5 enzyme properties you can improve with enzyme engineering

You already know the basics: enzymes are proteins with a catalytic function. This means they accelerate chemical reactions, but are not consumed during the process.

Although this definition is clear, it might raise more questions:

• • How fast can an enzyme transform a substrate into a product? Are they all equally fast?
  • Is there a biocatalyst for each substrate or are they not so specific?
  • Can enzymes “choose” among similar substrates?
  • To what extent the medium conditions and composition affect the performance of the biocatalyst?

All these questions reflect on different properties of enzymes that, as you may guess, vary widely among them. And these variations determine, let’s say, how well they “do their job”.

If you are searching for an existing biocatalyst for a very specific reaction –something like transforming a specific substrate into a specific product under specific temperature and pH conditions, for example– you may get lost and even not get anything. That’s why enzyme engineering is so important for the practical applications of biocatalysis: if we can modify enzyme properties, we can adapt every enzyme to every need.

Cool, right? Well, computational chemistry allows to do it even “cooler”.

Molecular modelling lets us design an enzyme atom by atom and predict how changes will affect its function.

Want to know about each enzyme property and how they can be improved? Keep reading:

1. Enzyme activity

Do you know what improving the activity of an enzyme means? It basically consists of increasing a lot the speed at which enzymes transform the substrate into a product.

A reaction that would take years in the absence of a catalyst, occurs in less than a second when it is catalyzed by the appropriate enzyme.

There are different ways of improving enzyme activity:

• • Modifying conditions of the reaction such as temperature, pH, enzyme concentration or substrate concentration.
  • Adding enzyme activators.
  • Modifying the protein structure with enzyme engineering, avoiding the need for those other changes.

Now imagine a company using enzymes in their industrial processes. Like, for example, a textile manufacturer that uses cellulases for fabric treatment.

By increasing enzyme activity, those processes become more efficient, thus improving aspects like resource management or environmental impact.

Why? Because, among other reasons, faster processes require spending less energy and other resources. And, in turn, these savings help to significantly reduce the environmental footprint (fun fact: did you know that “catalysis” is one of the 12 principles of green chemistry?).

2. Enzyme specificity

Did you know that enzyme and substrate structures must perfectly match within the active site of the enzyme for the substrate’s transformation to take place?

But, at the same time, biocatalysts can bind to more than one variety of substrates.

It all depends on its specificity.

• • An enzyme with high specificity can bind to a very limited range of substrates, maybe just one, like β-D-glucose for glucose oxidase.
  • An enzyme with low specificity can bind to a much broader range of substrates, like the Cytochrome P-450 family of enzymes, which are involved in detoxification processes within the cells of all kinds of organisms and have multiple targets.

We can improve an enzyme’s specificity by restricting the variety of substrates it can transform. However, this might not always be the goal.

There are cases where it is preferable that one enzyme transforms different substrates.  In that case, improving the specificity means actually reducing it. Or engineering the biocatalyst to transform new specific substrates apart from the original one(s).

When an enzyme is able to transform different substrates, we say it is promiscuous. In practical terms, having a very specific enzyme means that only one type of reaction can take place, resulting in just one type of product.

For an industrial process, high specificity implies higher quality of the final product, because no other side products are created. As a consequence, downstream waste management and purification also improve.

3. Enzyme selectivity

Special attention here! Specificity and selectivity are not the same.  Normally, enzymes that are able to transform several substrates –  low specificity – do not have the same preference for all of them; they prefer one substrate over another. This is a property known as enzyme selectivity.

It is very interesting when it comes to stereoisomers, which are very similar molecules that only differ in the spatial arrangement of their atoms.  Well, believe it or not, an enzyme can be selective on one stereoisomer over the other!

Enzymes selectivity will precisely determine the product of the reaction they catalyze. In this sense, we can talk about two different types of selectivity:

• • Regioselectivity is the preference for bonding a functional group to one atom over another atom in the substrate.
  • Stereoselectivity is the preference for a stereoisomer over another one.

Prioritizing the production of one stereoisomeric product over another is called asymmetric synthesis. This type of synthesis is essential for fine chemicals production in pharma and agrochemical sectors, because they need very specific compounds with very specific properties that differ among stereoisomers.

4. Enzyme pH & thermal stability

Remember when explaining enzyme activity we mentioned it can be modified by changing temperature or pH, among other reaction conditions?

Well, the truth is you cannot just do whatever you want when playing around with these variables, because enzymes only work at certain ranges. If you exceed the extremes a biocatalyst can tolerate, you can denature it, as it happens with any other protein.

That’s the stability of the enzyme.

Each enzyme has its own stability ranges of temperature and pH within which they perform optimally. You can imagine this can be quite restrictive for practical applications of enzymes in the industry. Working at extreme pH and temperatures is less convenient and safe than doing it at milder conditions. And it also affects energy saving, since working at high temperatures requires more energy.

The most common example of how convenient it is to modify the thermal stability of an enzyme is that of detergent enzymes. If today we are able to wash our clothes with cold water it’s because we found cold-active enzymes in nature, but also obtained and improved them with protein engineering.

But the opposite example occurs as well: some detergents contain enzymes that have been improved to tolerate higher temperatures than they do in nature.

5. Enzyme tolerance to organic solutions and complex matrices

One key thing about enzymes – and proteins in general –  is that their structure determines their function.

If a biocatalyst is inactivated or denatured, it partially or completely loses its activity. That’s why it’s so important that the structure of an enzyme is stable to preserve its function. And this is not easy when our reaction needs to take place within a solvent or matrix that interacts chemically with our enzyme.

This is a problem that mainly happens in organic (carbon-based) solutions and other complex matrices (like detergents), and not that much in water. But many substrates often are not water-soluble and need those other organic solvents.

In order to solve this tradeoff between enzyme stability and substrate availability, we can engineer an enzyme to be more tolerant to non-water solutions.

To sum it up…

Enzymes are capable of accelerating multiple types of reactions under very different conditions. However, it wasn’t until recent advances in enzyme engineering that we started to be able to fully adapt these proteins to our specific needs.

Understanding enzyme properties and how we can fine-tune them for every purpose has opened the door to a new era of biocatalysis; an era where sustainable chemistry and innovation are more accessible than ever before.