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This year, our team embarked on a journey of learning and had ambitious plans to complete a wide range of experiments. Unfortunately, due to time constraints and a lack of essential resources and components, we couldn't realize all our ideas. Nevertheless, we took on significant responsibilities and put in a substantial amount of work. We remain hopeful that our efforts will serve as a valuable foundation for future IGEM teams, enabling them to make groundbreaking discoveries and address pressing challenges.

As part of this commitment, we conducted an in-depth analysis of several articles related to our topic, aiming to provide a comprehensive overview of the effectiveness of specific mutations on PETase and MHETase enzymes. We also proposed new types of mutations that could introduce unique properties to these enzymes. Additionally, we've left behind a significant number of untested parts, and we wish the best of luck to future participants in their endeavors.


Mutations in PETase and MHETase enzymes, crucial for PET degradation, have shown significant potential in enhancing their catalytic activity, stability, and versatility.

In PETase, the S238F mutation, which replaces serine with phenylalanine in its active site, resulted in an up to 100-fold increase in catalytic efficiency, likely due to the enhanced hydrophobic character of the active site promoting improved PET binding. Similarly, mutations R61A, L88F, and I179F, which involved replacing charged amino acids with hydrophobic ones in the catalytic groove, also resulted in amplified catalytic activity. Additionally, the introduction of S121E, D186H, and R280A mutations in PETase increased its thermal stability, possibly due to enhanced hydrogen bonding networks with water molecules.

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Turning to MHETase, the Phe424D mutation, which replaced phenylalanine with smaller and more hydrophobic amino acids in its substrate-binding groove, demonstrated up to a 15-fold increase in BHETase activity. This improvement can likely be attributed to the expanded substrate-binding cleft, permitting the binding of larger molecules like BHET. Meanwhile, the R411K mutation, where an arginine is replaced by a lysine in MHETase's catalytic domain, led to heightened MHETase activity, potentially through the stabilization of the catalytic intermediate.

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Notably, combining PETase and MHETase into a chimeric protein through a Gly-Ser linker has presented a synergistic increase in PET degradation efficiency. By analyzing the efficiency of these mutations, one can conclude that the modified enzymes, especially with drastic improvements like the S238F mutation in PETase, hold immense potential for PET degradation.

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Mutations in the active center of enzymes can have significant impacts on their catalytic capabilities, a phenomenon explicitly observed in the enzyme MHETase, essential for PET degradation. In the discussed article, several mutations located in MHETase's active center all resulted in decreased MHET hydrolytic activity. The S131G mutation, which eliminates the hydroxyl group from Ser131, is particularly impactful. Without this group, Ser131 loses its nucleophilic properties, hampering its ability to attack MHET's carbonyl carbon. Similarly, the E226T mutation, substituting glutamic acid for threonine, could potentially interrupt the hydrogen bonding network, pivotal for stabilizing the transition state. The F495I mutation, which replaces phenylalanine with isoleucine, might hinder the proper positioning of the substrate within the active site. All these mutations seem to challenge the integrity and functioning of MHETase's active center, leading to decreased enzyme activity.

Meanwhile, PETase, another crucial enzyme in PET degradation, mirrors MHETase in its structure. While the discussed articles did not mention any direct mutations in PETase, it's worth noting the striking resemblance between the active centers of both enzymes. Both contain key residues, Ser131 and Phe415, which play pivotal roles in their respective hydrolytic actions. This similarity hints at a shared evolutionary lineage, suggesting they might have evolved from a common ancestor or possibly originated from the same ancestral enzyme through gene duplication.

Diving deeper into PETase's domain reveals that its active center is not a standalone entity but is complemented by other crucial residues. Notably, His528 activates the catalytic water molecule, while Asp492 stabilizes the transition state. Mutations in any of these supporting residues could compromise PETase's activity. Moreover, this active center is nestled in a cleft formed by various amino acids, dictating substrate binding and positioning. Any changes to this cleft's constituent residues could influence PETase's ability to bind to and hydrolyze PET.

In summation, while MHETase and PETase share similarities in their active centers, mutations in these domains can have profound impacts on their functionalities. The detailed exploration of PETase's domains provides a rich foundation for future studies, potentially leading to enhanced enzyme performance and innovative solutions for PET degradation.

Results and conditions

In the pursuit of sustainable solutions for PET recycling, two experiments shed pivotal insights into the capabilities of the enzymatic duo: PETase and MHETase.

The first study was an exploration into the combined might of PETase and MHETase in degrading commercial PET film. Conducted at 37°C for 24 hours with both enzymes at concentrations of 10 μg/mL, the experiment recorded a noteworthy 40% reduction in the initial 100 μm thick PET film. An interesting facet of this study was the assessment of specific mutations. Mutations such as S131G, E226T, and F495I were observed to diminish PET degradation activity, underscoring the critical role of these specific sites in enzymatic function.

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Venturing further, a second experiment juxtaposed the efficiency of the two-enzyme system against PETase in isolation. The combined system's supremacy was evident: PETase and MHETase together demonstrated superior degradation prowess compared to PETase alone.

Delving into the mechanism of this enhanced capability, it was discerned that a two-pronged breakdown occurs. PETase initiates the process, truncating the PET polymer into shorter chains. Following this, MHETase steps in, severing these shorter chains into their basic monomers - terephthalic acid and ethylene glycol.

Drawing parallels between the experiments, several conclusions emerge. Firstly, the synergistic effect of PETase and MHETase is incontrovertibly more potent than PETase's solo effort. The mechanistic revelation of a sequential breakdown further elucidates why the two-enzyme system holds an edge. Secondly, the optimal milieu for this enzymatic dance appears to be a temperature of 37°C coupled with enzyme concentrations of 10 μg/mL. Lastly, the significance of specific residues, notably Ser131, Glutamic Acid, and Phenylalanine, in the enzymatic degradation process cannot be overstated. Disruptions or mutations at these sites invariably curtail the enzymes' activity

In summation, this research is emblematic of the potential harbored by the PETase-MHETase tandem. By achieving efficient degradation under optimized conditions, this enzymatic pair presents a promising avenue for PET waste management, heralding a future with diminished environmental impact and judicious resource utilization.

To Future iGEM Teams

As you embark on the journey of research and innovation in biotechnology, a critical area of focus should be the potential modifications in the enzymes PETase and MHETase, vital for PET degradation. That is why our team has created quite a large part collection this year that we did not have enough time and resources to check. So, we would like to see future igemers using our constructs in their projects.

Our main goal was to clone the studied genes into appropriate vectors for expression in Pseudomonas putida. This is because one of the types of microbes associated with decomposing materials was identified as Pseudomonas bacteria. Plasmids compatible with Pseudomonas putida and containing appropriate regulatory elements for gene expression are usually used for this purpose. There are several dual expression system plasmids that can be compatible with Pseudomonas putida for co-expression of multiple genes. The J435500 vector was used for cloning the PETase and MHETase genes. The expression vector pSEVA-251 was used to express both PETase and MHETase enzymes.

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Figure 1: Map J435500 vector with differentially cloned genes.

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Figure 2. Map of pSEVA-251 dual expression plasmid with CJ-MHETase and PETase genes.

PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalate hydrolase (MNETase) are known to synergistically convert PET into its monomeric building blocks. Our team developed six bacterial expression vectors with PETase and six dual expression plasmids containing PETase and MHETase enzymes for optimal PET degradation using Pseudomonas putida, targeting scalability in PET recycling plants. Due to time constraints, we focused on comparing the mutant form of BBa_J435500_W159H_S238F enzyme with wildtype PETase, using p-nitrophenyl butyrate (pNPB) as a PET analog. Under conditions of 2500 nanomolar pNPB, pH 9.0, and 30°C, the modified PETase's activity exceeded the wildtype's by seven-fold. This highlights the potential of BBa_J435500_W159H_S238F for developing PET recycling strategies. Data from iGEM 2019 in Toronto further confirms our results indicating the superior performance of our construct, highlighting the critical role of enzyme modification in combating the plastic pollution problem. BBa_J435500_W159H_S238F (BBa_K4901036)


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In essence, the landscape of enzyme engineering, especially concerning PET degradation, presents vast opportunities. By focusing on the mentioned mutations and potentially discovering others, you could contribute significantly to creating efficient, biocatalytic solutions for PET recycling. Best of luck with your endeavors, and may your research pave the way for a more sustainable future!


1. Maity, W., Maity, S., Bera, S., & Roy, A. (2021). Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes. Applied Biochemistry and Biotechnology, 193. https://doi.org/10.1007/s12010-021-03562-4

2. Knott, B. C., Erickson, E., Allen, M. D., Gado, J. E., Graham, R., Kearns, F. L., Pardo, I., Topuzlu, E., Anderson, J. J., Austin, H. P., Dominick, G., Johnson, C. W., Rorrer, N. A., Szostkiewicz, C. J., Copié, V., Payne, C. M., Woodcock, H. L., Donohoe, B. S., Beckham, G. T., & McGeehan, J. E. (2020). Characterization and engineering of a two-enzyme system for plastics depolymerization. Proceedings of the National Academy of Sciences of the United States of America, 117(41), 25476-25485.https://doi.org/10.1073/pnas.2006753117