DESCRIPTION

Overview

● Discarded PET in the environment has caused serious ecological and health problems at present. Enzymatic recycling of PET has been extensively investigated in the past 20 years. PETase is a novel PET-degrading enzyme with the ability to degrade the highly crystallized PET. However, the heat-labile property seriously hinders its industrial applications.

● To over this limitation, we firstly rationally designed several PETase mutants through our combine bioinformatics strategies. Then we cloned and expressed these mutant genes by using the E. coli system. We next purified all the expressed PETase mutants and tested their thermostability at several conditions.

● We found 7 of PETase mutants with increasing thermostability. Excitingly, the enzyme activity of one mutant was 163 times that of the wild-type PETase at 60°C that was 20°C higher than the optimum temperature of the wild-type PETase.Our project sheds light on the industrial application of PETase.

BACKGROUND

--- why we chose our iGEM project

Plastics are essential materials in our lives due to their desirable properties, such as lightness, durability, low price, easy processibility into many different forms, and non-degradability.^[1] However, non-degradability, which had been considered to be a great advantage of employing plastics, has been reconsidered as a major cause of environmental problems, in particular due to the accumulation of waste plastics in landfills and ocean. ^[2,3,4]

Polyethylene terephthalate (PET) is a thermoplastic polyester (semicrystalline) of high strength, transparency, and safety. These unique properties enable PET to be used in the production of textiles, photographic films, high-strength fibers, bottles, and energy-storage materials. The graph shows PET worldwide demand, the number continually increase from 2010 to 2020, and might reach 42 million tons in 2030, that’s a huge number!^[5]

PET is relatively chemically inert and, therefore, is resistant to microbial degradation, which results in a huge accumulation of post-consumer PET waste in ecosystems across the globe, particularly in the oceans. Growing concerns about the depletion of fossil reserves, environmental pollution, and the need for more efficient and circular carbon economies have highlighted the importance of recycling PET^[6].

Usually, there are three ways for PET degradation: thermal-mechanical, chemical and biological method. ^[7,8,9] But thermal-mechanical degradation includes energy usage with very high demand. And for every industry, it is not feasible to generate a degradation plant with high energy demand and also it may involve the evolution of gases, which are probably poisonous for humans and the environment.

Therefore, we consider bio-degradation, which means using enzyme, it is eco-friendly but most PET degrading enzymes got low activity. Fortunately, an enzyme from a special kind of bacteria (Ideonella sakaiensis), PETase^[10], brings hope.

As we have known, PETase shows high activity relatively on PET under mild condition. However, it is quite thermal-unreliable and would be disabled within 24 hours even under 37 centigrade. If we want to put it into practical use, low thermostability is a big problem. SCIENCE [10] has republished a list of 125 cutting-edge questions, including eight related to ecology. Of these, two issues are closely related to our project, demonstrating the impact of plastic on the ecological environment. PET is a plastic, widely used in clothing, food, housing and other aspects. Our project is to achieve the degradation and recycling of PET, which is consistent with the requirements of The Times, and is also a theme that countless people have been committed to solving but failed to achieve over the years. Our aim is to improve the degradation rate of a novel PET degradation enzyme named PETase. According to the previous studies of PETase, its low degradation rate is largely due to poor thermal stability. Therefore, we tried to improve the thermostability of PETase and finally improve the degradation efficiency of PETase.

Figure4. The difference between Low and High stability of PETase

INSPIRATIONS

---what inspired us this year

Human Pratices give our inspiration
--- the reasons why we choose PETase.

Market research inspired us to choose those PET with large and wide demand. Through factory interviews, we understood the disadvantages of physical and chemical degradation, which inspired us to pick enzymatic degradation. It can ensure that the performance of PET is not lost during degradation. Therefore, we prefer PETase, which shows higher activity on hc-PET under mild condition compared to other PET degrading enzyme.
Articles give us inspiration
--- the reasons why we aimed to improve the thermostability of PETase.

Our aim is to improve the degradation performance of a novel PET degradation enzyme named PETase. According to the previous studies of PETase^[11-16], its low degradation rate is largely due to poor thermal stability. Therefore, we tried to improve the thermostability of PETase and finally improve the degradation of PET.

We were mainly inspired by a paper published in NATURE[16] in April 2020. This paper mentioned another PET depolymerase LCC that can decompose and recycle plastic. They successfully improved the degradation efficiency by improving the thermostability of the protein. Therefore, we also want to improve its degradation ability by improving the thermostability of PETase.

METHODS

--- how we will achieve our goal

Based on literature materials, we learned five methods to improve the thermostability of enzymes, salt bridges, hydrogen bonds, hydrophilic interaction, prolines, and disulfide bonds.^[17]

However, in most cases the positive mutant library may not cooperate to reach the target results owing to the universality of epistatic effects, the robustness of protein structure remains high uncertainty in practical engineering. To solve this problem, we tried to find out unstable regions in PETase by reading literature as well as doing structural analysis and visual screening via bioinformatics tools.

Figure5. Five methods to improve thermostability of enzymes

By performing thousands of rational combinations of mutations^[18] on residues in these unstable regions, we successfully designed a series of mutants whose thermostability was predicted by the software. After operating thousands of rational designs by combinations of residue mutations in these unstable regions, we have obtained many successful mutant models whose thermostabilities are predicted and proved by computational results.^[19]

Then, we tested and verified the thermostability of mutants through experiments.

Firstly, we got the mutant genes by overlap PCR^[20,21], which allows us to generate multiple point mutation genes. And then we built hundreds of genetic circuits containing our mutants. Subsequently, the mutant proteins are expressed in E·coli^[22]. Some of them could be purified successfully while others forming inclusion bodies, which are hard to use as enzymes. We made efforts on optimizing conditions to avoid forming inclusion bodies. We have added different tags in the genetic circuits, including GST, MBP, and SUMO. Meanwhile, we optimized the protein expression conditions, involving the concentration of IPTG, and the inducing time as well as temperature. We have tested more than 40 kinds of conditions in total. So far, 26 mutant proteins have been produced and purified as expected. Then we carried out experiments to check the thermostability of these mutants.

Then we measure the thermostability of enzymes by measuring the concentration of the main reaction product---MHET. We used HPLC (High Performance Liquid Chromatography) to analyze the reaction mixture and got the HPLC chromatogram of products. We can get its Peak Area by Integration, and then we can figure out its MHET concentration. Using this method, we tested our mutants and found 7 mutants with high thermostability. Among them, one mutant has 163 fold MHET product in comparison with wildtype and is still stable at 65°C, 25°C higher than the wildtype.

AIMS & MEANINGS

There is currently no applicable high-efficiency PET degradation method. Our project aimed to improved the thermostability of PETase, which helps to increase the possibility of achieving the biodegradation of highly crystallized PET, contributing to solving environmental and health problems caused by the abuse of PET.

REFERENCE

[1] Wayman C, Niemann H. The fate of plastic in the ocean environment - a minireview. Environ Sci Process Impacts. 2021 Mar 4;23(2):198-212. doi: 10.1039/d0em00446d. PMID: 33475108.

[2]Cressey D. Bottles, bags, ropes and toothbrushes: the struggle to track ocean plastics. Nature. 2016, 536(7616):263.

[3]Zhang X, Fevre M, Jones Gavin O, et al. Catalysis as an enabling science for sustainable polymers. Chemical Reviews. 2017, 118(2).

[4]Hahladakis J N, Velis C A, Weber R, et al. An overview of chemical additives present in plastics: Migration, release, fate and environmental impact during their use, disposal and recycling. Journal of Hazardous Materials. 2018, 344:179-199.

[5]http://www.statista.com/statistics/1128658/polyethylene-terephthalate-demand-worldwide/

[6]Ragaert K, Delva L, Van Geem K. Mechanical and chemical recycling of solid plastic waste. Waste Manag. 2017 Nov;69:24-58.

[7]Cecchini M, Signori F, Pingue P, Bronco S, Ciardelli F, Beltram F. High-resolution poly(ethylene terephthalate) (PET) hot embossing at low temperature: thermal, mechanical, and optical analysis of nanopatterned films. Langmuir. 2008 Nov 4;24(21):12581-6.

[8]Bach C, Dauchy X, Chagnon MC, Etienne S. Chemical compounds and toxicological assessments of drinking water stored in polyethylene terephthalate (PET) bottles: A source of controversy reviewed. Water Res. 2012 Mar 1;46(3):571-83.

[9]Papadopoulou A, Hecht K, Buller R. Enzymatic PET Degradation. Chimia (Aarau). 2019 Sep 18;73(9):743-749.

[10]Yoshida Shosuke, Hiraga Kazumi, Takehana Toshihiko, et al. A bacterium that degrades and assimilates poly(ethylene terephthalate).Science. 2016,351(Mar.11 TN.6278):1196-1199.

[11]Chen CC, Han X, Ko TP, Liu W, Guo RT. Structural studies reveal the molecular mechanism of PETase. FEBS J. 2018 Oct;285(20):3717-3723.

[12]Han X, Liu W, Huang JW, Ma J, Zheng Y, Ko TP, Xu L, Cheng YS, Chen CC, Guo RT. Structural insight into catalytic mechanism of PET hydrolase. Nat Commun. 2017 Dec 13;8(1):2106.

[13]Joo S, Cho IJ, Seo H, Son HF, Sagong HY, Shin TJ, Choi SY, Lee SY, Kim KJ. Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nat Commun. 2018 Jan 26;9(1):382.

[14]Son HF, Joo S, Seo H, Sagong HY, Lee SH, Hong H, Kim KJ. Structural bioinformatics-based protein engineering of thermo-stable PETase from Ideonella sakaiensis. Enzyme Microb Technol. 2020 Nov;141:109656.

[15]Liu C, Shi C, Zhu S, Wei R, Yin CC. Structural and functional characterization of polyethylene terephthalate hydrolase from Ideonella sakaiensis. Biochem Biophys Res Commun. 2019 Jan 1;508(1):289-294.

[16]Tournier V, Topham C.M, Gilles A, et al. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580, 216–219 (2020).

[17]Rigoldi F, Donini S, Redaelli A, Parisini E, Gautieri A. Review: Engineering of thermostable enzymes for industrial applications. APL Bioeng. 2018 Jan 11;2(1):011501.

[18]Hyeoncheol Francis Son, In Jin Cho, Seongjoon Joo, Hogyun Seo, Hye-Young Sagong, So Young Choi, Sang Yup Lee, and Kyung-Jin Kim

ACS Catalysis 2019 9 (4), 3519-3526

[19]Yinglu Cui, Yanchun Chen, Xinyue Liu, Saijun Dong, Yu’e Tian, Yuxin Qiao, Ruchira Mitra, Jing Han, Chunli Li, Xu Han, Weidong Liu, Quan Chen, Wangqing Wei, Xin Wang, Wenbin Du, Shuangyan Tang, Hua Xiang, Haiyan Liu, Yong Liang, Kendall N. Houk, and Bian Wu

ACS Catalysis 2021 11 (3), 1340-1350

[20]Vallejo A N , Pogulis R J , Pease L R . PCR mutagenesis by overlap extension and gene SOE[J]. Csh Protocols, 2008, 2008(3):pdb.prot4861.

[21]Zeng F , Zhang Y , Zhang Z , et al. Multiple-site fragment deletion, insertion and substitution mutagenesis by modified overlap extension PCR[J]. Biotechnology & Biotechnological Equipment, 2016, 31(2):339-348.