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PET hydrolase (also known as PETase) is an enzyme isolated from the bacteria Ideonella sakaiensis ^[1]. It is a type of enzyme called esterase that belongs to the α/β-hydrolase superfamily (EC 3.1.1.101.) ^[2].

It was in 2016, that Yoshida et al. discovered the bacterium Ideonella sakaiensis 201-F6 ^[1]. The enzyme PETase allows this bacterium to grow by degrading PET (Polyethylene Terephthalate) that is used as its main carbon and energy source.

PETase could also be a solution to one of the world's current biggest problems: plastic recycling. Indeed, the stability of the polymers, their crystallinity and their hydrophilic surface make recycling difficult. Polyethylene terephthalate (PET) is one of the most widely used plastics today (around 30 million tons per year) and its recycling is now possible thanks to PET hydrolase ^[3].

Function

PETase is a hydrolysing enzyme, capable of cleaving esters into acids and alcohols, with the help of water ^[3][4] according to the following reaction:

         (ethylene terephthalate)n + H2O = (ethylene terephtalate)n-1 + 4-[(2-hydroxyethoxy)carbonyl]benzoate

It is able to catalyse the hydrolysis of PET written as (ethylene terephthalate)n. As a polymer, PET is a complex structure with crystalline regions that feature tightly packed chains in parallel, and amorphous regions where the chains are disordered. However, PET has different degrees of crystallization, 35% for bottles and textiles and 6% for PET used in packaging (PET film). Without enzymes, the degradation of PET can take hundreds of years, whereas by using PETase, PET can be degraded in a matter of days ^[5][6]. The degradation of plastic thanks to PETase is recent. Indeed, scientists believe that PET degrading bacterias developed this faculty because of the accumulation of plastic in the environment ^[7]. Although there are different mutants, none of these enzymes are able to dissolve all forms of PET.

Structure

Primary and Secondary Structure

PETase (PDB 6ANE) weighs about 86,37 kDa and is a unique chain, made up of 787 amino acids. These amino acids fold themselves into 6 𝛼-helixes and 9 β-sheets ^[8].

Tertiary Structure

Catalytic site

The PETase discovered in I. sakaiensis is a cutinase-like serine hydrolase. As in every cutinase, the catalytic site is made of a serine, a histidine and an aspartate. Altogether, they are called the catalytic triad. In PETase, these three amino acids are ^[8]. The PETase follows the canonical serine hydrolase catalytic mechanism when PET binds to the enzyme. The serine performs a nucleophilic attack on the substrate, then the basic amino acid histidine polarizes the serine and the acidic amino acid aspartate stabilizes the histidine ^[9]. The reaction mechanism takes place in two steps, acylation and deacylation. Acylation consists of proton transfer from Ser133 to His210 and a nucleophilic attack by Ser133 on the substrate, leading to a tetrahedral transition state. Deacylation consists of deprotonation of a water molecule by His210, resulting in a hydroxide attacking the acylated Ser133 intermediate and breaking its bond to the substrate. His210 transfers the water’s proton to Ser133, with formation of MHET and enzyme regeneration ^[10]. Questions remain regarding the mobility of certain residues during the catalytic cycle.

Although the catalytic triad remains the same between cutinases, there are some residue substitutions in the PETase active site compared to its homologs. These substitutions play a major role in the substrate binding. Indeed, the H132W, F211S, H187S substitution modify the active site’s environment with their side chains leading to a “widening of the ends of the binding cavity” ^[8] and thus an enhancement of the PETase activity. For instance, in homologs, the phenylalanine’s side chain “interacts with the terephthalic ring of PET at the entrance of the binding cavity”^[8]. Because there is no phenylalanine in PET but a serine, there is no interaction with the substrate before its binding to the enzyme. The binding is therefore facilitated and the hydrolysis enhanced.

Disulfide bridges

PETase possesses two disulfide bridges, C246 - C262 common to all cutinases and C176 - C212 specific to PETase. The first one plays an important role in the stability of cutinases. The second one is the consequence of the substitution of two highly conserved alanines in other cutinases by cysteines. It links a strand of the enzyme to the loop of the active site containing the catalytic residue H210 ^[8] . This results in a rigidification of the catalytic site. Thereby, its stability is better and its hydrolysis activity is enhanced. In PETase, the loop containing the residue H210 has three more residues than its homologs. ^[8] In this way, the loop is longer and pushes away the side chain of other residues contained in the neighbouring helices and strands, allowing a better binding of the substrate to the binding cavity. It “keeps the active site flexible enough to compensate for substrate rigidity without compromising the enzyme’s structural integrity.” ^[8]

Applications

Degradation of PET

The most obvious use of PET hydrolase is the degradation of PET. PET hydrolase enzymes preferentially degrade the regions of PET that are amorphous in nature because of the flexibility and movement in these regions: the polymer chains are less restricted.To remedy this, the reaction takes place above the glass transition temperature of PET. Amorphous PET has a glass transition temperature of 67°C and for crystalline PET it is 81°C ^[9].

Bioremediation

Bioremediation refers to the use of living organisms or their enzymes to detoxify or restore contaminated sites, often by directing the natural capabilities of microbes towards environmental pollutants. PET hydrolase could be used in the environment, like the oceans, to harness its enzymatic ability to degrade plastic. ^[9].

Biological recycling

Thanks to bacteria’s rapid adaptation to their environment, we found a potential solution to our plastic solution problem. In mechanical recycling, collected and sorted PET waste can be powdered before melting and reprocessing to other forms. Chemical recycling degrades PET into its basic monomers which can then be polymerized again ^[9]. As biological recycling is cheaper, can be done with temperature conditions without the use of hazardous chemicals, by using microbial catalysis of polymer bond cleavage reactions and doesn’t use toxic reagents ^[9], it is a sustainable alternative to mechanical recycling. However, bio-recycling is limited by the organism used, inherent polymer properties and the choice of pre-treatment, so modifications of these factors are to be explored before the PET hydrolase can be used in recycling processes.

As a future challenge, we can think about the circular bioeconomy ^[9].