2.2 Enzymes Capable of Degrading PET
4. Ecological Significance
5. Current Research
Species: Sakaiensis (1)
Of the Bacterium: In 2016, a team of researchers (Miyamoto et al.) from Kyoto Institute of Technology and Keio University were attempting to isolate bacteria from outside of a plastic bottle recycling facility that were capable of degrading PET plastics, the most common of the thermoplastic polymer resin of the polyester family, and an ecological burden (2). The researchers cultivated natural samples from outside of the facility in test tubes with PET plastic as the primary carbon source (2). They noticed that after several weeks a distinct bacterial community had formed on the plastic from one of the samples from the Sakai, Osaka prefecture, and it was apparent that the PET plastics were being degraded by microbes (2). The researchers were then able to isolate a novel bacterium that was capable of degrading PET plastics. The bacterium was named Ideonella sakaiensis , named for Sakai, Osaka prefecture, Japan, the region from which the microbial environmental sample originated. (4).
Of the Enzymes Capable of the Degradation and Assimilation of PET: Once the research team had determined that this novel bacterium, I. sakaiensis , was the bacterium from their sample responsible for the degradation of PET plastics, they sought to discover how I. sakaiensis was able to do so (2). They were able to determine that I. sakaiensis was able to assimilate PET plastics to promote growth, so they performed a genome analysis in order to identify which enzymes are produced by I. sakaiensis that allow it to degrade and assimilate the PET plastics (2). In doing so, the researchers found that the genes responsible for the enzymes that degrade the PET polymer were similar to previously known enzymes that were able to hydrolyze PET (2). A key difference however is that the enzyme associated with PET hydrolysis in I. sakaiensis was much more robust (2). The novel enzyme isolated from I. sakaiensis was found to have a higher preference for PET and was also able to degrade at temperatures at which PET plastics are their most stable, i.e. most resistant to degradation (2). Due to this enzyme’s unique preference and ability for PET degradation, it was named PETase by the research team (2). The research team then found that once PET was degraded by PETase the bacterium was able to further break down MHET (mono(2-hydroxyethyl) terephthalic acid), a byproduct of PET degradation, into usable monomers that can be hydrolyzed and assimilated by I. sakaiensis (2). The research team suspected that MHET must be degraded by a similar enzyme to PET, so they performed a gene expression analysis and found a match in the genome for a similar enzyme (2). After finding a potential match, they performed a functional analysis of gene, and the corresponding enzyme was found to have a high affinity for MHET degradation; this confirmed the researchers’ suspicions, and this enzyme was named MHETase (2). The research team now had the key as to how I. sakaiensis is capable of degrading and assimilating PET plastic. They posed that I. sakaiensis degrades PET using PETase and MHETase to produce the monomers terephthalic acid and ethylene glycol, which are easily able to be degraded into carbon dioxide and water (2).
Ideonella sakaiensis is a Gram-Negative rod-shaped bacteria. It is highly motile due to one or sometimes two polar flagella (3). On a culture, I. sakaiensis colonies are 0.5-1mm in diameter and they present as circles on culture with a whitish color (4). I. sakaiensis optimally degrades strong materials like polyethylene terephthalate (PET) at temperatures around 30 degrees Celsius (5). Members of the Comamonadaceae family have the ability to perform solid-phase denitrification, a new technilogical process used to remove nitrogen from water (6). Using a polyethylene material to denitrify water in the presence of these bacteria make denitrification easier and more cost effective, in terms of energy lost and gained throughout the process (6). I. sakaiensis uses two enzymes that degrade PET to serve as its natural carbon source by converting PET into monomers, or building blocks, that can be metabolized (6). I. sakaiensis was discovered in a culture of degraded PET and seemed to have extra appendages that can be beneficial to the delivery and excretion of the powerful enzymes that degrade plastic (6). I. sakaiensis is positive for catalytic activity, which means it has chemicals that increase the rate of the plastic degrading reaction (6). Ideonella bacterial strains are positive for oxidase and catalase, meaning this bacteria needs to be in the presence of oxygen to metabolize materials (3) with circular colorless colonies when appearing on a plate (3). Ideonella bacterium are chemoorganotrophs, meaning they can utilize organic materials like carbohydrates as their main carbon source (3).
It has been discovered that the DNA strain 201-F6 (3), in these bacteria codes for the ability to degrade PET, gives these bacteria their physical characteristics in colonies and morphology (3). Specific bacteria that can produce PETase and degrade plastic have specific genes that are upregulated by the presence of PET itself, mostly when these bacteria form biofilms on the plastic (6). The actual gene responsible for the production of PETase is ISF6_0224, which is located near the Terephthalate (TPA) degradation, another polyethylene compound, gene cluster (6) in the coding region. The first enzyme responsible for degradation tough polymers is ISF6_F6, which uses water to perform degradation of PET (5).
Plastic waste has become a major influence on overall environmental health (7) . There are some microbes that are able to form biofilms on materials such as PET, and ultimately degrade it. I. sakaeiensis is one of the most successful microbes at performing this via enzymatic activity. Since I. sakaeiensis secretes the enzyme PETase and degrades PET, there is a beneficial use of this bacteria in an environment where there is a lot of plastic waste that can be metabolized as their carbon source, which in turn benefits the wastelands where this usually undegradable material lies (7). There is a gross accumulation of non-biodegradable plastics in our environment, which is having a negative effect on not only terrestrial but marine life (7). These bacteria can prove to be extremely useful if mass-engineered to function as PET degraders, and current research is in motion to execute that goal (7).
Current research focuses on assessing manipulation methods to use I . sakaeiensis, a microbe that use PETase to degrade PET found in plastic, in order to sustain the environment and keep plastic from having negative effects on the environment and on other organisms (8). By isolating the mechanism by which I. sakaeiensis degrades PET, a compound once thought to be resistant to microbial degradation, it may be possible to degrade large amounts of plastic and maintain a sustainable environment free of plastic pollutants (5). Increasing the use of Ideonella sakaiensis in plastic breakdown greatly reduces the amount of plastic that sits in landfills. Plastic thrown in landfills degrade at extremely slow rates, and introducing the use of the bacterial species greatly increases the sustainability of the environment and the breakdown of PET-based plastics (9). Other current research is searching for a variety of PETases and MHETases isolated from various microbial species due to the fact that different species’ PETases and MHETases (or enzymes that are similar) have different specificities (10). De Castro et. al. tested catalytic efficiency and activity for various sourced enzymes, as well as testing if they act synergistically at all (10). The authors report a variety of enzymes involved in PET hydrolysis, including cutinases, lipases, serine esterases and carboxylesterases. By testing activities on various substrate types, the authors were able to characterize enzymatic synergy and activity (10), as a step toward mass commercialization and as a step toward a more sustainable planet. The importance of having a variety of bio-tools (like mass-produced enzyme products) with different plastic specificities is linked to the fact that manufacturers across the world don’t use one type of plastic, and are typically mixed once at their final storage site. Through industrial optimization of these enzymes, the scientific community is hopeful that one day it will be able to develop a zero-waste plastic.
1. European Bioinformatics InstituteProtein Information ResourceSIB Swiss Institute of Bioinformatics. “European Bioinformatics Institute.” UniProt , www.uniprot.org/taxonomy/1547922.
2. Oberbeckmann S, Osborn A.M., and Duhaime M.B. 2016. Microbes on a Bottle: Substrate, Season and Geography Influence Community Composition of Microbes Colonizing Marine Plastic Debris 11(8): e0159289.
3. Glaser, John A. 2017. Polymer recycling using microbes. Clean Technologies and Environmental Policy 19:1817-1823.
4. Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., Toyohara, K., Miyamoto, K., Kimura, Y., and Oda, K. 2016. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351:1196-1199.
5. Miyamoto, K. 2016 . Discovery of a bacterium that degrades and assimilates poly(ethylene terephthalate) could serve as a degradation and/or fermentation platform for biological recycling of PET waste products. Keio University.
6. Khan , S. T., Horiba , Y., Yamamoto M., and Hiraishi , A. 2002. Members of the Family Comamonadaceae as Primary Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate)- Degrading Denitrifiers in Activated Sludge as Revealed by a Polyphasic Approach. American Society for Microbiology 68:3206-3214.
7. Alex Sivan. 2011. New perspectives in plastic biodegradation. Science Direct. http://www.sciencedirect.com/science/article/pii/S0958166911000292?via%3Dihub
8. Tanasupawat, S., Takehana, T., Yoshida, S., Hiraga, K., and Oda, K. 2016. Ideonella sakaiensis sp. nov. , isolated from a microbial consortium that degrades poly(ethylene terephthalate). International Journal of Systematic and Evolutionary Microbiology 66:2813-2818.
9. Prostak, S. 2016. Ideonella sakaiensis : newly-discovered bacterium can break down, metabolize plastic. Sci Mag 351:1196-1199.
10. de Castro, A.M., Carniel, A., Nicomedes Junior, J., et al. 2017. Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources. Journal of Industrial Microbiology and Biotechnology 44:835-844.