Cross-Linked Enzyme Aggregates: Immobilization & Applications

Instructor: Laura Foist

Laura has a Masters of Science in Food Science and Human Nutrition and has taught college Science.

Enzymes are used to speed up reactions, but can often be costly to use. In this lesson we will learn how cross-linked enzyme aggregates are used to optimize the use to enzymes in manufacturing.

Purpose and Restriction of Enzymes

Do you know anyone who is lactose intolerant? They may have been excited to learn about lactose-free milk, allowing the to drink milk without feeling ill. But how is lactose removed from milk without removing other desired products such as glucose? How are omega-3 fatty acids isolated for nutritional supplements?

These and many other applications in and out of the food industry utilize cross-linked enzyme aggregates to control the processes. Let's break down that term.

Enzymes are proteins that speed up (catalyze) reactions. Your body is full of enzymes to break down your food, move your muscles, and control your emotions. Enzymes have been used for millennia to make cheese and beer. But, sometimes it is difficult to control these enzymes.

They easily breakdown or are denatured, and only act within specific pH and temperature ranges. Also, once enzymes have been used, they typically can't be used again because they can't be removed from the product. This adds to the cost of production while adding possibly undesired proteins to the product.

One modern method used to help control enzymes is by immobilizing them. This makes the enzyme more resistant to pH or temperature changes, so the enzyme isn't destroyed. It also allows the enzyme to be held in place and recovered.

There are several methods to immobilize enzymes including binding the enzymes to a surface, trapping them in beads, forming covalent bonds with an insoluble medium, and cross-linkage. Here we'll just focus on cross-linkage.


A cross-linked enzyme aggregate (CLEAs) is when a bunch of the enzyme molecules group together and then form cross links between the enzymes to hold the group together.

If you had a stack of folded shirts, it would be pretty easy to unfold the entire stack. But, let's say that you linked those shirts together, even with just a small string connecting each shirt to each other. While you could probably figure out a way to at least partially unfold the shirts, it is suddenly a lot harder to do so.

In this same method, if we cross-link enzymes together it is a lot harder for them to unfold. Most denaturation of proteins (like enzymes) are simply the proteins unfolding. But, if there are links holding the proteins together, then they won't unfold as easily. This is how the cross linkages help protect the enzymes from denaturation.

Enzymes first group together (aggregate), and then form cross links
Enzymes aggregate then cross link

We must be careful however to not disturb the active site in the enzyme. Let's say that there were specific logos on the shirts you had folded, and you wanted those logos visible even when they were folded and linked together. You would need to put the string in a location that wouldn't hide the logo.

In the same way, the cross-linkages can't be between two amino acids that are involved in the active site of the enzyme. Because if the active site is changed even a little, then the enzyme won't be effective.

Another bonus of cross-linked enzymes is that the enzyme group is now a lot larger, making it easier to recover the enzymes to be used again.


There are many different applications of CLEAs. Ranging from food manufacturing, to oil processing, and detergents. Let's look at how CLEAs are used in making lactose-free milk.

Lactose is a major carbohydrate found in milk, but many people are not able to digest this carbohydrate because they are lacking the proper enzymes. If the enzyme beta-galactosidase is added to milk, it can break down the lactose into glucose and galactose, which the body can easily digest.

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