PETase; do not worry about plastic

PETase;
Do Not Worry About Plastic!
Supervisor: Dr. Barshan
Present by: Hoorie Sadat Sabet (hoorie.sabet@ut.ac.ir)
June 2019
‫علوم‬ ‫دانشکده‬‫فنون‬ ‫و‬
‫نوین‬
‫علوم‬ ‫مهندسی‬ ‫گروه‬
‫زیستی‬

Contents
1. What is plastic?
1.1 global plastics production
1.2 plastic waste generation
2. Environmental impacts of plastic pollution
3. Types of plastics
4. What is PET?
4.1 Applications of PET
5. PET disposal methods
5.1 landfill
5.2 incineration
5.3 recycling
6. Degradability of polymers
6.1 Degradability of PET
7. PET biodegradation
8. Discovery of PETase
9. How to work?
10. PETase VS three PET-hydrolytic enzymes
11. Protein engineering of PETase
12. Conclusion
13. References

What is Plastic?
• A synthetic material made from a wide range of organic polymers that
can be molded into shape while soft, and set into a rigid or elastic form
• Useful characteristics:
 resistant to corrosion and chemical decomposition
 lower density compared to other solid materials
 good stability to mechanical shock
 high strength-to-weight ratio
4
Farzi, A., Dehnad, A., & Fotouhi, A. F. (2019). Biodegradation of polyethylene terephthalate waste using Streptomyces species and
kinetic modeling of the process. Biocatalysis and Agricultural Biotechnology, 17, 25-31.
https://www.trendingpackaging.com

5
Geyer, R., Jambeck, Ja. R., & Law, K. L. (2017). Production, use, and fate of all plastics ever made. Science Advances, 3(7), e1700782.
Global Plastics Production

Plastic Waste Generation by Industrial Sector, 2015
6
Geyer, R., Jambeck, Ja. R., & Law, K. L. (2017). Production, use, and fate of all plastics ever made. Science Advances, 3(7), e1700782.

Environmental Impacts of Plastic Pollution
7
https://www.reusethisbag.com
https://www.britannica.com
http://blog.nus.edu.sg
http://blog.nus.edu.sg

Types of Plastics Used for Packaging
https://bizfluent.com/13657035/six-types-of-plastic-used-for-packaging. 22 January 2019.
8
• PET – Polyethylene Terephthalate
• PVC – Polyvinyl Chloride
• HDPE – High density Polyethylene
• LDPE – Low-density Polyethylene
• PP – Polypropylene
• PS – Polystyrene

What is PET?
Farzi, A., Dehnad, A., & Fotouhi, A. F. (2019). Biodegradation of polyethylene terephthalate waste using Streptomyces species and kinetic
modeling of the process. Biocatalysis and Agricultural Biotechnology, 17, 25-31.
9
• Since 1977
• Thermoplastic polyester
 high durability
 low price
• Obtained from condensation polymerization reaction of:

Applications of PET
10
• Products packaged in PET include:
 health and beauty products
 household cleaners
 salad dressings
 peanut butter
 beverages
 water
 candy
https://napcor.com/about-pet/

Plastic Wastes Disposal Methods
11
Landfill (55%)
Incinerated (25%)
Recycled (20%)
Geyer, R., Jambeck, J. R., & Law, K. L. (2017). Production, use, and fate of all plastics ever made. Science Advances, 3(7), e1700782.

1. Landfill
Webb, H., Arnott, J., Crawford, R., & Ivanova, E. (2013). Plastic degradation and its environmental implications with special reference to poly
(ethylene terephthalate). Polymers, 5(1), 1-18.
12
• Disadvantages:
Occupy space that could be
utilized for more productive
means, such as agriculture
Plastic debris acts as a source
for a number of secondary
environmental pollutants
http://www.europarl.europa.eu

2. Incineration
Singh, R. K., Ruj, B., Sadhukhan, A. K., & Gupta, P. (2019). Thermal degradation of waste plastics under non-sweeping atmosphere: Part 1: Effect of
temperature, product optimization, and degradation mechanism. Journal of Environmental Management, 239, 395-406.
13
• Disadvantages:
Formation of numerous harmful compounds
PAHs, PCBs, heavy metals, toxic carbon- and
oxygen-based free radicals
Contributes about 400 million tons of CO2
in the atmosphere
https://www.back-tobasics.org

3. Recycling
14
• Two approaches currently in use for the recycling of PET:
 Chemical processing (depolymerization by chemolysis)
 Mechanical processing
• Disadvantages:
Relatively expensive
Inefficient (NAPCOR, 2011: only %29 of 2.48 million ton of PET jars and bottles)
The presence of additives and impurities complicate the recycling procedure
yield and quality of the recovered product
https://www.dreamstime.com

Degradability of Polymers
15
polymers with pure carbon backbones are
polymers that include heteroatoms in the
backbone (e.g., polyesters, polyamines)
aromatic polymers
resistant to most methods of degradation
higher susceptibility to degradation
be resistant to degradation, despite the presence
of bonds that are normally readily hydrolysed

Degradability of PET
16
• The ester bonds could normally be quite easily broken by a number of mechanisms
• However, due to its aromatic groups:
“The polymer is essentially non-degradable under normal conditions”
• Microbial communities are capable of
utilising diethylene glycol terephthalate
(DTP), a subunit of PET, as a sole carbon and
energy source

Biodegradation
17
• The metabolic diversity of bacteria makes them a useful resource for
remediation of pollution in the environment
• Advantages:
 A cheaper process
 More efficient
 Does not produce secondary pollutants
 To obtain useful end products with economic benefit (ethanol for use in biofuels)

PET Biodegradation by Enzymatic and Microbial Methods
18
1981: Hydrolysis of copolyesters of aromatic and aliphatic polyesters
such as polycaprolactone and PET
by Rhizopus delemar lipase
2004: Biodegradation of PET fiber and diethylene glycol terephthalate (DTP)
by microbes prepared from activated sludge, and lipase enzyme

19
2006: Enzymatic biodegradation of PET films
by Aspergillus Niger
2011: Biodegradation degree of PET films modified with a polyester
by filamentous fungi Penicillium funiculosum extracellular hydrolytic
enzymes

20
2012: Biodegradation of PET films
by naturally growing microbes on degrading PET
such as different microbes from Actinomycetes family and some fungi
 slow degradation of PET by Nocardia with help of esterase enzyme
2015: Biodegradation of PET films
by Bacillus Subtilis
 week biodegradation ratio, but also the bacteria acted on PET films

Discovery of PETase
Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., ... & Oda, K. (2016). A bacterium that degrades and assimilates poly
(ethylene terephthalate). Science, 351(6278), 1196-1199.
21
• Collected 250 PET debris–contaminated environmental samples
from a yard of PET bottle-recycling factory in Sakai city, Osaka, Japan
• Screened for microorganisms could use PET film as the major C source for growth
• One sediment sample contained a distinct
microbial consortium that formed on the PET film
upon culturing (fig.A)
• Induced morphological change in the PET film
(fig.B)
B

Discovery of PETase
22
• The consortium on the film (no. 46) contained: bacteria + yeast-like cells + protozoa
• Using limiting dilutions of consortium no. 46:
Isolated a bacterium capable of degrading and assimilating PET
Ideonella
sakaiensis

Discovery of PETase
23
• Cells were appeared to be connected to each other by appendages (fig.E)
• Shorter appendages were observed between the cells and the film;
 These might assist in the delivery of secreted enzymes into the film (fig.F)

Discovery of PETase
24
• The PET film (60 mg, 20 × 15 × 0.2 mm) was damaged (fig.G)
• Completely degraded after 6 weeks at 30°C (fig.H)
 In the course of sub-culturing no.46:
found a sub-consortium that lost its PET degradation
this sub-consortium lacked I. sakaiensis
I. sakaiensis is functionally involved in PET degradation

How to Work?
25
 PETase hydrolyzes PET to MHET and TPA
 MHETase hydrolyzes MHET to TPA and EG
• When I.S grown on PET produces 2 enzymes
• Both enzymes are required to convert PET efficiently
into its two environmentally benign monomers,
terephthalic acid and ethylene glycol

How to Work?
Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., ... & Mykhaylyk, V. (2018). Characterization and engineering of a
plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19), E4350-E4357.
26
• PETase does the hardest part
 which is breaking down the crystal structure
 depolymerizing PET into MHET
• The work done by the second enzyme
 which converts the MHET into terephthalic acid
 is much simpler
 For this reason, research has focused on PETase

PETase VS Three PET-hydrolytic Enzymes
27
 TfH from a thermophilic actinomycete
 Cutinase homolog from leaf-branch compost
metagenome (LC cutinase, or LCC)
 F.solani cutinase (FsC) from a fungus
The activity of the PETase
against the PET film, was 120, 5.5, and 88
times as high as that of TfH, LCC, and FsC

PETase VS Three PET-hydrolytic Enzymes
• Effect of temperature on enzymatic PET film hydrolysis:
28
LCC, TfH and FsC are limited by their requirement
of a considerably high temperature to carry out
their maximum activity

Production Challenges of PETase
Son, H. F., Cho, I. J., Joo, S., Seo, H., Sagong, H. Y., Choi, S. Y., ... & Kim, K. J. (2019). Rational Protein Engineering of Thermo-Stable PETase
from Ideonella sakaiensis for Highly Efficient PET Degradation. ACS Catalysis.
• One major defect of this enzyme for PET-degradation is that it acts only
at mild temperature due to its low thermal stability
29
Need to design variants of PETase
Because of Tg of PET > 65°C

Result of PETase Protein Engineering
30
Son, H. F., Cho, I. J., Joo, S., Seo, H., Sagong, H. Y., Choi, S. Y., ... & Kim, K. J. (2019). Rational Protein Engineering of Thermo-Stable PETase from
Ideonella sakaiensis for Highly Efficient PET Degradation. ACS Catalysis.

Protein Engineering of PETase
1. First:
• PETase belongs to the α/β hydrolase superfamily
• The central twisted β-sheet is formed by 9 mixed β-strands surrounded by 7 α-helices
• The central β-sheet is interrupted due to abnormal conformation of the β6 strand:
31

 Considered that this disruption of the β-sheet severely affects the
thermal stability of PETase
 Generated the PETaseP181A variant:
32

1. Second:
• The β6-β7 connecting loop in PETase:
33
• The β6-β7 connecting loop in Tf CUT2:

• Reason of the high stability of the loop in Tf CUT2:
 Generated the PETaseS121D/D186H variants:
34
the His156 located
on the loop
forms a hydrogen bond with
the Asp94 residue on
the α2 helix

Result of PETase Protein Engineering
35

Conclusion
https://www.bbc.com/news/science-environment-43783631, “2018”
36
• Polyesters, such as PET, are widely used in plastic bottles and clothing
• PETase:
 Reverses the manufacturing process
 Most activity compared with other enzymes
• Industrial production:
 New variants with high thermal stability
 Hydrolyze PET faster than now

37
PROFESSOR JOHN MCGEEHAN
UNIVERSITY OF PORTSMOUTH
Department: School of Biological Sciences
Faculty: Faculty of Science
"There is an urgent need to
reduce the amount of
plastic that ends up in
landfill and the
environment, and I think if
we can adopt these
technologies we actually
have a potential solution in
the future to doing that"
https://www.bbc.com/news/science-environment-43783631

References
38
1. Farzi, A., A. Dehnad, and A.F. Fotouhi, Biodegradation of polyethylene terephthalate waste using Streptomyces species and kinetic modeling of the process.
Biocatalysis and Agricultural Biotechnology, 2019. 17: p. 25-31.
2. Geyer, R., J.R. Jambeck, and K.L. Law, Production, use, and fate of all plastics ever made. Science advances, 2017. 3(7): p. e1700782.
3. Webb, H., et al., Plastic Degradation and Its Environmental Implications with Special Reference to Poly(ethylene terephthalate). Polymers, 2012. 5(1): p. 1-18.
4. Son, H.F., et al., Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation. ACS Catalysis, 2019. 9(4): p.
3519-3526.
5. Singh, R., et al., Thermal degradation of waste plastics under non-sweeping atmosphere: Part 1: Effect of temperature, product optimization, and degradation
mechanism. Journal of Environmental Management, 2019. 239: p. 395-406.
6. Aryasomayajula, N., et al., A System for Degrading PET in the Environment.
7. Tokiwa, Y. and T. Suzuki, Hydrolysis of copolyesters containing aromatic and aliphatic ester blocks by lipase. Journal of Applied Polymer Science, 1981. 26(2): p.
441-448.
8. Zhang, J., et al., A study on the biodegradability of polyethylene terephthalate fiber and diethylene glycol terephthalate. Journal of Applied Polymer Science,
2004. 93(3): p. 1089-1096.
9. Marqués-Calvo, M.S., et al., Enzymatic and microbial biodegradability of poly (ethylene terephthalate) copolymers containing nitrated units. Polymer degradation
and stability, 2006. 91(4): p. 663-671.
10. Nowak, B., et al., Biodegradation of poly (ethylene terephthalate) modified with polyester" Bionolle®" by Penicillium funiculosum. Polimery, 2011. 56(1): p. 35-44.
11. Sharon, C. and M. Sharon, Studies on biodegradation of polyethylene terephthalate: A synthetic polymer. Journal of Microbiology and Biotechnology Research, 2012.
2(2): p. 248-257.
12. Nakkabi, A., et al., Biodegradation of poly (ethylene terephthalate) by Bacillus subtilis. Int J Recent Adv Multidiscip Res, 2015. 2: p. 1060-1062.
13. Yoshida, S., et al., A bacterium that degrades and assimilates poly (ethylene terephthalate). Science, 2016. 351(6278): p. 1196-1199.
14. Austin, H.P., et al., Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 2018. 115(19): p.
E4350-E4357.
15. https://www.bbc.com/news/science-environment-43783631

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