Plastic waste is one of the most pressing environmental challenges globally. Polyethylene terephthalate (PET), widely used in food and drink packaging, accounts for around 18% of global plastic production, roughly 70 million metric tonnes. However, an estimated 85 to 90% of produced PET is either incinerated or sent to landfill, posing significant environmental challenges.

This has caused a growing interest in using enzymes to recycle PET. Among the most promising candidates are cutinases, enzymes that naturally evolved to degrade the waxy cutin layer in plants but are now being repurposed to break down plastic polymers.

Unfortunately, many cutinases operate most efficiently at ambient temperatures, limiting their industrial application. Recent attempts at improving their thermostability can increase structural rigidity, but often at the expense of the flexibility needed for effective substrate binding.

A recent study published in the Open Access journal Crystals provides new structural insight into how one thermostable cutinase (CtCut) balances stability and flexibility. By capturing multiple structural states of the enzyme, the researchers provide new insights into how stability and flexibility can coexist, an essential feature for the bioremediation of plastics.

Prof. Tatsuya Nishino, lead author of the study, highlights the potential impact of the work:

“Our aim was to contribute to the development of practical recycling technologies by clarifying the molecular basis of enzymes that function even under high-temperature conditions.”

Adding more insights into enzyme catalysis

Enzymes are nature’s catalysts, speeding up countless biochemical reactions essential for life. The use of enzymes in industry is highly sought after, as they align with many of the principles of green chemistry, offering more sustainable and energy efficient alternatives to traditional processes. However, the precise mechanism of how they achieve such remarkable efficiency is still not fully understood and remain a matter of active research.

This knowledge is crucial for industry. By understanding how enzymes operate at a molecular level, researchers can engineer variants that perform better under harsh industrial conditions.

For PET degradation, thermal stability is especially important. PET is a semi-crystalline material with both crystalline (ordered, dense) and amorphous (random, disordered) regions. PET-degrading enzymes primarily target the more flexible, amorphous regions which become increasingly accessible near 70 °C.

However, most enzymes have evolved to operate optimally at lower temperatures and lose activity at higher temperatures. This has led researchers to explore thermostable enzymes, which naturally function at higher temperatures and may be better suited for industrial applications.

More than just an active site

Enzymes are large, complex molecules with specialised active sites where reactions occur. These sites create unique chemical environments that help weaken bonds and stabilise reaction intermediates.

However, enzyme activity is not limited to the active site. Structural features elsewhere in the enzyme can also play an important role, influencing both catalytic efficiency and stability.

Understanding how these features work together, particularly how enzymes maintain stability while retaining activity, can help future researchers design biocatalysts. Insights from studies like this could help guide the development of more effective enzymes for plastic recycling and other industrial processes.

Capturing the enzyme structure

To understand how CtCut functions, the researchers used X-ray crystallography to determine its structure in multiple states, including both the inactive (apo) and ligand bound forms.

In the absence of a substrate, the enzyme adopts a configuration with a closed “lid” loop. A key catalytic component, histidine (H204), is also positioned in an inactive conformation in this state.

During substrate binding, changes within the enzyme structure occurred:

• The active site reorganises into a catalytically competent state.
• The flexible “lid” loop opens.
• The catalytic histidine moves into an active position.

Interestingly, the histidine (H204) adopts eight different conformations across the structure, suggesting that the enzyme active site is highly dynamic.

A dynamic catalytic mechanism

These findings support a mechanism where the enzyme switches between inactive and active states during its catalytic cycle.

Rather than being permanently arranged for catalysis, part of the enzyme remains flexible and only adopts the correct configuration when the substrate is present. This flexibility may be particularly important for accommodating a large polymer chain, as found in other PET-degrading enzymes.

At the same time, the overall structure of the enzyme remains highly stable. Differential scanning calorimetry was used to analyse how the enzyme absorbs heat, denaturation of a protein is associated with increased heat absorption. Thermal analysis revealed that CtCut unfolds in two distinct steps, suggesting that flexible regions (such as the lid loop) can move independently from the more rigid core of the enzyme.

Prof. Tatsuya Nishino explains:

“Our findings suggest the possibility of functional division within the enzyme. We observed that the mobile region near the active site undergoes structural changes in response to ligand binding, and that thermal denaturation proceeds in multiple stages.”

Balancing stability and flexibility

This study provides key structural insights into how a thermostable cutinase balances global stability with active site flexibility:

• A rigid core helps the enzyme remain stable at high temperatures.
• Flexible regions near the active site allow substrate binding and catalysis.

These features could make CtCut a potential model for designing improved plastic-degrading enzymes as Prof. Tatsuya Nishino explains:

“Our study may lead to the development of technologies for efficiently decomposing and recycling PET in the future by providing design guidelines for enzymes that possess both heat resistance and potential catalytic capabilities for polymer degradation.”

Bridging the gap to industrial enzyme applications

This study provides important structural insights into how the thermostable enzyme CtCut functions. Notably, the findings agree with recent advances in PET-degrading enzyme engineering which highlights the importance of a flexible active site for accommodating bulky polymer substrates.

The major insight from this work is the combination of a rigid structural core to maintain stability at high temperatures, with a dynamic lid loop that reorganises the active site upon substrate binding. This balance between stability and flexibility is a common feature among efficient PET-degrading enzymes. However, whether these design principles can be broadly applied to structurally diverse enzymes remains unanswered.

Future work should also explore important residues and regions within the enzyme responsible for these properties. Such insights would enable more rational enzyme engineering and could help transfer these features into other enzyme scaffolds.

Overall, this study expands the known structural diversity of cutinases and provides insights that may guide the development of more efficient, thermostable enzymes for plastic recycling.

As our understanding of enzyme function continues to grow, and with promising advances in AI-driven protein design, the widespread use of enzymes in industrial processes may be closer than ever.

More studies on enzyme dynamics and the biological degradation of plastics can be found across the Open Access journals Catalysts and Crystals. Alternatively, you can access the full MDPI journal list here.