The structure of PET plastic

The plastic bottles we drink from are typically made of the plastic polymer polyethylene terephthalate (PET). The vast majority of PET used in packaging is produced to be used only once, and there is therefore great potential for better recycling.

Watch the video below and learn more about plastic.

PET is made from the molecules ethylene glycol (EG) and dimethyl terephthalate (DMT), both of which are produced from substances from crude oil.

Figure 1: The polymer polyethylene terephthalate (PET) is produced from the components ethylene glycol (EG) and dimethyl erephthalate (DMT).

PET is a linear polymer and is often just called polyester, although there are several different polyesters. The monomer of PET is called mono-(2-hydroxyethyl) terephthalate, MHET for short. MHET can be broken down into two molecules; terephthalic acid (TPA) and EG (see Figure 2). Terephthalic acid is an aromatic acid as it consists of an aromatic ring on which two carboxylic acids are attached. Note that TPA is not the same molecule as DMT, as the methyl groups (-CH₃) are replaced with hydroxy groups (-OH) during polymerization. Neither TPA nor EG are environmentally harmful in themselves.

Figure 2: Ethylene glycol (EG) and terephemic acid (TPA) form the monomer MHET. They are bound together by an ester bond. The monomer MHET is bound by ester bonds to form the polymer PET.

The two molecules, TPA and EG, form the monomer by being bound together by an ester bond. The MHET monomers are also bound to each other via ester bonds in PET and thereby form the polymer. These ester bonds can be broken by hydrolysis, and thus PET can become its smaller constituents.

The enzymes PETase & MHETase

In 2016, a team of Japanese researchers discovered a brand new bacterium capable of using carbon from PET as its primary carbon source. A carbon source is a carbon-containing molecule (carbohydrate, amino acid,_CO2, fatty acid) that living organisms use for energy and cell growth. The bacterium, which was later named Ideonella sakaiensis, was isolated from samples from a landfill with PET plastic bottles. It turns out that the bacterium is able to break down PET using two different hydrolases that break the ester bonds.

When the bacterium sits on the surface of PET, it secretes the extracellular enzyme – PETase. This enzyme catalyzes the hydrolysis of the PET polymers on the surface of the plastic bottle into its monomers MHET. MHET is now absorbed into the cell, where the intracellular hydrolase – MHETase – hydrolyses MHET into its two components, TPA and EG. The two components, TPA and EG, are further broken down and are part of the cell’s metabolism, where the bacterium uses carbon from these as an energy source and source material for growth. TPA and EG are thereby degraded into_H2O, and_CO2.

Figure 3: I. sakaiensis is seen as purple bacteria on the surface of a PET plastic bottle. First, PETase is excreted from the bacterium and breaks down PET into MHET by hydrolysis. MHET is then absorbed into the cell, where it is broken down by MHETase into EG and TPA.

See below and learn more about how bacteria break down plastic.

The active site in PETase

If we look at the active site in PETase, it is close to the surface. This is a great advantage, as it means that the plastic can more easily make contact and form intermolecular bonds with the active site. The active site in PETase consists of two binding parts and one catalytic part. In the first bonding part, PETase binds to the PET polymer with a hydrophobic cleavage. The hydrophobic chasm can be seen in Figure 4b as the blue color.

Figure 4: 4-MHET (green) bound to PETase. a) Subsite I is shown in pink. b) subsite II is shown in blue. c) The catalytic triad is shown in yellow.

The active site of PETase is able to bind a total of 4 MHET parts (in the PET polymer). In Figure 4 it can be seen that the MHET chain is rotated so that it fits in the enzyme. The two binding parts are called subsite I and II (Figures 4a and 4b, respectively). Between the two subsites is the catalytic part, which consists of 3 amino acids.

Figure 5 shows how subsite I binds the first MHET monomer, while subsite II binds the 3 remaining monomers. Subsite II is divided into 3 parts, a, b, and c, each of which binds to one MHET part. The hydrolysis of the ester bond occurs in the catalytic part between the two subsites. At the first hydrolysis, the PET polymer chain is divided into 2. Then the next MHET part moves up to subsite I, and another ester bond is broken. This then continues through the polymer.

Figure 5: The figure shows how the PET polymer is cleaved into individual MHET monomers. 1) PET polymer is bound to the active site and first hydrolysis precursor. 2) After the first hydrolysis, the polymer advances one unit and the second hydrolysis proceeds. 3) After second hydrolysis, an MHET monomer is released.

Amino acids that are part of a polypeptide are named based on the abbreviation of the amino acid and the number it has. E.g. H128 or His128. This means that this is the amino acid histidine (H), which is number 128 in the polypeptide chain from the N-terminal.

Subsite I consists of Met161, Ile208, and the aromatics Trp185, and Tyr87, which form hydrophobic interactions with the benzene ring in the first monomer of PET and thereby contribute to the stability of the complex in Fig. 5. The intermolecular bonds and interactions between PET and subsite II are mainly hydrophobic interactions. Subsite II consists of the amino acids Thr, Ala, Trp, Ile, Asn, Ser, and Arg.

Figure 6: The active site of PETase, which is bound to a PET polymer consisting of 4 MHET monomers (green). The amino acid side chains in the different subsites are highlighted in their respective colours. The pink amino acids belong to subsite I and the blue amino acids belong to subsite IIa, IIb, and IIc. The catalytic part is shown in yellow. The polypeptide chain of PETase is marked in grey.

Ser236 and Asn246 are located in subsite IIc, and form polar intermolecular bonds with the PET substrate (see Figure 7). The nitrogen atom in Ser236 forms a hydrogen bond with the carbonyl group on one side of the aromatic in the MHET part, where the nitrogen atom in the side chain of asparagine forms a hydrogen bond with the carbonylene on the other side of the aromatist.

Figure 7: Subsite IIc where Ser236 and Asn246 form polar intermolecular bonds with the last MHET monomer bound to the active site.

It can also happen that the PETase binds in the polymer, so that there are only 2 MHET monomers in subsite II. Because of this, PETase is able to break the ester bonds so that 2 different monomers are formed: bis(2-hydroxyethyl), terephthalate (BHET) and MHET. BHET is similar to MHET but has an additional –_CH2CH₂OH group. However, MHETase can only break down MHET and not BHET.

Figure 8: The structure of the 3 products PETase forms by the breakdown of PET.

The enzymes catalyze hydrolysis

Some enzymes have a catalytic triad in their active site. A catalytic traid is a set of three amino acids that are included in the catalysis for reactions where it is extra demanding for the reaction to take place. They are typical of hydrolases. The three amino acids in the triad consist of an acid, base, and a nucleophile. Nucleophiles are electron-rich atoms that are capable of donating an electron since they have a free electron pair (e.g., oxygen). Nucleophiles react with electrophiles, which are electron-poor atoms that want to absorb an electron. Electrophiles are typically positively charged or are a positive dipole in polar bonds. In our example, i.e. in PETase and MHETase, the catalytic triad consists of aspartate, histidine and serine (see Figure 9). This is common for hydrolases that modify esters.

Figure 9: The catalytic triad in PETase is shown in yellow, the enzyme itself is colored gray.

In PETase, the catalytic triad consists of Asp206, His237, and Ser160. Although the three amino acids are located far away from each other in the primary structure of the enzyme, they are located together, locally, in the tertiary structure and thus form the cleavage site. Aspartate is an acid as it contains a carboxylic acid group. Histidine is a base at neutral pH, since its NH group in the aromatic can act as a proton receptor. Serine is nukelophilic and can donate an electron from its -OH group. In a catalytic triad, the acid and base work together by forming hydrogen bonds and transferring electrons back and forth between each other to the serine. This makes the serine a stronger nucleophile, allowing it to form covalent bonds with the substrate so that the reaction can proceed more easily.

Ester hydrolysis

PETase and MHETase belong to the subgroup of hydrolases called esterases. Esterases catalyze the hydrolysis of ester bonds that break and turn into a carboxylic acid and an alcohol group.

Figure 10: Hydrolysis of an ester yields a carboxylic acid and an alcohol.

In the background theory, we found that the oxygen in the carbonyl in the ester is electronegative, and the carbon in the carbonylene is electropositive. Therefore, the carbon atom can act as an electrophile. The electrophile reacts with the nucleophile, Ser160, in the ester hydrolysis. Ser160 forms a covalent bond with the carbon atom from the carbonylene, which helps to absorb an -OH group and hydrogen from water, respectively.

The ester hydrolysis needs an acid for it to be catalyzed. This comes from the catalytic triad.

Watch the video below and learn more about how PETase breaks down PET.

The mechanism of hydrolysis in a more advanced form is illustrated in Figure 11.

Ester Hydrolysis (Advanced):

Figure 11: The figure shows the mechanism behind the ester hydrolysis that occurs in the active site of PETase and MHETase.

The black arrows in Figure 12 show how the electrons move and form new bonds between the atoms in the catalytic triad.

1. Aspartate first removes a hydrone (H⁺) from histidine, then histidine removes a hydrone from serine. This makes the oxygen in serine nucleophilic, and it therefore forms a bond with the electrophile in the carbonylene. The oxygen atom in the carbonylene is deprotonated and becomes an anion (oxyanion). The transition astate intermediate is stabilized by hydrogen bonds to aminins that are also present in the active site.

2. A transition state intermediate has now been formed, where the oxyanion is stabilized by the oxyanion hole.
3. Then the one oxygen atom in the intermediate hydron is removed from histidine. One product of the reaction with the alcohol group is now formed, and the ester bond is broken.
4. Water is now entering the active site. The oxygen atom in the water is nucleophilic as it has two free pairs of electrons. It therefore attacks the electrophilic carbon atom in the carbonylene.
5. The oxygen atom in turn becomes an oxyanion that is stabilized by the oxyanion hole. A transition state intermediate has again formed.
6. Aspartate loses its hydrone to histidine, and serine removes a hydrone from histidine. The bond between serine and the intermediate is thereby broken. The second product with the carboxylic acid end is now formed, and the reaction is over.

The mechanism of degradation of MHET in MHETase is the same as the above. Both PETase and MHETase are serine hydrolases, which are hydrolases in which there is a nucleophilic serine present in the active site.

Other PET degrading enzymes

We have now become acquainted with an enzyme that has been evolutionarily developed to break down PET. But before the bacterium I. sakaiensis was found, researchers had already been looking out in nature for enzymes that could nebdry PET. Right now, we know of several enzymes from both bacteria and fungi that are able to break down the PET polymer by hydrolyzing the ester bonds. The enzymes that have been shown to be able to break down PET are also serine hydrolases (cutinases, lipases and carboxylesterases). The enzymes were found by looking for esterases, whose substrates are structurally similar to PET.

Cutinases

It turns out that the hydrolases that have been found that show that can break down PET best are cutinases. As the name reveals, the substrate of cutinases is cutin. Cutin is a waxy polymer that sits in the upper cells on the surface of plants.

Cutin is a hydrophobic polyester, which it has in common with PET. Cutinases have developed active sites that lie on the surface of the protein, making it easier to come into contact with the hydrophobic molecule. We will look at two cutinases, one from the fungus Humicola insolens and one from the bacteriumThermobifida fusca. The enzymes are called HiCut and TfCut2, respectively. Both enzymes are serine hydrolases, and their catalytic triad also consists, as in PETase and MHETase, of a serine, histidine, and aspartate side chain. They also have an oxyanion hole. The amino acid sequence of the two enzymes is approximately 50% identical to that of PETase. Since the catalytic triad is the same in all three enzymes, it is assumed that their catalytic mechanism is also similar. The overall structure of PETase is similar to cutinase. Figure 12 shows the structure of the three enzymes.

Figure 12: The structure of the enzymes PETase from I. sakaiensis, HiCut from H. insolens of T. fusca.

Biodegradation in practice:

To break down PET plastic in practice, you do not necessarily need to use the microorganisms themselves, but only the enzymes. By using only the enzymes, you avoid having to support its growth. The germs of the enzymes have therefore been found by gene sequencing and have them expressed in a good production organ, such as the Enzyme Enzyme. E. coli. The production organism then makes similar enzymes, which are then extracted and purified in order to work with a pure enzyme solution.

Researchers are trying to optimize PET-degrading enzymes by changing individual amino acids in or around the active site. This is called enzyme engineering and is a major field in biotechnology. Companies like Novozymes^® optimize enzymes by enzyme engineering, so that they can become even more attractive as products by being more efficient. Among other things, researchers want to make PETase even better at binding to PET and thereby improving enzyme activity. There is also interest in designing enzymes that are more stable and thus robust in industrial use.

The special thing about Idoenella sakaiensis is that it has the extra enzyme MHETase, and it is therefore also able to break down the monomer of PET. No microorganisms other than I. sakaiensis have been found that can metabolize PET. However, this does not mean that it is not possible for them to exist.

The discovery of the system from I. sakaiensis may be the start of a new innovative technology that could potentially be a solution for recycling plastic. However, in order to be able to roll out biodegradation of PET plastic on a large scale, the process must be 100-1000 times faster than it is right now. It is therefore a significant improvement that is needed. But researchers are optimistic and see great potential in the research field, where progress is expected within the next few years.