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PET Polymer Recycling
Mary O’Reilly and JoAnne Stubbe*
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ur group was recently asked to write a Viewpoint on “An
O catalysts. Two new esterases designated PETase and MHETase
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engineered PET depolymerase to break down and
recycle plastic bottles”.1 Our lab focuses on understanding
the catalysis and specificity of enzymatic reactions and
biosynthesis/degradation of biodegradable polyhydroxyalka-
noate plastics, providing the basis for this commentary.
Downloaded via 193.203.10.208 on June 20, 2020 at 10:33:54 (UTC).
“Plastics” were first imprinted in my mind in the mid-1960s
based on two experiences. The first was the DuPont exhibit at
the World’s Fair held in New York City in 1964 [see the
YouTube video The Wonderful World of Chemistry-DuPont-
1964 New York World’s Fair (https://www.youtube.com/
watch?v=wQCKqDJksuE)]. The second was the famous quote
from the 1967 movie The Graduate starring Dustin Hoffman:
“One word. Are you listening? Plastics!” As the DuPont video
advertised, plastics are the answer to many of society’s
problems: they are inexpensive, lightweight, malleable, and
durable with a broad range of applications from clothing and
auto parts to electronics. Since that time, the ability of chemists
to make new plastics inexpensively has provided ingenious and
creative solutions to an ever-expanding range of problems and
cemented their importance to society. However, due to their
designed persistence, some types of plastics have been
publicized as an environmental nightmare.
As of 2009,2 4% of oil and gas feedstocks were used to make
plastic materials such as polyethylene terephthalic acid (PET)
(Figure 1). PET is the major constituent of plastic packaging
materials, which are essential for food safety in today’s world,
but also of ever increasingly abundant, non-essential, single-use
beverage water bottles. PET, with aromatic ester linkages, is
not readily degraded by microorganisms and has proven to be
challenging to recycle cost-effectively. Further, as a conse-
quence of the increase in the global population and our
lifestyle changes, the pollution/waste from this material has
become very visible in landfills and natural habitats every-
where.
To tackle the PET waste problem, Yoshida et al. in 20163
reported their results from screening soil samples in the
vicinity of a plant that recycles PET. This strategy led to their
discovery of the Gram-negative bacterium Ideonella sakatensis
201-F6 that can use PET as its sole carbon source that is
essential for its growth. Complete genome analysis of this
organism revealed sequence signatures of α/β-hydrolase
superfamily enzymes such as lipases and cutinases, which
possess esterase activity. It was expected that some of these
enzymes might have low levels of PET hydrolysis activity that
could then be engineered and/or evolved into more robust
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(Figure 1) were reported.3
Identification of enzymes to degrade PET has been
challenging as these aromatic ester polymers have a high
degree of crystallinity (Figure 1, bottom) with a glass transition
at 70 °C. Chain flexibility facilitates binding of the polymer to
the enzyme, which is required for degradation. The hydrolysis
products of PET are, furthermore, challenging to monitor
(Figure 1, MHET, TPA, and EG). Thus, to find appropriate
PETases as a starting point to engineer more efficient catalysts,
the key hurdle is to identify an enzyme with even very low
activity and then to develop a sensitive, quantitative assay to
monitor polymer degradation. X-ray structures soon followed
of the PETases4,5 and phylogenetically linked proteins,
including cutinase family members. Not surprisingly, the active
sites of these catalysts possess the canonical catalytic triad of
Ser, His, and Asp (Figure 1, center, Protein Data Bank entry
5XH3) that are used in the genome search for α/β hydrolase
superfamily members.3 The structures are monomeric and
reveal an extended surface groove adjacent to the catalytic triad
(Figure 1, active site “canyon” in the rectangular box) where
the aromatic substrates are proposed to bind. Efforts to obtain
structures of bound PET oligomers, however, have failed. The
problem of understanding the basis for substrate specificity and
binding is one encountered with all polymeric materials and is
almost always exacerbated by flat and often hydrophobic
surfaces, multiple binding areas, and disordered protein surface
loops. Binding of the enzyme to crystalline materials is also a
common problem encountered, requiring knowledge of the
glass transition of the polymer when the chain becomes more
accessible, and engineering of the enzyme to increase its
thermal stability.
The paper of Tournier et al.1 offers a glimpse into the
challenges and success of their engineering process. Their goal
was to set up a recycling system that could cost-effectively, in
high yield, and on the ton scale convert PET into TPA (Figure
1, top) to be purified and reused to remake PET at low cost,
high quality, and high recovery. They developed quantitative,
sensitive assays, and the paper describes a tour de force for
Received: June 1, 2020
https://dx.doi.org/10.1021/acs.biochem.0c00457
Biochemistry pubs.acs.org/biochemistry Viewpoint

Figure 1. Engineered PETases to degrade PET polymers to TPA that can be recycled to PET in high yield, in high quality, and cost-effectively. The
PETases, serine hydrolases, have been engineered for increased activity and thermal stability to make crystalline polymer accessible for binding and
degradation. MHET denotes monohydroxyethyl terephthalic acid, and EG ethylene glycol. Orange pacman is the engineered PET esterase. The
protein structure is the LLC serine hydrolase with the catalytic triad and MHET bound (Protein Data Bank entry 5XH31), and the active site
surface groove “canyon” that binds the polymer is within the rectangular box.

optimization of the protein catalyst. They used the PETase
(leaf-branch composite cutinase, LLC esterase) to generate
and study more than 200 esterase mutants and the rates of
their reactions, analyzing the recovered products, including the
amount of crystalline polymer remaining (Figure 1, bottom).
They used structure, molecular docking, and surface algorithms
to generate a bound 2-HE-(MHET)2 model substrate. They
also used molecular dynamics analysis to optimize the catalytic
potential, based on understanding the function of the catalytic
triad (Figure 1). To remove crystalline PET, they engineered
the PETase to have increased thermal stability by adding a
disulfide in a region identified to have a metal binding site in
survival of our planet and our limited resources. There are
many ways to solve problems associated with plastic waste,
including water bottles. The latter may require the will to find
alternatives! We would argue that to tackle these challenging
problems, it is important to understand more about the basic
biology and biochemistry of how natural polymers are made
and recycled and to develop new methods to evolve new
specificities with acceptable activity. Enzymes continue to play
a central role used in conjunction with continually changing
technologies.

some cutinase superfamily members. This disulfide approach
avoided problems associated with metals. They engineered the
enzyme to be stable at temperatures close to the PET glass
transition and for the times required for almost complete
degradation of the polymeric material. They optimized
catalytic activity in bioreactors mimicking conditions for the
tons of material to be processed. In the end, they were able to
achieve their goal: to recycle PET to TPA that could
regenerate in high yield a PET polymer with properties
identical to those of virgin PET. The paper describes the
importance of a multidisciplinary approach for success: a basic
understanding of catalysis, protein design, analytical chemical
methods, and engineering to scale in conjunction with an
integrated understanding of cost-effectiveness.
Given the importance of plastics in today’s society and the
fact that all materials have “lifetimes”, the types of approaches
described in this and other recent papers1−4 are required for
B https://dx.doi.org/10.1021/acs.biochem.0c00457
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AUTHOR INFORMATION
Corresponding Author
JoAnne Stubbe − Department of Chemistry, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139,
United States; orcid.org/0000-0001-8076-4489;
Email: stubbe@mit.edu
Author
Mary O’Reilly − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biochem.0c00457
Notes
The authors declare no competing financial interest.
Biochemistry XXXX, XXX, XXX−XXX
Biochemistry

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pubs.acs.org/biochemistry Viewpoint

ABBREVIATIONS
PET, polyterephthalic acid; TPA, terephthalic acid; MHET,
monohydroxyethyl-terephthalic acid; EG, ethylene glycol;
PETase, esterase that hydrolyzes PET.

■ REFERENCES
(1) Tournier, V., Topham, C. M., Gilles, A., David, B., Folgoas, C.,
Moya-Leclair, E., Kamionka, E., Desrousseaux, M.-L., Texier, H.,
Gavalda, S., Cot, M., Guemard, E., Dalibey, M., Nomme, J., Cioci, G.,
Barbe, S., Chateau, M., Andre, I., Duquesne, S., and Marty, A. (2020)
An engineered PET depolymerase to break down and recycle plastic
bottles. Nature 580, 216−219.
(2) Hopewell, J., Dvorak, R., and Kosior, E. (2009) Plastics
recycling: challenges and opportunities. Philos. Trans. R. Soc., B 364,
2115−2126.
(3) Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H.,
Maeda, Y., Toyohara, K., Miyamoto, Y., Kimura, and Oda, K. (2016)
A bacterium that degrades and assimilates poly(ethylene terphtalate).
Science 351, 1196−1199.
(4) Joo, S., Cho, I. J., Seo, H., Son, H. F., Sagong, H.-Y., Shin, T. J.,
Choi, S. Y., Lee, S. Y., and Kim, K.-J. (2018) Structural insight into
molecular mechanism of poly(ethylene terephthalate) degradation.
Nat. Commun. 9, 382.
(5) Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A.,
Kearns, F. L., Silveira, R. L., Pollard, B. C., Dominick, G., Duman, R.,
El Omari, K., Mykhaylyk, V., Wagner, A., Michener, W. E., Amore, A.,
Skaf, M. S., Crowley, M. F., Thorne, A. W., Johnson, C. W.,
Woodcock, H. L., McGeehan, J. E., and Beckham, G. T. (2018)
Characterization and engineering of a plastic-degrading aromatic
polyesterase. Proc. Natl. Acad. Sci. U. S. A. 115, E350−4357.

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