The enzyme that cracks nature’s toughest materials

Every year, vast quantities of biological resources – from forestry, agriculture and marine systems – remain underused. One way of converting these materials into fuels, chemicals and other useful products relies on enzymes, but the processes are often slow and costly. Even small improvements in efficiency can have a major economic impact.

At first, it looked like a protein most researchers would classify and move on from – small, almost featureless, binding to chitin in crustacean shells, fungi and insects. Useful, perhaps. But not revolutionary.

When Eijsink’s group added it to chitin-degrading enzymes, something unexpected happened.

“The reaction accelerated. Dramatically. Materials that had resisted breakdown began to give way. But the real surprise was not just that it worked better, but how,” explains Professor Vincent Eijsink from the Norwegian University of Life Sciences.

These proteins help unlock value in materials we struggle to use – from wood and crop residues to shrimp shells. What seemed like a minor helper turned out to be part of a broader strategy nature uses to pry open resistant materials.

He fell for the shape of proteins

Before Vincent Eijsink became associated with enzymes that crack open some of nature’s toughest materials, he was fascinated by the shape of proteins – a fundamental feature of biology.

He grew up in the Netherlands and studied molecular sciences in Wageningen just as biotechnology was beginning to take shape. For the first time, researchers were not only describing biological molecules – they were starting to change them.

“I was completely fascinated by that,” Eijsink says.

What captivated him was that proteins do chemistry by folding into three-dimensional structures. “A protein is just a chain of amino acids,” he says, “yet somehow that chain folds into a structure that can carry out remarkable reactions.”

He moved to the University of Groningen for a PhD programme in protein engineering in the mid 1980s, when even changing a single amino acid was a major undertaking. The required reagents were expensive, and computer tools were only just emerging.

“Every mutant was a celebration,” Eijsink says.

“It was fascinating – but also very new. We did not always know what we were doing – we were exploring.”

Wood chips are made of plant fibers built of tightly packed cellulose, one of nature’s most abundant and stubborn materials. Breaking down this structure is a key challenge in producing biofuels and other sustainable products.

Understanding came first – and changed what he noticed

Eijsink’s PhD research focused on a practical problem: how to make enzymes more stable so they keep working under industrial conditions. Step by step, a growing understanding of protein stability led to an article entitled “Engineering an enzyme to resist boiling” – an early career highlight.

His PhD work also shaped how he thought about biotechnology.

“If we want to do sensible applied research – if we want to contribute to solutions in society – it all starts with understanding,” he says. Not just what an enzyme does, but why it works at all – what makes a structure stable or fragile?

That habit stayed with him – linking detailed understanding to practical use.

“It has been fantastic to look at a protein structure in the morning and a reactor in the afternoon,” he says. “For us, the two belong together.”

Across various projects, the balance between fundamental insight and application has remained central.

“The underlying idea has remained the same: understanding and use move together.”

Over time, that approach sharpened into something more specific: a way of looking at proteins that others might ignore.

This instinct – to take a second look at what others passed over – would turn out to matter. One of the proteins that would change his career had been overlooked for years.

The protein that did not seem to matter

In the 1990s, a visit from a Norwegian researcher to Groningen changed both Vincent Eijsink’s research direction – and his life.

“There was a Norwegian guest researcher – a female Norwegian guest researcher – coming to the lab, who caught my interest, if I may phrase it like that,” he says. “And, well, the end of the story is that we are still together.”

Scientifically, the visit centred on expressing chitinases in lactic acid bacteria to give the bacteria antifungal properties. But it also led Eijsink to Norway, where his research gradually shifted from individual proteins to how biology breaks down tough materials.

One of those materials is chitin. Like cellulose in plants, chitin is an abundant, rigid and hard-to-degrade polysaccharide. It forms fungal cell walls, insect exoskeletons and crustacean shells – structures built to last.

The textbook explanation for its degradation seemed simple. Enzymes called chitinases cut these polymers by hydrolysis, adding water to break bonds. It was a reasonable model – but was it complete?

In reality, these materials are tightly packed and often crystalline. Breaking them down is energetically difficult.

“For years, people assumed hydrolytic enzymes were the main tools,” Eijsink says. “But that did not quite fit with what we saw in nature.”

If hydrolytic enzymes struggled to attack these materials, how could microorganisms break them down so efficiently? Hints began to appear. Some systems worked better than expected. Certain proteins kept showing up – binding to chitin but not clearly breaking it down.

These proteins were easy to overlook. Classified as helpers. Not enzymes. Useful, perhaps – but not central.

A breakthrough that changed the field

CBP21, short for Chitin-Binding Protein (21 kDa), had been seen before. Other researchers had looked at it and moved on.

It showed no clear activity and did not fit the hydrolytic model for chitin degradation.

“In the beginning, we were just curious,” Eijsink says.

In the early 2000s, a PhD student in his group, Gustav Vaaje-Kolstad, wanted to take a closer look.

“When Gustav said he wanted to work on this protein, I said: are you sure?” Eijsink says. “I did not think it would help us break down chitin.”

But the protein kept showing up in systems that degraded chitin.

“Fact is, it was consistently produced by bacteria growing on chitin,” he says. “If nature keeps something, it is there for a reason.”

So the work on CBP21 continued and then there was a surprise.

“When CBP21 was added to chitinases, chitin degradation improved.”

This suggested that the protein was doing something – not cutting the material itself but changing how other enzymes could act on it.

“We did not expect this,” Eijsink says, “and we did not yet fully understand the impact of what we were seeing. The referees were more enthusiastic than we were.”

At first, the result still fit the existing model. Maybe the protein helped to loosen the structure – many people were looking for such “loosening” proteins at the time. A reasonable explanation – but not a complete one. Because the observations did not fully fit. Something was off. Gradually, a different picture began to emerge.

“The protein was not just binding to chitin. It was changing it. Something else was at work.”

Gustav Vaaje-Kolstad and Vincent Eijsink.

The experiment that revealed a different chemistry

The breakthrough came when Eijsink and his team stopped assuming that they understood the reaction and began testing conditions.

“We started throwing metals at the reaction,” Eijsink says. “Then we added vitamin C and suddenly realised that we were feeding the system with electrons. The control experiment turned out to be the breakthrough.”

That was the moment everything shifted. The reaction depended on electrons – bonds were being cleaved not by water but by oxidation.

“We realised that we were looking at something completely different,” he says. “Not a binding protein – but an enzyme doing chemistry no one had seen before.”

Chitin fibres (shown as long chains of circles) are tightly packed and difficult for enzymes to break down. Traditional enzymes (blue shapes, such as ChiA and ChiB) can only cut the chains where they are already exposed, gradually releasing small sugar units. LPMOs (shown as the triangle in the centre) work differently. They attack the surface of the fibre directly, breaking open the tightly packed structure. This creates new entry points that allow other enzymes to access the chains more easily and speed up the overall breakdown. Source: The chitinolytic machinery of Serratia marcescens--a model system for enzymatic degradation of recalcitrant polysaccharides. 

A protein that changed the paradigm

Further experiments confirmed this. Under the right conditions, the protein could even break down chitin on its own. The role of CBP21 snapped into focus. Rather than simply assisting hydrolysis, it actively promoted the process.

“It attacks the surface of the material and creates openings – entry points that other enzymes can use. What we thought was just a helper turned out to be essential.”

In 2010, the team published the discovery in Science. A new class of enzymes was defined: LPMOs, for Lytic Polysaccharide Monooxygenases.

Around the same time, similar observations were emerging in systems that break down cellulose. Proteins with no clear hydrolytic function appeared to boost degradation in the same unexpected way.

What first had been observed for chitin turned out not to be an isolated case. In 2011, several groups described oxidative cleavage of cellulose.

For decades, the breakdown of materials like chitin and cellulose had been understood as purely hydrolytic. That picture was no longer complete. An oxidative chemistry had been there all along.

The discovery forced scientists to rethink biomass conversion. LPMOs use oxidation to attack the surface of tightly packed, crystalline materials – regions that classical enzymes cannot reach.

The process turned out to be a two-step strategy: oxidation opens the structure, and hydrolysis finishes the job.

“People needed to start rethinking their strategies for biomass conversion,” Eijsink says.

The enzyme that turned up everywhere

In the years that followed, the evidence began to converge.

LPMOs were found across biology – encoded in bacteria and fungi that break down plant biomass and chitin-rich materials. In some organisms, dozens of LPMO genes pointed to a more complex picture: different enzymes likely targeting different parts of these composite materials.

“It took some time before people realised how important these enzymes are,” Eijsink adds.

As more data came in, the picture became clearer. These enzymes had a distinctive copper-based active site, and their reactions left oxidised products. Independent lines of evidence pointed to the same conclusion.

What had seemed like an exception began to look like a rule.

“Everything is new about these enzymes – the mechanism, the active site, the reaction – and they are industrially important,” he says.

For decades, researchers had suspected that hydrolytic enzymes alone could not explain how materials like cellulose were broken down in nature. As early as the 1950s, a famous cellulase researcher, Elwyn Reese, had proposed a missing component – a “C1 factor” – that made the material accessible.

No one had found it. LPMOs turned out to be exactly that.

“The C1 factor had finally been found,” Eijsink says.

The field began to reorganise around that idea.

From discovery to industry

Proteins once classified as inactive were reassigned as enzymes, and researchers began looking not just for hydrolytic enzymes but also for their oxidative partners. The real impact came in industry, in which large quantities of plant biomass must be processed efficiently.

“Breaking down plant biomass at industrial scale is difficult because its structure is tightly packed and resistant. Classical enzymes can break down accessible regions, but they struggle with the most crystalline parts – driving up costs and limiting efficiency.”

LPMOs changed this. By attacking these regions oxidatively, they made the material more accessible to the enzymes already used in industry.

Over the past two decades, developments like these have helped make biomass conversion more efficient, bringing processes such as biofuel production closer to industrial viability.

The oxygen surprise

But the story did not end there. If LPMOs had changed how biomass was broken down, a new question emerged: what actually powered them?

“In 2010 we were wrong, as was everyone else in those early years,” Eijsink says. “Our data indicated that LPMOs use molecular oxygen; in fact, we did quite advanced experiments that seemed to show that, like other monooxygenases, LPMOs use oxygen.”

But something did not fit.

“The enzyme system did not behave as expected; we could not understand the kinetics,” he says.

The results were inconsistent. The reaction rates were lower than expected.

One detail had been overlooked: small amounts of hydrogen peroxide were forming in the background – and driving the reaction.

Careful experiments, mostly done by a talented post-doc called Bastien Bissaro, gave radical new insight: LPMOs do not use oxygen directly; they use hydrogen peroxide. In standard reactions, hydrogen peroxide forms and drives the reaction – but it can also be added directly.

A correction that changed the model

The discovery of hydrogen peroxide’s role explained why the reactions behaved unexpectedly and why the earlier data did not make sense.

“We believe that these are not monooxygenases at all,” Eijsink says. “They are peroxygenases. The LPMO name stuck – but the chemistry is different.”

The idea met resistance. The oxygen-based model was well established, and replacing it meant reinterpreting years of data.

Hydrogen peroxide also posed a problem. It is reactive and unstable – and can damage enzymes if not carefully controlled.

Atomic force microscopy images show cellulose fibres before and after treatment with an LPMO. The tightly packed structure (a) becomes progressively disrupted (b, c), as the enzyme opens up the surface and makes the material more accessible. Quantification (d) shows a reduction in fibre width over time. Real-time imaging reveals that lytic polysaccharide monooxygenase promotes cellulase activity by increasing cellulose accessibility. Source: Real-time imaging reveals that lytic polysaccharide monooxygenase promotes cellulase activity by increasing cellulose accessibility.

Why hydrogen peroxide changed the story

The shift from oxygen to hydrogen peroxide did more than correct the chemistry. If hydrogen peroxide drives the reaction, controlling it becomes key. In industrial settings, that mattered immediately: controlled dosing increased reaction rates and improved yields.

“There was quite a bit of debate,” Eijsink says. “But once people saw the data – and especially what happens when you add hydrogen peroxide – it became hard to argue against.”

As more groups reproduced the results, the new model took hold. It did not replace the old one entirely – it refined it.

“At the same time, multiple studies by several research groups showed that LPMOs use very powerful chemistry.”

These findings began to reshape both research and application.

Not a miracle boost – but still a big deal

LPMOs do not break down complex biomass on their own. But combined with existing enzymes, they improve the process. Even modest efficiency gains can have significant economic impact.

“They are important tools for process efficiency,” Eijsink says. “Even improvements of a few percent can have a huge economic impact.”

Adding LPMOs reduced the amount of enzyme needed and increased yields. In some cases, enzyme use dropped by half, while sugar release rose by as much as 60%.

“It may seem disappointing that it is not a factor of two or more,” he says. “It is tens of percent – but that still matters a lot. And we may not have seen the best of LPMOs yet.”

Powerful enzymes with a built-in weakness

The same chemistry that makes LPMOs effective also makes them fragile.

“These are very powerful enzymes,” Eijsink says. “But that also makes them difficult to control.”

Under the wrong conditions, LPMOs damage themselves.

If you put an LPMO in the wrong environment, it will just damage itself,” he says. “And we think that is happening in quite a few bioreactors around the globe.”

This creates a practical paradox: the enzymes that improve biomass conversion are also among the hardest to manage. Too little hydrogen peroxide, and the reaction slows. Too much, and the enzyme breaks down.

“You need to tightly control the conditions,” Eijsink says.

Hard to control in the real world

In practice, that control is difficult. When acting on biomass, LPMOs operate in a messy system of reactive molecules.

“This is a very messy system,” Eijsink says. “There are electrons and reactive oxygen species moving around everywhere, and you have to control these.”

One major factor is lignin, which participates in reactions that both generate and break down hydrogen peroxide, interfering with the process in multiple ways.

What works in defined laboratory conditions is much harder to control in real industrial systems.

“That is why performance is hard to predict – and even harder to optimise. Nature has had millions of years to get that balance right. Industrial biotechnologists are still learning.”

Vincent Eijsink

From biomass to a broader principle

This chemistry is challenging – but it also opens possibilities beyond biomass. LPMOs may be useful wherever surfaces need to be disrupted or modified, rather than simply cut.

“By modifying surfaces rather than just cutting chains, these enzymes can change the properties of materials like cellulose and chitin. That opens up possibilities in areas like material science and waste utilisation beyond just breaking things down.”

Within a decade, the discovery had reshaped both research and industrial practice.

“The question now is how far this principle can be pushed,” Eijsink says. “How far that chemistry can be extended – and where its limits lie. We would love to make LPMOs catalyse other difficult reactions, such as the selective oxidation of small molecules that are important in the chemical industry.”

What scientists still do not know

A durable contribution does not close a subject – it opens it. As the field expanded, new questions emerged.

Why do some microbes produce so many different LPMOs? Plant cell walls are complex composites of different polymers, and different LPMOs may have evolved to target different parts of this structure – not only cellulose. Which, then, are the best LPMOs to use when processing plant biomass?

The questions are both practical and fundamental: which structures do LPMOs act on? Under what conditions do these enzymes work best? How is their activity controlled in living systems – and how do they avoid damaging themselves?

“Too little, and the reaction slows. Too much, and the enzyme destroys itself. Understanding how that balance is maintained – in nature and in industrial systems – remains an ongoing challenge.”

Beyond biomass: an unknown biological role

The questions extend beyond biomass.

“For some microbial pathogens, these enzymes are absolutely crucial,” Eijsink says.

“If you remove them, the organism becomes much less effective at causing disease.”

LPMOs have now been identified in pathogenic organisms, including bacteria, fungi and oomycetes.

“In certain cases, targeting the LPMOs of these microbial pathogens can dramatically reduce virulence,” he says. “That makes LPMOs very interesting from a medical perspective.”

What they are doing in these systems is still unclear.

“The fascinating thing is that we still do not really know what they are doing there,” Eijsink says.

What began as a correction to an existing model for degrading robust polysaccharides has become something larger: a way of understanding how nature uses controlled redox chemistry to solve difficult problems.

What this changed in biology

At first glance, the discovery of LPMOs might seem like a technical refinement – an additional enzyme and a clearer mechanism.

But the shift was broader: how nature deploys very powerful redox chemistry to its advantage.

“LPMOs show how such chemistry can be used in a controlled and targeted way to attack highly resistant materials,” Eijsink says.

Not uncontrolled, but directed: confined to specific sites and tightly regulated.

LPMOs are striking in their structural simplicity: small enzymes that require only a single copper ion to catalyse a difficult reaction. By contrast, other enzymes that perform similarly demanding chemistry are often more complex and harder to use in practice.

“Other natural enzymes catalysing equally challenging reactions, such as the controlled oxidation of methane to methanol, tend to be much more complex and hard to produce,” Eijsink notes.

For polysaccharide degradation, the key new insight is the integration of fundamentally different types of chemistry.

What once seemed like a minor addition turned out to be a missing piece.

What this makes possible

This shift in perspective suggests that proteins dismissed as inactive may have roles we do not yet understand – and that redox chemistry may be present where we have not looked for it.

“Sometimes, we are not just learning from nature,” Eijsink says. “We are trying to teach nature how to do something new.”

One ambition is to extend this chemistry to harder synthetic materials, including plastics – using enzymes designed to attack surfaces that biology did not evolve to handle.

“I am not driven by grand visions,” Eijsink says. “I try to do good science – and follow where it leads.”

“I have always been fascinated by enzymes,” he says. “They can do complicated chemistry, very precisely, at 37 degrees, in water – that is just remarkable.”

Today, LPMOs are standard components of industrial enzyme systems used in converting biomass. But their behaviour is still not fully understood.

Researchers continue to explore how their activity is regulated, how they interact with other enzymes and how they can be stabilised in complex, real-world conditions.

The protein that once seemed insignificant has become something else: not an exception but a clue.

“Sometimes, the way forward is not to refine the existing model – but to recognise that something fundamentally different has been there all along.”