream fermentation without any buffer or dilution step.

That compatibility underpins the team’s larger vision: a simultaneous enzymatic depolymerization and fermentation process, or SEDF, modeled on the simultaneous saccharification and fermentation framework used in lignocellulosic biofuel production, but conducted entirely in seawater. The missing piece is a microbial chassis that can consume terephthalic acid and ethylene glycol under high-salinity conditions; existing PET-metabolizing strains such as Pseudomonas and Rhodococcus pyridinivorans have only been demonstrated in freshwater. The authors point to Halomonas bluephagenesis, a salt-loving bacterium already scaled to commercial fermenters producing tens of thousands of tons of bioplastic annually, as the most promising candidate for engineering a complete seawater-based recycling loop.

The enzyme’s robustness extends beyond the reactor. M8 retained more than 80 percent of its activity after four months of incubation under high-salinity, low-temperature conditions, suggesting potential for treating PET microplastic contamination in industrial wastewater or even in-situ marine remediation, though the authors caution that open-environment deployment would require engineered microbial carriers, rigorous biocontainment strategies, and thorough ecological risk assessment. They are equally candid about the work’s limitations: the experiments were milliliter-scale, and only a techno-economic analysis at pilot or cubic-meter scale, weighing slower reaction kinetics against eliminated heating and freshwater costs, can establish true industrial competitiveness. Computational tools including FoldX, Pythia, ThermoMPNN and CataPro failed to predict further improvements over M8, underscoring that experimental screening remains indispensable. Still, with a single engineered enzyme now capable of chewing through real plastic waste in real seawater at body temperature, the prospect of freshwater-free, low-energy plastic bio-recycling has moved from concept to demonstrable reality.

Subject of Research: Protein engineering of the PET-degrading enzyme IsPETase for seawater-based plastic depolymerization at ambient temperature

Article Title: Engineering a robust IsPETase for energy-efficient PET depolymerization in natural seawater at ambient temperatures

Article References: Huang, X., Jia, Q., Li, G., Yang, X., Xu, S., Liu, J., Li, W., Liu, Y., Xie, W., & Cao, L. (2026). Engineering a robust IsPETase for energy-efficient PET depolymerization in natural seawater at ambient temperatures. Advanced Biotechnology, 4(2), Article 14. https://doi.org/10.1007/s44307-026-00104-z

Image Credits: AI Generated

DOI: 10.1007/s44307-026-00104-z

Keywords: PET recycling, IsPETase, enzyme engineering, plastic pollution, seawater biocatalysis, thermostability, protein engineering, biodegradation, molecular dynamics, industrial biotechnology, plastic upcycling, Halomonas