Development of biocatalysts for plastic degradation

Abstract

It is impossible to conceive our modern world without plastics. Since the mass production of plastics began, humanity has produced around 8 billion tons of plastic waste, and only 9% has been recycled. Terephthalic acid polymers such as poly(ethylene terephthalate) (PET) have been massively produced over the last years thanks to their characteristics and applications. Due to their limited biodegradability, they have accumulated in landfills and oceans, posing an environmental threat. In 2016, a PET hydrolase (PETase) from Ideonella sakaiensis was identified and suggested to degrade PET into terephthalic acid (TPA), mono-2-hydroxyethyl terephthalate (MHET) and ethylene glycol. 

The results reported here aim to contribute to the development of engineered biocatalysts that degrade terephthalic acid polyesters. An assessment of the recombinant PETase and Cut5b lipase ability to degrade not only PET but also other terephthalic acid polymers into TPA is reported. Based on experimental data regarding enzymatic degradation and computational analysis, I give the first insight into the recombinant PETase and Cut5b interactions with various polyesters. Also reported here, a recombinant PETase with a fusion domain had better production yield and augmented enzymatic activity on PET than the wild type. The experimental data and computational analysis confirm the attachment of the fusion domain as a solubility enhancer of recombinant PETase. The molecular structure of Cut1b was simulated, and molecular dynamics analysis confirmed the experimental results suggesting that the fusion domain does not interact with PETase’s active site. Observations showed a corresponding catalytic triad, π-π stacking interactions in subsite I, and hydrophobic interactions in subsite II. Finally, the phylogenetic analysis confirmed the conserved sequence of Cut5b when compared to other PET hydrolases and its evolutionary relationship to I. sakaiensis.

Popular Abstract

It is impossible to conceive our modern world without plastics. They shape everything: the way our food is processed, how our technology is made, medicine, transportation, clothing, furniture, entertainment, and communications [1]. Plastics save millions of lives every year through pacemakers, car seat belts, defibrillators, plastic hearing aids, and disposable plastic syringes. Plastics have even reached the space [2], as they are used every day in the International Space Station, they are in the flag in the Moon and the lander InSight in Mars.

Global plastics production reached approximately 360 million tonnes in 2018 [3]. The PET bottle became the most popular form of packaging in 1975 when The Coca Cola Company and Pepsi Company decided to change their glass bottles to plastic bottling. It was not until the late 80s that we realized plastics are creating an environmental catastrophe [4-6], since the estimated time in which a PET bottle naturally degrades is 450 years. In the late 1980s, recycling was subject to economic and feasibility studies [4], and 40 years later, we know that PET bottles can be recycled to make T-shirts and sneakers. It takes 88% less energy to recycle plastics than to produce new ones from raw materials. Nevertheless, globally only 20% of plastics are recycled from waste [3].

It was thought that no living organism could degrade plastic, until very recently in 2016, a PET-degrading bacteria was found that could use it as a carbon and energy source. This bacteria, Ideonella sakaiensis, has enzymes (a type of protein) that help break down PET [7]. With the advances in biotechnology and protein engineering, biocatalysts like enzymes are continuously developed to reach optimal biodegradation activity for industrial scale-up.

In this thesis, experiments with the PET-degrading enzyme (called PETase because it degrades PET) from I. sakaiensis resulted in the fact that PETase could degrade not only PET but also other plastics that are similar to it, although not as efficiently. Also, a new way to increase the production of PETase in the lab was found by attaching small domain to PETase, increasing its solubility. This way, both PETase production and activity increased. Finally, a recently found enzyme with the potential to degrade plastic had been tested with PET but not with other plastics, so it was produced and experimented with the same plastics aforementioned as PETase. The results were compared, resulting in the degradation of PET with less efficiency than PETase but higher activity with the other two plastics.

This master thesis provides an insight into the development of biocatalysts for plastic degradation in an attempt to extend the knowledge and provide a basis for future research.

• author: Wagner Egea, Paula
• supervisor: Javier Linares-Pastén; Baozhong Zhang
• organization: Biotechnology (MSc); Biotechnology (M.Sc.Eng.)
• course: KBTM01 20201
• year: 2020
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences; Technology and Engineering
• keywords: Biotechnology, Bioteknik
• language: English
• id: 9024668
• date added to LUP: 2020-07-31 14:07:26
• date last changed: 2020-07-31 14:07:26