Plastics are everywhere. Once they get into the environment
as trash, they stay there for years, decades, or even centuries. That's
because most plastic is chemically inert and immune to the enzymatic
processes involved in biodegradation. We've tried to curtail plastic
pollution through recycling and by creating plastics that are
biodegradable or compostable. But what about all the plastic litter
that's already out there and could persist long after our grandchildren
are gone?
Life may be coming to our aid. A team of scientists in Japan, led by
Shosuke Yoshida of Kyoto University, has recently discovered a species
of bacteria that can degrade a plastic called PET.
Identifying microbes that degrade PET
PET stands for polyethylene terephthalate, a plastic with
good mechanical, barrier, and optical properties. Bottles for water and
soft drinks are just a couple of PET's many, many uses. PET is a
polyester compound with a high aromatic content, which makes it
chemically inert. As a result, it is typically considered resistant to
microbial degradation, although certain fungi grow on a mineral medium
containing PET. Roughly 56 million tons of PET are produced each year,
and a lot of that ends up in the environment.
To see whether organisms other than a few fungi can manage
to digest this plastic, Dr. Yoshida and his team screened 250 PET
debris-contaminated environmental samples. These samples originated in
everything from sediment to wastewater. The scientists looked for
microorganisms that could use low-crystallinity (1.9 percent) PET film
as a major carbon source for growth.
The team identified a distinct microbial consortium that,
once cultured, was able to grow on PET. The PET film surface degraded at
a rate of 0.13mg per square centimeter each day at 30°C. Under similar
conditions, the organisms turned 75 percent of the carbon it obtained
from the PET into CO2.
In order to identify the particular organism that was using
PET as a carbon source, the scientists diluted the microbial consortium
before growing it on PET. The team isolated a novel bacterial species of
the genus Ideonella, which they gave the catchy name Ideonella sakaiensis 201-F6. When they discovered a subconsortium that lost the ability to degrade PET, further analysis revealed it lacked I. sakaiensis.
Using the newly identified bacteria, the team almost completely degraded a PET film in just six weeks.
Identifying the enzymes that break down PET
Currently, there are few known enzymes capable of breaking
down PET through a chemical process known as hydrolysis. In order to
determine what enzymes I. sakaiensis uses, the scientists
sequenced its genome. They identified one gene, ISF6_4831 that encodes a
protein that shares half of its amino acids with another enzyme that
hydrolyzes PET. The area of similarity includes the parts of the enzyme
that are used for catalytic activity.
The scientists purified the recombinant protein from I. sakaiensis and
incubated it with a PET film at 30°C for 18 hours. The incubation
resulted in pitting at the film surface, which is a good indication of
degradation. They also performed chemical analysis of the surface, which
revealed the presence of certain chemicals including
mono(2-hydroxyethyl) terephthalic acid, which is an intermediate for PET
hydrolysis.
Using the DNA sequence of the enzyme ISF6_4831, the team
built a phylogenetic tree based on enzymes that are known to degrade
PET. Using this tree, they identified three other enzymes that they
hypothesized could catalyze PET hydrolysis. They then tested their
ability to hydrolyze several polymers.
Compared to the three other enzymes, ISF6_4831 had a high
preference for PET vs. other polymers with aliphatic esters. As a
result, the team called this enzyme “PETase.” They assessed the ability
of the PETase to break down the PET found in a typical soda bottle,
which has higher crystallinity than their original PET samples—again, it
was more active than other enzymes.
While the PETase could break open the PET polymer, it didn't
take the polymer all the way to its original starting components, so
the team wanted to know whether another enzyme was responsible. Through
further gene analysis, the scientists discovered the enzyme ISF6_0224,
which has a protein sequence matching those of another enzyme family
that is known to hydrolyze the ester linkage of aromatic compounds.
The team purified recombinant ISF6_0224 and found that it
was able to efficiently hydrolyze mono(2-hydroxyethyl) terephthalic
acid. But when tested against PET, ISF6_0224 did not show any hydrolytic
activity. The results strongly suggest that ISF6_0224 protein is
responsible for the conversion of mono(2-hydroxyethyl) terephthalic acid
(MEHT) into PET’s two environmentally benign monomers, terephthalic
acid and ethylene glycol. As such, the team decided that ISF6_0224
should be termed a MEHT hydrolase abbreviated to MEHTase.
This investigation has opened the possibility for a viable
remediation strategy for PET. Through further research and development,
we could have bacteria that efficiently clean up PET waste.
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