Antarctic Polyester Hydrolases Degrade Aliphatic and Aromatic Polyesters at Moderate Temperatures

Abstract

Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics in the packaging industry, and consequently has become one of the main components of plastic waste found in the environment. However, several microorganisms have been described to encode enzymes that catalyze the depolymerization of PET. While most known PET hydrolases are thermophilic and require reaction temperatures between 60°C and 70°C for an efficient hydrolysis of PET, a partial hydrolysis of amorphous PET at lower temperatures by the polyester hydrolase IsPETase from the mesophilic bacterium Ideonella sakaiensis has also been reported. We show that polyester hydrolases from the Antarctic bacteria Moraxella sp. strain TA144 (Mors1) and Oleispira antarctica RB-8 (OaCut) were able to hydrolyze the aliphatic polyester polycaprolactone as well as the aromatic polyester PET at a reaction temperature of 25°C. Mors1 caused a weight loss of amorphous PET films and thus constitutes a PET-degrading psychrophilic enzyme. Comparative modeling of Mors1 showed that the amino acid composition of its active site resembled both thermophilic and mesophilic PET hydrolases. Lastly, bioinformatic analysis of Antarctic metagenomic samples demonstrated that members of the Moraxellaceae family carry candidate genes coding for further potential psychrophilic PET hydrolases. IMPORTANCE A myriad of consumer products contains polyethylene terephthalate (PET), a plastic that has accumulated as waste in the environment due to its long-term stability and poor waste management. One promising solution is the enzymatic biodegradation of PET, with most known enzymes only catalyzing this process at high temperatures. Here, we bioinformatically identified and biochemically characterized an enzyme from an Antarctic organism that degrades PET at 25°C with similar efficiency to the few PET-degrading enzymes active at moderate temperatures. Reasoning that Antarctica harbors other PET-degrading enzymes, we analyzed available data from Antarctic metagenomic samples and successfully identified other potential enzymes. Our findings contribute to increasing the repertoire of known PET-degrading enzymes that are currently being considered as biocatalysts for the biological recycling of plastic waste.

Keywords: Antarctica; Moraxella sp.; Oleispira antarctica; Polyethylene terephthalate (PET); plastic biodegradation; polyester hydrolases; psychrophilic enzymes.

FIG 1

Hydrolysis of PCL by Mors1 and OaCut. (A) Agar plate containing PCL with E. coli expressing Mors1. (B) Agar plate containing PCL with E. coli expressing OaCut. (C) Time course of PCL hydrolysis (decrease of turbidity of a PCL nanoparticle suspension) by Mors1 (blue) and OaCut (black). (D) Relative initial hydrolysis rates of PCL nanoparticles by Mors1 at different reaction temperatures; 100% = hydrolysis rate at 25°C. (E) Determination of the thermal inactivation temperature (T₅₀ = 48.7°C) of Mors1.

FIG 2

Enzymatic degradation of amorphous PET films. (A) Weight loss of PET films (in dark green) and amounts of TPA and MHET (in pink) released by Mors1 and IsPETase after a reaction time of 24h at 25°C. In gray, negative control without enzyme. (B) Weight loss of PET films after a reaction time of 6 days at 25°C with Mors1 and OaCut. NC: Negative control without enzyme. (C) Weight loss of PET films after a reaction time of 1, 7, and 10 days at 25°C with Mors1. (D) Surface changes of amorphous PET films. A film used as negative control with no enzyme is shown on the left, whereas a film treated with Mors1 for 10 days at 25°C is shown on the right. Scanning electron microscopy images of the surface of an untreated PET film (E) and a film treated with Mors1 for 10 days at 25°C (F).

FIG 3

Comparison of the sequence, structure, and active site dynamics of Mors1, OaCut, IsPETase, TfCut2, and LCC. (A) Multiple sequence alignment. Residues numbered according to the full protein sequences with signal peptide. Strictly conserved residues are highlighted in green background, with yellow triangles indicating cysteine pairs that form disulfide bonds and orange stars indicating catalytic residues. A secondary structure topology based on the structure of TfCut2 (PDB 4CG1) is shown on top of the sequence alignment. (B) Cartoon representation of the modeled structure of Mors1, showing its three disulfide bridges (DB) in yellow sticks. (C) Active site of Mors1 (blue), with catalytic residues in bold. (D) Active site of IsPETase (green), showing residues equivalent to Mors1 and catalytic residues in bold. (E) Average backbone RMSF for Mors1 and IsPETase. The secondary structure is indicated as lines in the background, with α-helices in pink and β-sheets in gray.

FIG 4

Sequence variability of potential polyester hydrolases from Antarctic metagenomes. A multiple sequence alignment between Mors1 and homologous enzyme candidates with high sequence coverage from Antarctic metagenomes from Chile Bay. Blue boxes indicate columns with either strict (red background) or 75% (red characters) sequence conservation between all enzymes. Green stars indicate conserved catalytic residues, whereas blue spheres indicate active site residues.