Investigating immobilised enzymes
Iona Twaddell finds out what these catalysts are and what they are used for
Enzymes catalyse biological reactions in our body, but they can also be used to catalyse industrial reactions outside the body. These enzymes are often bound to a support (‘immobilised’) and can be used for a wide range of purposes.
What are the advantages of immobilised enzymes?
Using enzymes instead of other molecules in reactions is useful because enzymes catalyse specific reactions and work at much lower temperatures than chemical catalysts.
The molecule that an enzyme acts on is called a substrate. Enzymes can either be mixed freely with the substrate in solution or immobilised to a solid support so they do not mix freely.
There are many advantages of immobilisation, one of which is that the enzymes can be reused – catalysing the same reaction many times. Binding the enzymes to a surface also makes them more stable and less likely to denature (lose their shape). In addition, there will be no enzyme left in the product at the end, so purification is not necessary.
What are the disadvantages of immobilised enzymes?
There are some disadvantages: immobilisation requires extra time, equipment and work; there may be a reduction in reaction rates if enzymes cannot mix freely with the substrate; and immobilised enzymes cannot be used if one of the substrates is insoluble.
How are immobilised enzymes made?
There are several techniques used to immobilise enzymes. Which one is used depends on the enzymes, substrates and products.
One method is adsorption, in which enzymes are attached to a support (eg charcoal, glass beads or clay) through hydrophobic interactions and ionic bonds. Other methods involve different types of bonds and support.
How are immobilised enzymes used in food?
Making lactose-free milk
The enzyme lactase breaks down the sugar lactose, which is found in milk, into the sugars glucose and galactose. Most people produce this enzyme in their bodies, but some people (and most cats) don’t, meaning that they are lactose intolerant. Because they can’t break down lactose, it builds up in their digestive system where bacteria feed on it, causing digestive problems.
Immobilised lactase can be used to produce lactose-free milk: normal milk is poured down a column containing the immobilised lactase enzymes, which break down the lactose. After the milk has passed through this system, it will only contain the products of the reaction (glucose and galactose), so lactose-intolerant people (and cats) can drink it.
Fruits contain pectins, carbohydrates found in the cell wall that hold the plant together. Immobilised pectinase can be used to break down these pectins, loosening the connections between cells. This increases the amount of juice you can get from the fruit, makes the juice runnier and gets rid of the cloudiness that pectins can cause.
High fructose corn syrup
Production of high-fructose corn syrup (HFCS) is the biggest industrial use of immobilised enzymes. The enzyme glucose isomerase is immobilised, and this enzyme converts the glucose in the corn syrup to fructose, which makes it sweeter.
HFCS is used as a sweetener in place of sucrose, especially in the USA where sugar prices are high. It is not widely used in Europe, because sucrose is cheaper there and there are concerns about the health risks of HCFS.
How are immobilised enzymes used in biosensors?
The specificity of enzymes means that they can be used to test for a unique substance, which is exactly what a biosensor does.
Glucose test strips
People with type 1 diabetes lack the hormone insulin, so they have to test their blood sugar levels regularly to ensure they stay within a healthy range. They do this by measuring the amount of glucose in their blood with a glucose test strip. On the test strip is the immobilised enzyme glucose oxidase; when glucose is present, the enzyme catalyses a reaction that changes glucose into hydrogen peroxide and gluconic acid.
There is also another mediator molecule on the test strip, which catalyses a reaction involving the products of the enzyme reaction. In the early test strips, this second reaction caused a colour change, with the colour indicating the amount of glucose present. In most modern tests, this second reaction produces electrical current, which can be measured by a meter to give the exact concentration of glucose in the blood.
Some pesticides and other toxins are harmful because they inhibit the enzyme acetylcholinesterase (AChE). AChE breaks down the neurotransmitter acetylcholine, which is a very important brain chemical. Without AChE, acetylcholine builds up in the nervous system, interfering with neural signals and causing muscle, breathing and heart problems.
Biosensors for these AChE-inhibiting toxins contain immobilised AChE and its substrate (acetylcholine). The AChE breaks down the acetylcholine and, through another reaction, this produces an electrical current. When a toxin that inhibits AChE is present, this current is reduced or absent. The biosensor detects this reduction in current and therefore the presence of the toxin.
How else are immobilised enzymes used?
Increasing levels of carbon dioxide in the atmosphere is a pressing environmental problem. A large proportion of carbon dioxide emissions come from power plants. Many scientists are working on techniques to capture this carbon dioxide and either store it underground or convert it into something useful.
The enzyme carbonic anhydrase, which is found in the human respiratory system, may be the answer. Carbonic anhydrase catalyses the breakdown of carbon dioxide into bicarbonate (HCO3-) and protons (H+). If the enzyme is immobilised within the power plant chimney, it will extract the carbon dioxide from the smoke and turn it into bicarbonate. Consequently, the smoke that leaves the power plant will not contain any carbon dioxide, and the bicarbonate can be transformed back into carbon dioxide elsewhere and compressed for storage or used elsewhere (e.g. in fire extinguishers).
Carbonic anhydrase denatures at high temperatures, but researchers are working to modify the enzyme so it can work in the heat of the power plant.Lead image:
Anthea Sieveking/Wellcome Images CC BY NC ND
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Questions for discussion
- Can you find any more advantages and disadvantages of immobilised enzymes?
- What other immobilisation techniques are there? What are the advantages and disadvantages of the different methods of enzyme immobilisation?
- What health risks are associated with high-fructose corn syrup?
About this resource
This resource was first published in ‘Proteins’ in January 2014.
- Cell biology, History, Health, infection and disease, Biotechnology and engineering
- Education levels:
- 16–19, Continuing professional development
I did an experiment in school last week investigating the effect of temperature on both free and immobilized lipase. We used temperatures of 40 and 80 degrees C. At 40, both were active but at 80 only the immobilized lipase was active. However, its activity was greatly reduced to that at 40. I understand that the immobilisation process makes the enzymes more stable but why is this and why, if it is more stable, is the activity reduced at all at a higher temperature? In other words, what is the explanation for the decrease in activity in immobilised lipase between 40 and 80 degrees C when with free lipase the enzyme is simply denatured and activity is completely halted?
Any ideas would be greatly appreciated!! Thanks
The immobilisation process makes the enzyme MORE stable, not completely stable. So, the activity of the immobilized enzyme will be greater than that of the free enzyme. Basically, just because its more stable it does not mean that it wont have reduced activity at certain temperatures.
The alginate beads (I presume) stabilise the bonds within the tertiary structure of the immobilised lipase, thus makes them more stable. However, SOME of the immobilised lipase will still denature, and so there is less activity at 80 degrees. The lipase which does not denature is still active and so can still hydrolyse whatever substrate it may be, so there is still some activity.