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One of the world's current biggest problems is the recycling of plastic. 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, an enzyme isolated from the bacteria Ideonella sakaiensis ^[1].

Function

PET hydrolase (PETase) is a type of enzyme called an esterase and it belongs to the α/β-hydrolase superfamily. These enzymes are hydrolysing enzymes capable of cleaving esters into an acid and an alcohol, with the help of water ^[2][3]. This enzyme is able to catalyse the hydrolisis of PET. Naturally, without enzymes, the degradation of PET can take hundreds of years. However, by using PETase, this degradation can be done in a mater of days ^[4][5]. Some scientists believe that PET degrading bacteria have developped only in recent years due to the accumulation of plastics, including PET, in the environment ^[6]. In 2016, Yoshida et al. ^[2] discovered the bacterium Ideonella sakaiensis 201-F6. This bacterium uses PET as its main carbon and energy source. The enzyme PETase is essential to the bacterium's growth, as its primary function is to create molecules that can be assimilated by the micro organism. Thanks to bacteria's rapid adaptation to their environment, we found a potential solution to our plastic polution problem.

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 ^[7].

Tertiary Structure

Catalytic site

As in every cutinase, the catalytic site is made of a serine, a histidine and an aspartate. Altogether, they are called the . In PETase, these three amino acids are S133, H210 and D179 ^[7]. When a substrate, PET or homologs, binds to the enzyme, the serine performs a nucleophilic attack on the substrate, then the basic amino acid histidine polarises the serine and the acidic amino acid aspartate stabilises the histidine. ^[8]

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” ^[7] 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”^[7]. 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 , 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 ^[7] . 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. ^[7] 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.” ^[7]

Applications

Degradation of PET

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).The most important field of application for the use of PET hydrolase is the degradation of PET. Although there are different mutants, none of these enzymes are able to dissolve all forms 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 less restricted. To remedy this, the reaction takes place above the glass transition temperature of PET. Amorphous PET have a glass transition temperature of 67°C and for crystalline PET it is of 81°C ^[8]

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. ^[8].

Biological recycling

In mechanical recycling, collected and sorted PET waste can be powdered before melting and reprocessing to other forms. Chemical recycling leads to degrade PET into its basic monomers which can then be repolymerized ^[8]. This method is unfavorable because mechanical recycling is much more cost effective. Moreover, chemical methods require the maintenance of high temperature and pressure as well as employing toxic reagents and several preceding unit operations. Therefore, biological recycling is emerging as a more sustainable solution as it can be done with low temperature conditions, without the use of hazardous chemicals, by using microbial catalysis of polymer bond cleavage reactions, which results in the recovery of monomers ^[8]. 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.

=== Circular bioeconomy === ^[9]

Circular economy is creating loops which feed resources back into the economy to make the same or new products. In general, the low production cost of plastic shows that the reuse does not offer an economic advantage ^[8]. However, a combination of biodegradation and biosynthesis, bio-based PET economy could contribute to an environmental advantage. A biotechnology leading to introduce PET hydrolase in the circular economy, will create PET waste and reduce its release into the environment. Bio-PET, which refers to a PET polymer that is at least partially derived from biological sources, can be produced through the microbial synthesis of terephthalic acid TPA and ethylene glycol EG ^[8]. This method could make a significant contribution to a sustainable and circular PET economy. However, some complexities are associated with biological TPA production and therefore, it is only EG that is produced biologically from renewable feedstocks to give bio-PET ^[8].