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This Sandbox is Reserved from 26/11/2020, through 26/11/2021 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1643 through Sandbox Reserved 1664.

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PET Hydrolase

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.) [?].

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 ^[2].

1 Function
2 Structure
- 2.1 Primary and Secondary Structure
- 2.2 Tertiary Structure
  - 2.2.1 Catalytic site
  - 2.2.2 Disulfide bridges
3 Applications
- 3.1 Degradation of PET
  - 3.1.1 Bioremediation
  - 3.1.2 Biological recycling

Function

PETase is a hydrolysing enzyme, capable of cleaving esters into acids and alcohols, with the help of water ^[2]^[3] 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 ^[4]^[5]. 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 ^[6]. 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 ^[7].

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 . Altogether, they are called the catalytic triad. In PETase, these three amino acids are S133, H210 and D179 ^[7]. 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 ^[8]. 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. [???] 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” ^[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 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 ^[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

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 ^[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.

References

↑ ^1.0 ^1.1 Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, Toyohara K, Miyamoto K, Kimura Y, Oda K. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science. 2016 Mar 11;351(6278):1196-9. doi: 10.1126/science.aad6359. PMID:26965627 doi:http://dx.doi.org/10.1126/science.aad6359
↑ ^2.0 ^2.1 Danso D, Schmeisser C, Chow J, Zimmermann W, Wei R, Leggewie C, Li X, Hazen T, Streit WR. New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes. Appl Environ Microbiol. 2018 Apr 2;84(8). pii: AEM.02773-17. doi:, 10.1128/AEM.02773-17. Print 2018 Apr 15. PMID:29427431 doi:http://dx.doi.org/10.1128/AEM.02773-17
↑ Panda T, Gowrishankar BS. Production and applications of esterases. Appl Microbiol Biotechnol. 2005 Apr;67(2):160-9. doi: 10.1007/s00253-004-1840-y. , Epub 2005 Jan 4. PMID:15630579 doi:http://dx.doi.org/10.1007/s00253-004-1840-y
↑ P. Dockrill, « Scientists Have Accidentally Created a Mutant Enzyme That Eats Plastic Waste », ScienceAlert. https://www.sciencealert.com/scientists-accidentally-engineered-mutant-enzyme-eats-through-plastic-pet-petase-pollution Retrieved 2021-01-11.
↑ Kim JW, Park SB, Tran QG, Cho DH, Choi DY, Lee YJ, Kim HS. Functional expression of polyethylene terephthalate-degrading enzyme (PETase) in green microalgae. Microb Cell Fact. 2020 Apr 28;19(1):97. doi: 10.1186/s12934-020-01355-8. PMID:32345276 doi:http://dx.doi.org/10.1186/s12934-020-01355-8
↑ Austin HP, Allen MD, Donohoe BS, Rorrer NA, Kearns FL, Silveira RL, Pollard BC, Dominick G, Duman R, El Omari K, Mykhaylyk V, Wagner A, Michener WE, Amore A, Skaf MS, Crowley MF, Thorne AW, Johnson CW, Woodcock HL, McGeehan JE, Beckham GT. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc Natl Acad Sci U S A. 2018 Apr 17. pii: 1718804115. doi:, 10.1073/pnas.1718804115. PMID:29666242 doi:http://dx.doi.org/10.1073/pnas.1718804115
↑ ^7.0 ^7.1 ^7.2 ^7.3 ^7.4 ^7.5 ^7.6 Fecker T, Galaz-Davison P, Engelberger F, Narui Y, Sotomayor M, Parra LP, Ramirez-Sarmiento CA. Active Site Flexibility as a Hallmark for Efficient PET Degradation by I. sakaiensis PETase. Biophys J. 2018 Mar 27;114(6):1302-1312. doi: 10.1016/j.bpj.2018.02.005. PMID:29590588 doi:http://dx.doi.org/10.1016/j.bpj.2018.02.005
↑ ^8.0 ^8.1 ^8.2 ^8.3 ^8.4 Carr CM, Clarke DJ, Dobson ADW. Microbial Polyethylene Terephthalate Hydrolases: Current and Future Perspectives. Front Microbiol. 2020 Nov 11;11:571265. doi: 10.3389/fmicb.2020.571265., eCollection 2020. PMID:33262744 doi:http://dx.doi.org/10.3389/fmicb.2020.571265

Retrieved from "http://52.214.119.220/wiki/index.php/Sandbox_Reserved_1643"

@@ Line 27: / Line 27: @@
 ==== '''Catalytic site''' ====
-<font color='#cc80ff'>serine, </font><font color='#c88033'>histidine</font>
-<scene name='86/868176/Petase_catalytictriad_ba1/1'>serine, a histidine and an aspartate</scene>
 The PETase discovered in I. sakaiensis is a cutinase-like serine hydrolase. As in every cutinase, the catalytic site is made of <scene name='86/868176/Petase_catalytictriad_ba1/1'>a serine, a histidine and an aspartate</scene>. Altogether, they are called the catalytic triad. In PETase, these three amino acids are S133, H210 and D179 <ref name="structure">DOI:10.1016/j.bpj.2018.02.005</ref>. 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 <ref name="current and futur perspectives">DOI:10.3389/fmicb.2020.571265</ref>. 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. [???]
 Questions remain regarding the mobility of certain residues during the catalytic cycle.
-As in every cutinase, the catalytic site is made of a serine, a histidine and an aspartate. Altogether, they are called the <scene name='86/868176/Petase_catalytictriad_ba1/1'>catalytic triad</scene>. In PETase, these three amino acids are S133, H210 and D179 <ref name="structure">DOI:10.1016/j.bpj.2018.02.005</ref>. 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. <ref name="current and futur perspectives">DOI:10.3389/fmicb.2020.571265</ref>
 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” <ref name="structure">DOI:10.1016/j.bpj.2018.02.005</ref> 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”<ref name="structure">DOI:10.1016/j.bpj.2018.02.005</ref>. 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 <scene name='86/868176/Petase_disulfidebridges_ba1/1'>disulfide bridges</scene>, 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 <ref name="structure">DOI:10.1016/j.bpj.2018.02.005</ref> . 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. <ref name="structure">DOI:10.1016/j.bpj.2018.02.005</ref> 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.” <ref name="structure">DOI:10.1016/j.bpj.2018.02.005</ref>
+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 <ref name="structure">DOI:10.1016/j.bpj.2018.02.005</ref> . 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. <ref name="structure">DOI:10.1016/j.bpj.2018.02.005</ref> 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.” <ref name="structure">DOI:10.1016/j.bpj.2018.02.005</ref>
@@ Line 50: / Line 42: @@
 === '''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 <ref name="current and futur perspectives">DOI:10.3389/fmicb.2020.571265</ref>
+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 <ref name="current and futur perspectives">DOI:10.3389/fmicb.2020.571265</ref>.
 ==== '''Bioremediation''' ====
@@ Line 60: / Line 53: @@
 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 <ref name="current and futur perspectives" />. 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 <ref name="current and futur perspectives" />. 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''' ===
-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 <ref name="current and futur perspectives" />. 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 <ref name="current and futur perspectives" />. 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 <ref name="current and futur perspectives" />.
 </StructureSection>
 == References ==
 <references/>