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Better milk for cats: immobilised lactase used to make lactose-reduced milk | www.scienceinschool.org

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Dean Madden from the National Centre for Biotechnology Education (NCBE), University of Reading, UK, suggests an experiment to make lactose-free milk - useful both for cats and for the 75% of the world's human population that are intolerant to this type of sugar.

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Class practical

Enzymes are essential catalysts for the chemical processes of life. We exploit the properties of enzymes in research and industry, using them to catalyse chemical reactions or to synthesize products. Sometimes we catalyse a reaction by using entire microscopic organisms (algae, yeast or bacteria) rather than a purified enzyme.

Immobilising enzymes or microscopic organisms involves trapping them in a matrix of an inert material or binding them to its surface. This makes it easier to remove the active catalysts from the reaction mixture, and so makes it easier to purify the products. It also allows us to set up systems for continuous processing, packing the immobilised catalysts in a vessel through which a steady stream of reactants can flow - collecting useful products at the outlet.

This procedure describes how to make alginate beads containing yeast. These are used to catalyse the fermentation of glucose to ethanol. The method can be applied to other catalysts in other reactions, or to more sophisticated investigations of enzyme action. See links below.

Lesson organisation

Students will probably find making alginate beads quite entertaining, at least the first time. Allow each group to make some beads. Set up a flask containing free yeast as a demonstration. Each group could either set up the beads in a conical flask with glucose solution, or pack a column with beads and try to set glucose flowing through suitably slowly.

Apparatus and Chemicals

Clamp stand, 1

Boss and clamp, 1

Syringe barrel, 1

Health & Safety and Technical notes

Avoid skin contact with enzymes in solution and with calcium chloride solution.

Avoid exposure to enzyme as dry powder - by using a fume cupboard if preparing enzyme solutions, or by wiping up spills of enzyme solutions so they do not dry out.

1 Preparing solutions to make alginate beads (See CLEAPSS Recipe card): Dissolve sodium alginate in 100 cm ³ of cold, distilled or deionised water. Warm gently if it does not dissolve readily, or start again with warm water. Stir with a spatula every half hour or so. Leave overnight and stir again in the morning. There are variations in alginate from different suppliers and in different batches. It is worth carrying out a trial run, and adjusting the concentration of alginate if necessary. If the alginate gel is too dense, the substrate cannot enter the bead. Use 4% alginate to mix with equal volumes of yeast suspension. Use 2% alginate to mix 4:1 with enzyme solution.

2 Calcium chloride solution: Dissolve 4 g of calcium chloride-2-water in 200 cm ³ of pure water in a 250 cm ³ beaker. The CLEAPSS Hazcard describes calcium chloride as an IRRITANT, so wear eye protection when making up solution. Once made, this 4% solution is LOW HAZARD. Anhydrous calcium chloride (only 3 g in 200 cm ³) could be used, but take care as it could make the water boil. The calcium ions need to be in excess for this to work well. They form cross-links of calcium alginate in the gel, trapping the enzyme or yeast within the matrix.

3 Enzymes: the CLEAPSS Hazcard suggests that many enzymes are harmful as powders. Solutions at a concentration of 1% and above are an IRRITANT, but below 1% enzyme solutions are LOW HAZARD.

4 If your Pasteur pipette tip produces beads that are too big, use instead a drawn-out glass capillary tube, attached to the syringe with about 2 cm of flexible rubber or plastic tubing.

Procedure

SAFETY: Take care handling the calcium chloride. Do not consume any of the products of a reaction carried out in a laboratory where chemical or biological contamination could occur, or using non-food-grade reagents, or apparatus that has been used for other chemicals or biological materials.

Preparation

a Make up the alginate solution and the calcium chloride solution. (See Notes 1 and 2.)

b As close to the time of use as possible, make up the yeast suspension or enzyme solution with distilled water.

c Set up the apparatus as shown in the diagram to the right. Put calcium chloride solution in the beaker. A Pasteur pipette tip is attached to a syringe supported in a clamp, so that the tip of the pipette is 5 cm above the surface of the calcium chloride.

Investigation

d Mix equal volumes of alginate solution and yeast suspension to give a final concentration of 2% alginate. ( Note 1.)

e Pour the mixture into the syringe barrel and allow it to drip through into the calcium chloride solution as in the diagram above.

f Separate the alginate beads from the calcium chloride solution using a small sieve or tea-strainer and rinse with a little distilled water.

g Use the beads as a source of catalytic enzyme in a batch fermentation process. Alternatively, pack the beads into a column and run the solution of reactants slowly past the beads to demonstrate a continuous reaction process. You can use a syringe barrel or a longer burette to make a column. A useful tip is to place some nylon mesh in the bottom of the syringe barrel; otherwise the alginate beads will block the outlet.

h Collect the products of the reaction.

Teaching notes

You could run this practical in a variety of ways.

Set up batch fermentation of glucose with yeast using free yeast in some vessels and immobilised yeast in others

Quantify the rate of fermentation using a bubble logger as described by NCBE. After a few days, separate the yeast from the reactants and products - by filtration, centrifugation, or simply straining out the beads.

Compare the ease of removal of yeast in each process. Evaluate the process in terms of how long it takes, how clear the final mixture is, and how easy it would be to use the yeast again.

Quantifying the success of the reaction in terms of production: extract the ethanol by distillation to estimate yield, or compare the specific gravity (density) of the resulting solution with that of 8% glucose to estimate yield. Density should change from 1008 g per litre to 1004 g per litre. The hydrometer from a home brewing/ wine-making kit would be most appropriate for measuring this quantity.

Co-entrap brewer's yeast (Saccharomyces cerevisiae) and the enzyme β galactosidase (lactase)

The yeast can ferment sugars such as glucose and sucrose to produce ethanol and carbon dioxide, but it cannot usually ferment lactose. If it is entrapped with lactase, it will be able to ferment the sugars produced by the action of the lactase.

Compare the rate of fermentation of lactose by yeast immobilised in alginate beads with and without lactase. More details of this NCBE procedure.

Immobilise an enzyme to change one chemical into another

Milk contains significant quantities of the carbohydrate lactose, a disaccharide made from linked glucose and galactose. Adult humans and cats often like drinking milk, but many of them do not produce enough active lactase in their digestive systems to process the lactose. Non-digestion or mal-digestion of lactose can cause digestive upsets.

Treating milk with the enzyme lactase removes the lactose and makes the carbohydrates digestible. This can be done by adding lactose to milk or by running the milk through a column containing immobilised lactose and collecting the 'improved' (lactose-reduced) milk at the outlet.

In this case, use 8 cm ³ of 2% sodium alginate solution, mixed with 2 cm ³ of lactase (β-galactosidase) such as Novozymes lactozym® from NCBE. More details of this NCBE procedure.

Health & Safety checked, May 2009

Web links

www.ncbe.reading.ac.uk
The National Centre for Biotechnology Education has a huge number of protocols for practical biotechnology activities and some associated biology practicals. They also supply enzymes and consumables for biotechnology practical work.

There are several useful practicals on fermentation downloadable from the NCBE website at www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/fermentation.html. This booklet includes details of logging bubble rate.

NCBE protocol for immobilised yeast: http://www.eurovolvox.org/Protocols/immobilisedyeast.html

NCBE protocol for immobilised β-galactosidase: http://www.eurovolvox.org/Protocols/catmilk.html

http://www.nationaldairycouncil.org/Pages/Home.aspx
Information about lactose intolerance and the nutritional value of dairy foods.

www.lsbu.ac.uk/biology/enztech/
London South Bank University site with more information about the use of immobilised enzymes in industry. Includes Enzyme technology by Martin Chaplin and Christopher Bucke (Cambridge University Press, 1990). The book is out of print, but the text is available here. 'Methods of immobilization' in Chapter 3 includes diagrams of the different ways an inert matrix can contain or simply bind an enzyme. Chapter 5 includes more technical information about the design of enzyme reactors and examples of enzymes used in industry - particularly the food industry.

(Websites accessed October 2011)

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Working with immobilised enzymes or microscopic organisms

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Class practical Enzymes are essential catalysts for the chemical processes of life. We exploit the properties of enzymes in research and industry, using them to catalyse chemical reactions or to synthesize products. Sometimes we catalyse a reaction by using entire microscopic organisms (algae, yeast or bacteria) rather than a purified enzyme.

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Immobilised Enzymes | A-level Biology | OCR, AQA, EDEXCEL

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https://goo.gl/31T06Y to unlock the full series of AS, A2 & A-level Biology videos created by A* students for the new OCR, AQA and Edexcel specification. In this video we'll look at immobilized enzymes and their importance in industrial processes.

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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.

Fruit juices

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.

Detecting pesticides

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?
Carbon capture
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 (HCO ₃⁻) 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

References

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?

Digestive enzymes can be useful

Iona Twaddell looks at the science of the waterborne disease cholera

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