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Shosuke Yoshida , et al.
Ideonella sakaiensis vs Polyester



https://www.theguardian.com/environment/2016/mar/10/could-a-new-plastic-eating-bacteria-help-combat-this-pollution-scourge

Could a new plastic-eating bacteria help combat this pollution scourge?
Scientists have discovered a species of bacteria capable of breaking down commonly used PET plastic but remain unsure of its potential applications
Karl Mathiesen

PET makes up almost one sixth of the world’s annual plastic production but only about half is ever collected for recycling.

Nature has begun to fight back against the vast piles of filth dumped into its soils, rivers and oceans by evolving a plastic-eating bacteria – the first known to science.

In a report published in the journal Science, a team of Japanese researchers described a species of bacteria that can break the molecular bonds of one of the world’s most-used plastics - polyethylene terephthalate, also known as PET or polyester.

The Japanese research team sifted through hundreds of samples of PET pollution before finding a colony of organisms using the plastic as a food source.

Further tests found the bacteria almost completely degraded low-quality plastic within six weeks. This was voracious when compared to other biological agents; including a related bacteria, leaf compost and a fungus enzyme recently found to have an appetite for PET.

“This is the first rigorous study – it appears to be very carefully done – that I have seen that shows plastic being hydrolyzed [broken down] by bacteria,” said Dr Tracy Mincer, a researcher at Woods Hole Oceanographic Institution.

The molecules that form PET are bonded very strongly, said Prof Uwe Bornscheuer in an accompanying comment piece in Science. “Until recently, no organisms were known to be able to decompose it.”

In a Gaian twist, initial genetic examination revealed the bacteria, named Ideonella sakaiensis 201-F6, may have evolved enzymes specifically capable of breaking down PET in response to the accumulation of the plastic in the environment in the past 70 years.

Such rapid evolution was possible, said Enzo Palombo, a professor of microbiology at Swinburne University, given that microbes have an extraordinary ability to adapt to their surroundings. “If you put a bacteria in a situation where they’ve only got one food source to consume, over time they will adapt to do that,” he said.

“I think we are seeing how nature can surprise us and in the end the resiliency of nature itself,” added Mincer.

The bacteria took longer to eat away highly crystallised PET, which is used in plastic bottles. That means the enzymes and processes would need refinement before they could be useful for industrial recycling or pollution clean-up.

“It’s difficult to break down highly crystallised PET,” said Prof Kenji Miyamoto from Keio University, one of the authors of the study. “Our research results are just the initiation for the application. We have to work on so many issues needed for various applications. It takes a long time,” he said.

A third of all plastics end up in the environment and 8m tonnes end up in the ocean every year, creating vast accumulations of life-choking rubbish.

PET makes up almost one-sixth of the world’s annual plastic production of 311m tons. Despite PET being one of the more commonly recycled plastics, the World Economic Forum (WEF) reports that only just over half is ever collected for recycling and far less actually ends up being reused.

Advances in biodegradable plastics and recycling offer hope for the future, said Bornscheuer, “but [this] does not help to get rid of the plastics already in the environment”.

However the potential applications of the discovery remain unclear. The most obvious use would be as a biological agent in nature, said Palombo. Bacteria could be sprayed on the huge floating trash heaps building up in the oceans. This method is most notably employed to combat oil spills.

This particular bacteria would not be useful for this process as it only consumes PET, which is too dense to float on water. But Bornscheuer said the discovery could open the door to the discovery or manufacture of biological agents able to break down other plastics.

Palombo said the discovery suggested that other bacteria may have already evolved to do this job and simply needed to be found.

“I would not be surprised if samples of ocean plastics contained microbes that are happily growing on this material and could be isolated in the same manner,” he said.

But Mincer said breaking down ocean rubbish came with dangers of its own. Plastics often contain additives that can be toxic when released. WEF estimates that the 150m tonnes of plastic currently in the ocean contain roughly 23m tonnes of additives.

“Plastic debris may have been less toxic in the whole unhydrolyzed form where it would ultimately have been buried in the sediments on a geological timescale,” said Mincer.

Beyond dealing with the plastic already fouling up the environment, the bacteria could potentially be used in industrial recycling processes.

“Certainly, the use of these microbes or enzymes could play a role in remediation of plastic in a controlled reactor,” said Mincer.

Miyamoto’s team suggested that the environmentally-benign constituents left behind by the bacteria could be the same ones from which the plastic is formed. If this were true and a process could be developed to isolate them, Bornscheuer said: “This could provide huge savings in the production of new polymer without the need for petrol-based starting materials.” According to the WEF, 6% of global oil production is devoted to the production of plastics.

But the plastics industry said the potential for a new biological process to replace or augment the current mechanical recycling process was very small.

“PET is 100% recyclable,” said Mike Neal, the chairman of the Committee of PET Manufacturers in Europe. “I expect that a biodegradation system would require a similar engineering process to chemical depolymerisation and as such is unlikely to be economically viable,” he said.



http://science.sciencemag.org/content/351/6278/1196
Science  11 Mar 2016: Vol. 351, Issue 6278, pp. 1196-1199
DOI: 10.1126/science.aad6359

A bacterium that degrades and assimilates poly(ethylene terephthalate)
Shosuke Yoshida, et al.

Bacteria isolated from outside a bottle-recycling facility can break down and metabolize plastic. The proliferation of plastics in consumer products, from bottles to clothing, has resulted in the release of countless tons of plastics into the environment. Yoshida et al. show how the biodegradation of plastics by specialized bacteria could be a viable bioremediation strategy (see the Perspective by Bornscheuer). The new species, Ideonella sakaiensis, breaks down the plastic by using two enzymes to hydrolyze PET and a primary reaction intermediate, eventually yielding basic building blocks for growth.

Abstract
Poly(ethylene terephthalate) (PET) is used extensively worldwide in plastic products, and its accumulation in the environment has become a global concern. Because the ability to enzymatically degrade PET has been thought to be limited to a few fungal species, biodegradation is not yet a viable remediation or recycling strategy. By screening natural microbial communities exposed to PET in the environment, we isolated a novel bacterium, Ideonella sakaiensis 201-F6, that is able to use PET as its major energy and carbon source. When grown on PET, this strain produces two enzymes capable of hydrolyzing PET and the reaction intermediate, mono(2-hydroxyethyl) terephthalic acid. Both enzymes are required to enzymatically convert PET efficiently into its two environmentally benign monomers, terephthalic acid and ethylene glycol.



WO2015025861
AROMATIC POLYESTER DECOMPOSITION ENZYME AND METHOD FOR DECOMPOSING AROMATIC POLYESTER USING SAID ENZYME
    [ PDF ]
Inventor: MIYAMOTO KENJI,  et al.

Provided is an enzyme for hydrolyzing an aromatic polyester resin such as PET resin, and provided is a method for decomposing an aromatic polyester resin such as PET resin using said enzyme. An aromatic polyester such as polyethylene terephthalate (PET) can be decomposed by an aromatic polyester decomposition enzyme composed of an amino acid sequence represented by sequence nos. 2 or 4 of the sequence listing. Monohydryoxy ethyl terephthalate (MHET) produced by enzymatic decomposition of an aromatic polyester such as polyethylene terephthalate (PET) can be furthermore decomposed to completely form a monomer using an enzyme having MHET hydrolytic activity composed of the polyester decomposition enzyme and an amino acid sequence represented by sequence nos. 10 or 12 of the sequence listing.



https://www.ncbi.nlm.nih.gov/pubmed/26291558
Biochim Biophys Acta. 2015 Nov;1850(11):2308-17.
doi: 10.1016/j.bbagen.2015.08.009.

Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential.
Dimarogona M.,  et al.

Abstract
BACKGROUND:
Cutinases are serine hydrolases that degrade cutin, a polyester of fatty acids that is the main component of plant cuticle. These biocatalysts have recently attracted increased biotechnological interest due to their potential to modify and degrade polyethylene terephthalate (PET), as well as other synthetic polymers.

METHODS:
A cutinase from the mesophilic fungus Fusarium oxysporum, named FoCut5a, was expressed either in the cytoplasm or periplasm of Escherichia coli BL21. Its X-ray structure was determined to 1.9Å resolution using molecular replacement. The activity of the recombinant enzyme was tested on a variety of synthetic esters and polyester analogues.

RESULTS:
The highest production of recombinant FoCut5a was achieved using periplasmic expression at 16°C. Its crystal structure is highly similar to previously determined Fusarium solani cutinase structure. However, a more detailed comparison of the surface properties and amino acid interactions revealed differences with potential impact on the biochemical properties of the two enzymes. FoCut5a showed maximum activity at 40°C and pH 8.0, while it was active on three p-nitrophenyl synthetic esters of aliphatic acids (C(2), C(4), C(12)), with the highest catalytic efficiency for the hydrolysis of the butyl ester. The recombinant cutinase was also found capable of hydrolyzing PET model substrates and synthetic polymers.

CONCLUSIONS:
The present work is the first reported expression and crystal structure determination of a functional cutinase from the mesophilic fungus F. oxysporum with potential application in surface modification of PET synthetic polymers.

GENERAL SIGNIFICANCE:
FoCut5a could be used as a biocatalyst in industrial applications for the environmentally-friendly treatment of synthetic polymers.



https://en.wikipedia.org/wiki/Ideonella_sakaiensis

Ideonella sakaiensis

Ideonella sakaiensis is a bacterium from the genus Ideonella and family Comamonadaceae capable of breaking down PET plastic which was isolated from outside a plastic bottle recycling facility.[2]

Discovery
Ideonella sakaiensis was identified in 2016 by a team of researchers from Kyoto Institute of Technology and Keio University after collecting samples of PET debris in search for bacteria that relied on the plastic for carbon growth. The bacterium first uses PETase, an enzyme that works with water, to break down the PET plastic. It then breaks it down further using MHETase,[2] another enzyme that further reacts with water to break down the plastics into terephthalic acid and ethylene glycol.[3][2]

Impact
The discovery of Ideonella sakaiensis has potential importance for the recycling process of PET plastics. Prior to its discovery, the only known consumers of PET were a small number of fungi including Pestalotiopsis microspora, and knowledge of the new species has spurred discussion about biodegradation as a method of recycling.[4] The bacterium can currently break down a thin film of PET in a little over six weeks, so it is thought that any prospective applications in mass recycling programs will have to be preceded by enhancement of its abilities through genetic modification.[5]

Characterization
I. sakaiensis is Gram-negative, aerobic, and rod-shaped. It does not form spores. The individual cells of the organism are motile and have a single flagellum. I. sakaiensis tests positive for oxidase and catalase. The bacterium grows at a pH range of 5.5 to 9.0 (optimally 7 to 7.5) and a temperature of 15-42 °C (optimally at 30-37 °C). Through phylogenetic analysis, the species was shown to be affiliated to the genus Ideonella, and also related to Ideonella dechloratans and Ideonella azotifigens, justifying its scientific classification.[6]

Morphology
Colonies of I. sakaiensis are colorless, smooth, and circular.[6]



https://en.wikipedia.org/wiki/PETase

PETase
 
PETase
Identifiers
EC number     3.1.1.101
Alt. names     PET hydrolase, poly(ethylene terephthalate) hydrolase
Databases
IntEnz     IntEnz view
BRENDA     BRENDA entry
ExPASy     NiceZyme view
KEGG     KEGG entry
MetaCyc     metabolic pathway
PRIAM     profile
PDB structures     RCSB PDB PDBe PDBsum
 
PETase is an enzyme discovered in 2016 from a Japanese rubbish dump from Ideonella sakaiensis bacteria.[1][2] PETase breaks down PET-plastic to monomeric mono-2-hydroxyethyl terephthalate (MHET) molecules. MHET is broken down in these bacteria to hydroxyethyl terephthalate with the help of MHETase enzyme. Hydroxyethyl terephthalate breaks down in water to terephthalic acid and ethylene glycol, which are environmentally harmless as they are broken down further to produce carbon dioxide and water.[1]

The reaction catalyzed by PETase is (n is the number of monomers in the PET polymer):[3]...

Genetic engineering
In 2018, John McGeehan co-led an international team (the University of Portsmouth and the U.S. Department of Energy's National Renewable Energy Laboratory) that managed to genetically alter PETase making it 20% more efficient, and raising the possibility of further efficiency gains.[4][5][6] Their aim is to use PETase to recycle the coloured polyethylene terephthalate (PET) plastic used in soft drink bottles and turn it back into easily reusable clear plastic.[5]

Professor John McGeehan, Professor of Structural Biology at the University of Plymouth and one of the scientists leading the study, made the following remarks on the enzyme improvement:

"Although the improvement is modest, this unanticipated discovery suggests that there is room to further improve these enzymes, moving us closer to a recycling solution for the ever-growing mountain of discarded plastics."[7]

References
Yoshida, Shosuke; Hiraga, Kazumi; Takehana, Toshihiko; Taniguchi, Ikuo; Yamaji, Hironao; Maeda, Yasuhito; Toyohara, Kiyotsuna; Miyamoto, Kenji; Kimura, Yoshiharu (2016-03-11). "A bacterium that degrades and assimilates poly(ethylene terephthalate)". Science. 351 (6278): 1196–1199. doi:10.1126/science.aad6359. ISSN 0036-8075. PMID 26965627.

Tanasupawat, Somboon; Takehana, Toshihiko; Yoshida, Shosuke; Hiraga, Kazumi; Oda, Kohei (August 2016). "Ideonella sakaiensis sp. nov., isolated from a microbial consortium that degrades poly(ethylene terephthalate)". International Journal of Systematic and Evolutionary Microbiology. 66 (8): 2813–2818. doi:10.1099/ijsem.0.001058. ISSN 1466-5034. PMID 27045688.

"BRENDA - Information on EC 3.1.1.101 - poly(ethylene terephthalate) hydrolase". www.brenda-enzymes.org. 

Austin, Harry P.; Allen, Mark D.; Donohoe, Bryon S.; Rorrer, Nicholas A.; Kearns, Fiona L.; Silveira, Rodrigo L.; Pollard, Benjamin C.; Dominick, Graham; Duman, Ramona (2018-04-17). "Characterization and engineering of a plastic-degrading aromatic polyesterase". Proceedings of the National Academy of Sciences. doi:10.1073/pnas.1718804115. ISSN 0027-8424. PMID 29666242.

Carrington, Damian (16 April 2018). "Scientists accidentally create mutant enzyme that eats plastic bottles". the Guardian.

Editorial, Reuters. "Plastic-eating enzyme holds promise in fighting pollution - scientists". reuters.com. 
"Scientists accidentally discovered a mutant enzyme that could help the world eliminate plastic waste".



http://www.microbiologyresearch.org/docserver/fulltext/ijsem/66/8/2813_ijsem001058.pdf?expires=1524372986&id=id&accname=guest&checksum=BBF1EEA2EA1EC7FB5C19D60FF74DAF92

Ideonella sakaiensis sp. nov., isolated from a microbial consortium that degrades poly(ethylene terephthalate)
Somboon Tanasupawat, et al
[ PDF ]
 


Related:
US7341857
Novel, esterase, fungus capable of producing the same and method for producing the same

Inventor(s): SHINOHARA MAKOTO, et al.

Abstract
The present invention provides a novel esterase derived from Ideonella sp. 0-0013 strain (FERM BP-08660) having the following properties: (1) function, substrate specificity: hydrolyzes methyl 3-hydroxypalmitate to generate 3-hydroxypalmitic acid and methanol; (2) optimal temperature for functioning: 37 DEG C.; (3) optimal pH and stable pH range: pH 7 or more to pH 10 or less; (4) temperature stability: 97% of the enzyme is stable at 43 DEG C.; (5) inhibition, activation, and stabilization: activated by sodium ion and potassium ion, and inhibited by strontium ion, iron ion (divalent), and methyl palmitate; (6) molecular weight: about 46,500 Da (by SDS-PAGE), about 41,000 Da (by a gel filtration method); and (7) isoelectric point: pI 4 (by polyacrylamide gel isoelectric focusing method); a microorganism producing the enzyme; and a method of producing the enzyme.



https://www.sciencedaily.com/releases/2018/01/180131133404.htm

Structural insight into molecular mechanism of PET degradation

KAIST team newly suggests a molecular mechanism showing superior degradability of PET

A KAIST metabolic engineering research team has newly suggested a molecular mechanism showing superior degradability of poly ethylene terephthalate (PET).

This is the first report to simultaneously determine the 3D crystal structure of Ideonella sakaiensis PETase and develop the new variant with enhanced PET degradation.

Recently, diverse research projects are working to address the non-degradability of materials. A poly ethylene terephthalate (PET)-degrading bacterium called Ideonella sakaiensis was recently identified for the possible degradation and recycling of PET by Japanese team in Science journal (Yoshida et al., 2016). However, the detailed molecular mechanism of PET degradation has not been yet identified.

The team under Distinguished Professor Sang Yup Lee of the Department of Chemical and Biomolecular Engineering and the team under Professor Kyung-Jin Kim of the Department of Biotechnology at Kyungpook National University conducted this research. The findings were published in Nature Communications on January 26.

This research predicts a special molecular mechanism based on the docking simulation between PETase and a PET alternative mimic substrate. Furthermore, they succeeded in constructing the variant for IsPETase with enhanced PET-degrading activity using structural-based protein engineering.

It is expected that the new approaches taken in this research can be background for further study of other enzymes capable of degrading not only PET but other plastics as well.

PET is very important source in our daily lives. However, PET after use causes tremendous contamination issues to our environment due to its non-biodegradability, which has been a major advantage of PET. Conventionally, PET is disposed of in landfills, using incineration, and sometimes recycling using chemical methods, which induces additional environmental pollution. Therefore, a new development for highly-efficient PET degrading enzymes is essential to degrade PET using bio-based eco-friendly methods.

Recently, a new bacterial species, Ideonella sakaiensis, which can use PET as a carbon source, was isolated. The PETase of I. sakaiensis (IsPETase) can degrade PET with relatively higher success than other PET-degrading enzymes. However, the detailed enzyme mechanism has not been elucidated, hindering further studies.

The research teams investigated how the substrate binds to the enzyme and which differences in enzyme structure result in significantly higher PET degrading activity compared with other cutinases and esterases, which make IsPETase highly attractive for industrial applications toward PET waste recycling.

Based on the 3D structure and related biochemical studies, they successfully predicted the reasons for extraordinary PET degrading activity of IsPETase and suggested other enzymes that can degrade PET with a newly-classified phylogenetic tree. The team proposed that 4 MHET moieties are the most properly matched substrates due to a cleft on structure even with the 10-20-mers for PET. This is meaningful in that it is the first docking simulation between PETase and PET, not its monomer.

Furthermore, they succeeded in developing a new variant with much higher PET-degrading activity using a crystal structure of this variant to show that the changed structure is better to accommodate PET substrates than wild type PETase, which will lead to developing further superior enzymes and constructing platforms for microbial plastic recycling.

Professor Lee said, "Environmental pollution from plastics remains one of the greatest challenges worldwide with the increasing consumption of plastics. We successfully constructed a new superior PET-degrading variant with the determination of a crystal structure of PETase and its degrading molecular mechanism. This novel technology will help further studies to engineer more superior enzymes with high efficiency in degrading. This will be the subject of our team's ongoing research projects to address the global environmental pollution problem for next generation."



https://phys.org/news/2018-02-insight-molecular-mechanism-pet-degradation.html
https://www.nature.com/articles/s41467-018-02881-1
doi:10.1038/s41467-018-02881-1
Nature Communicationsvolume 9, Article number: 382 (2018)

Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation
Seongjoon Joo, et al.

Abstract
Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis, was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase (IsPETase) at 1.5 Å resolution. IsPETase has a Ser–His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins.
PETase
[ Click to enlarge ]




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