Image of a tropical beach with palm trees littered with seaweed and plastics. Uwe Bornscheuer is biotechnologist and holds the chair for Biotechnology and Enzyme Catalysis at the University of Greifswald in northeastern Germany. Gert Weber, a structural biologist and biochemist, is affiliated with the Helmholtz-Zentrum-Berlin, Bessy II Synchrotron. They have teamed up to improve the catalytic properties of plastic-degrading enzymes for use in sustainable recycling – iteratively engineering proteins on the basis of molecular structure.

Summary

We’ve long celebrated plastics for their strength and simple manufacture, but their high production rates and uncontrolled disposal have turned them into a global environmental burden. The amount of industrially-produced plastics increases year-on-year, and their production depends on an ever-declining resource – fossil fuels. For us to limit environmental pollution and prepare for the reduction in crude oil, we need to introduce more sustainable (and biodegradable) polymers into the supply chain and stop wasting our existing oil-based plastics – ensuring that they enter a circular and sustainable economy.

Only about 20% of plastics currently in production can be recycled sustainably.

Our current chemical and thermal recycling processes suffer from their high energy costs and the fact that they also rely on crude oil; however, in recent years, the application of enzymes has been recognized as a promising alternative. Admittedly, they don’t yet qualify as industrial recycling processes, but they have nonetheless received significant public attention since the discovery of two naturally-occurring bacterial hydrolases (enzymes) that degrade the polyester polyethylene terephthalate (PET).

To understand these enzymes and how they work, we first need to see their molecular structures while bound to PET fragments. These structures then serve as a basis for iterative biotechnological improvement, where we engineer enhancements to the capabilities of these enzymes.

The current situation

Over the past 100 years, plastics have become an integral part of daily life. Unfortunately, their durability – while convenient for carrying items home from the shops or keeping food items fresh – has turned disposed plastics into a global environmental burden. Their environmental mark is pervasive, ranging from larger, more easily collected debris down to micro- and nano-scale particles in the soil, water, and air (as well as their respective food chains).

As polymers break down, so too does our ability to collect them. While larger pieces can be readily retrieved, the distribution of smaller fragments is harder to control. This fragmentation has led to nigh-irreversible pollution of our oceans and will likely take terrestrial habitats several hundred years from which to recover. It’s critical that we stop the uncontrolled release of plastics into the environment and introduce novel sustainable technologies to replace the current ones.

To cut down on waste, we need to channel crude oil-derived plastics currently in circulation into sustainable, closed materials cycles that don’t rely on crude oil to function. We also need to prioritize the production of biodegradable plastics from renewable sources; however, these alternatives often suffer from insufficient material properties, comparatively high production costs, and (often overlooked) a biodegradability that depends on the environment into which we release them.

Requirements for a sustainable recycling

Unlike conventional plastic recycling methods, recycling sustainably means that the process uses little energy and is separate from crude oil supplies. Over the past 20 years, engineered enzymes have received increased attention for their ability to break down plastics to their initial building blocks. The biotechnological potential of these enzymes depends on the similarity between the synthetic polymer we’re manipulating, and the organic polymer which the enzymes target naturally. As such, it’s important to start with the best possible naturally-occurring foundation.

In the case of plastics, for example, enzymes from fungi and bacteria (cutinases), typically used to hydrolyze the ester bonds of a plant’s cuticula, were engineered to break down polyethylene terephthalate (PET). PET is the most prominent and abundant type of polyester – used in drinking bottles, textiles, and films – and is, therefore, an important plastic to move into sustainable recycling practices. However, these natural enzymes as well as the engineered ones are yet low in activity and depend on elevated temperatures, so aren’t ideal for general use.

Given that PET was only created about 70 years ago, it came as a big surprise to learn that nature has already evolved enzymes that specifically address it. In 2016, Japanese researchers discovered a bacterium, Ideonella sakaiensis, that has two enzymes called PETase and MHETase; these enzymes break down the polyester by cutting off certain chemical (ester) bonds of PET and then promoting bacterial growth on its breakdown products.

How bacterial enzymes could work in PET recycling

The first enzyme, called PETase, converts PET into a molecule called mono-(2-hydroxyethyl) terephthalate (or MHET for short). MHETase, the second key enzyme, then breaks down this MHET even further to produce terephthalic acid and ethylene glycol – the building blocks from which we can then re-synthesise PET.

Since PET has been released into the environment only for the last 70 years, the PETase / MHETase system has undergone a rapid evolutionary process. PETase and MHETase already possess a higher activity at ambient temperature than the aforementioned engineered cutinases, and so provide an ideal foundation for biotechnological enhancement. Unlike previous attempts, these could prove likely candidates to qualify for industrial applications. What’s more, as we will elaborate on below, the amount of biotechnological engineering we need to do is reduced as we already have access to the structure of these ligand-bound enzymes. This is shown for MHETase in our concurrent study. Excitingly, it’s quite possible that other such enzymes exist. By continuing to screen marine and terrestrial habitats, we may uncover suitable enzymes with specificities towards different synthetic polymers.

Few synthetic polymers currently qualify for a sustainable recycling

Plastic polymers have a large spectrum of chemical properties. Given the plethora of synthetic polymers currently in production and the fact that enzymes work best when applied to a plastic type that resembles organic material, it begs the question as to which plastics can even enter enzyme-based recycling loops. Depending on the type of plastic, breaking down these polymers will either produce valuable building blocks for recycling or low-value products. Thus, we have to differentiate between polymers that are suitable for closed-loop recycling, and ‘dispensable’ ones that need thermal or crude oil-dependent chemical recycling processes. As it turns out, only about 20% of plastics currently in production can be recycled sustainably.

About 70% of the synthetic polymers we know – including the most abundant types (polyethylene and polypropylene) – belong to a group with branched hydrocarbon scaffolds. Even if we harnessed enzymes to degrade these plastics, the product of the reaction wouldn’t be suitable for any subsequent re-synthesis.

Similarly, polyvinyl chloride (PVC), which is often used in construction, has a high content of chlorine and differs from all known naturally occurring polymers. It isn’t biodegradable and it even causes problems when recycled by traditional methods. Polystyrene (and expanded polystyrene) has a hydrocarbon fibre backbone with branched-off phenyl moieties. Interestingly, it was shown to be degradable by mealworms, but it’s currently unclear which enzymes are involved in the process and whether any products extracted are suitable for recycling.

The aforementioned polymer types amount to about seven of the ten billion tons of plastics ever made and are unfortunately inaccessible to sustainable closed-loop recycling. Replacing these polymers with biodegradable alternatives in the near future will increase the proportion of sustainable plastics before current polymers are forced to decline with the depletion of crude oil supplies.

The 20% of plastics that are suitable for closed materials loops fall in three main groups: polyesters, polyamides, and polyurethanes. Polyurethanes (such as kitchen sponges or building foam) are, despite their mixed structure and diverse chemistry, biodegradable. In fact, several bacterial and fungal enzymes have been isolated that are found to cleave the polymer’s urethane or ester bonds– a process that releases potentially valuable building blocks. Unfortunately, while the potential is certainly there, these enzymes still require biotechnological optimization to be used on a technical scale.

Polyesters typically have two moieties that are linked via an ester bond. In the case of PET, these moieties are ethylene glycol and terephthalate (TPA), which are currently derived from crude oil, and then purified by vacuum distillation (ethylene glycol) and repeated crystallization (TPA). Once formed, it’s impossible to purify PET any further, so the purity of the two base molecules is crucial. Enzymes such as cutinases, PETase and MHETase could play a key role in securing this.

Other synthetic polymers such as polyamides represent only about 1% of all plastics and resemble the backbone of proteins found in nature. Polyamides (nylon, for example) are biodegradable by enzymes called proteases in a process that, fortunately, yields appropriate building blocks for re-synthesis.

As polyurethanes, polyamides, and polyesters are accessible to enzymatic breakdown, they are promising candidates for a circular plastic economy. Hopefully, this economy will one day parallel that of renewable resources.

Future perspectives of enzymatic plastic recycling exemplified by PET

Unlike the aforementioned examples, PET has significant potential for closed-loop sustainable recycling; it’s synthesized from two building blocks which can both be readily regained from the polymer at low energy costs with the help of enzymes. Theoretically, this technology also applies to compound plastics and alleviates the removal of additives (like colourants, fillers, or plasticizers). But there are still problems with the system, and they need to be overcome before large-scale enzymatic plastic recycling is adopted more widely.

One significant drawback right now is the comparatively low activity of PETase and MHETase, especially towards highly crystalline (or dense) PET. With our structure and bioengineering work on MHETase, we have made an important step towards improving the system in PET recycling. Technically speaking, the improvement is based on an iteration of structural biology and bioengineering.

Why do we need the enzymes’ molecular structures?

Theoretically, we could use known molecular structures to extrapolate the architecture of PETase and, with higher uncertainty, MHETase – a common practice termed homology modelling. However, those modelled – and even experimentally-determined structures – will yield little useful information if said structures don’t contain the plastic ligands or substrates, it remains unclear as to exactly how these enzymes bind to plastics and operate.

We can integrate plastic ligands (typically, snippets of the polymer) in the experiment as non-convertible derivatives of the original compound, or we can mutate the enzyme so that no catalysis (reaction) occurs. In both scenarios, the enzyme remains in a ‘frozen’ state bound to the ligand and can be structurally characterized. Quite logically, only with a ligand-bound MHETase structure will we see which parts of the enzyme position the ligand for catalysis.

From there, bioengineers can target and modify these sections of the molecule, and then assess the mutant enzyme’s activity. As our own work shows, the effects of modification aren’t restricted to just the activity, substrate binding, or substrate release. We were able to alter MHETase to affect a compound (BHET) much closer to the recalcitrant polyester PET that the initial bacterial enzyme couldn’t deal with. Thinking further along this line, MHETase (and also PETase) could now be altered to target other polyesters than PET.

Every step opens up new avenues for further bioengineering. We have already achieved a twofold increase of MHETase activity versus its substrate MHET; continuing to enhance MHETase and PETase, as well as extending their substrate specificities, will soon render this system a viable alternative to conventional PET recycling. The strategies that we and others apply can then be transferred to other plastic polymers with different sets of enzymes, opening up a range of new and sustainable circular materials cycles.

Admittedly, low oil prices currently outcompete – economically – sustainable strategies for plastics recycling. However, as we look to reverse the environmental repercussions of mismanaging plastics in our move toward a low-impact future, our need for sustainable recycling will only continue to grow.


Photo by Dustan Woodhouse on Unsplash

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