Immobilised Enzymes

AS · 15 min

Enzymes are expensive to produce and purify, and if they are simply dissolved in a reaction mixture they end up in the product and are thrown away at the end. Industry gets round this by immobilising the enzyme: trapping or fixing it so that it stays in one place while the substrate flows past. The syllabus asks you to investigate the difference in activity between an enzyme immobilised in alginate and the same enzyme free in solution, and to state the advantages of immobilisation. It is a favourite topic for Paper 3 practical questions and for short "state two advantages" questions on Paper 2.

What immobilisation means

Definition

An immobilised enzyme is an enzyme that is fixed to, or trapped within, an inert, insoluble material (a matrix), so that it does not dissolve in the reaction mixture and can be separated from the products and reused.

Enzymes can be immobilised in several ways:

Entrapment in a gel, such as calcium alginate beads. The enzyme molecules are trapped in the mesh of the gel, but small substrate and product molecules can diffuse in and out. This is the method on the syllabus.
Adsorption onto the surface of an insoluble material such as glass beads, clay or charcoal.
Covalent bonding to an insoluble support such as cellulose.
Encapsulation behind a partially permeable membrane.

Making alginate beads

Sodium alginate is a soluble polysaccharide extracted from brown seaweeds. When drops of sodium alginate solution fall into a solution of calcium chloride, the calcium ions form cross-links between the alginate chains, producing insoluble calcium alginate gel. If the alginate is first mixed with an enzyme, the enzyme molecules are trapped inside the gel beads that form.

Immobilising an enzyme in alginate beads

Mix a known volume of enzyme solution (e.g. $2\ \text{cm}^3$ of lactase or sucrase) with an equal volume of sodium alginate solution (about 2–3%). Stir gently until evenly mixed.
Draw the mixture into a syringe (without a needle).
Release the mixture drop by drop into a beaker of calcium chloride solution (about 1.5%), from a constant height. Each drop forms a bead as calcium alginate gel sets on its surface.
Leave the beads to harden in the calcium chloride for about 10–15 minutes.
Strain the beads with a tea strainer and rinse them with distilled water, to remove calcium chloride and any free enzyme on the surface.
Keep the beads in distilled water until used.

The same syringe, the same drop height and the same alginate concentration give beads of similar size, which matters because bead size affects the rate (see the worked example on bead size below).

Using immobilised enzymes in a column

The beads are packed into a column, such as the barrel of a large syringe or a burette with glass wool at the bottom to hold them in. The substrate solution is poured into the top and flows slowly over the beads. Substrate diffuses into the beads, binds to active sites, and products diffuse out and drain from the bottom of the column. The enzyme stays in the column.

Two classic school investigations use this:

Lactase and milk. Lactase hydrolyses lactose (a disaccharide) into glucose and galactose. Milk is passed down a column of lactase beads, and the liquid collected is tested with a glucose test strip (which is specific for glucose). Benedict's solution cannot be used to show the reaction has happened, because lactose is itself a reducing sugar.
Sucrase (invertase) and sucrose. Sucrase hydrolyses sucrose into glucose and fructose. Because sucrose is a non-reducing sugar, a positive Benedict's test or glucose test strip on the liquid collected shows that hydrolysis has taken place.

The flow rate of the substrate through the column controls how long each portion of substrate is in contact with the enzyme. A slower flow rate gives more time for substrate to diffuse into the beads and be converted, so a higher proportion of substrate is converted to product.

Comparing immobilised and free enzymes

When the same amount of enzyme is used immobilised and free in solution, the results usually show:

Feature	Free enzyme in solution	Immobilised enzyme (alginate)
Initial rate at optimum conditions	usually faster	usually slower: substrate must diffuse into the bead, and some active sites inside the bead are hard to reach or are partly blocked by the matrix
Stability at high temperature	denatures at a lower temperature	more stable: the matrix holds the enzyme molecules in shape and limits their vibration, so they denature at higher temperatures
Stability to pH changes	narrow working pH range	wider pH range; the matrix partly shields the enzyme
Separation from product	enzyme mixed with the product; must be removed by purification, or product is contaminated	product flows out of the column free of enzyme
Reuse	lost at the end of each batch	reused many times
Type of process	batch process	can be a continuous process

Key result

Advantages of immobilised enzymes

The enzyme can be reused many times, which saves money (enzymes are expensive to extract and purify).
The product is not contaminated with enzyme, so no costly separation or purification step is needed.
Immobilised enzymes are more stable: they tolerate a wider range of temperature and pH before denaturing, so the process can be run at a higher temperature (faster) and is less affected by small changes in conditions.
The process can run continuously (substrate added at the top, product collected at the bottom), rather than in batches.
Enzymes are easily recovered and the reaction is easily stopped (remove the substrate supply), so the process is easier to control.
Several enzymes can be immobilised together, or in sequence in different columns, for multi-step processes.

The main disadvantages are the extra cost and time of immobilisation, a lower rate than the free enzyme because of slower diffusion to the active sites, and the possibility of enzyme leaking out of the matrix or the column becoming blocked.

Uses

Lactose-free milk: lactase immobilised in columns hydrolyses lactose in milk for people who are lactose intolerant. The product also tastes sweeter because glucose and galactose are sweeter than lactose.
High-fructose syrup: glucose isomerase converts glucose to fructose (which is sweeter) for food manufacture.
Semi-synthetic penicillins: penicillin acylase modifies penicillin to make antibiotics that bacteria have not yet become resistant to.
Biosensors: glucose oxidase immobilised on test strips is used to measure glucose concentration in blood or urine.

Stating advantages (routine, 3 marks)

State three advantages of using immobilised lactase, rather than lactase in solution, to produce lactose-free milk.

Solution

Any three of:

Lactase can be reused, reducing cost;
the milk is not contaminated with lactase, so it does not need to be purified / no enzyme in the product;
the immobilised enzyme is more stable / less easily denatured at higher temperatures or changes in pH;
it allows a continuous process;
easy to separate enzyme from product.

"Cheaper" alone is not enough: say why (reuse, or no purification step).

Temperature stability data (moderate)

Samples of free and immobilised sucrase were kept at different temperatures for 30 minutes, then their activity was measured at $30\ ^\circ\text{C}$ . Activity is given as a percentage of the activity of an unheated sample.

Temperature / °C	30	40	50	60	70
Free enzyme / %	100	95	60	15	0
Immobilised enzyme / %	100	100	90	65	30

(a) Calculate how much more activity, as a percentage of the free enzyme's activity, the immobilised enzyme retains at $50\ ^\circ\text{C}$ . (b) Describe the effect of immobilisation on the stability of the enzyme. (c) Suggest an explanation for the results.

Solution

(a) $\dfrac{90 - 60}{60} \times 100 = 50\%$ more.

(b) At $40\ ^\circ\text{C}$ and above, the immobilised enzyme retains more activity than the free enzyme at every temperature: e.g. at $60\ ^\circ\text{C}$ , 65% compared with 15%. The free enzyme is completely inactive at $70\ ^\circ\text{C}$ , while the immobilised enzyme retains 30%. Immobilisation makes the enzyme more stable at high temperatures / it is denatured at higher temperatures.

(c) The alginate matrix surrounds and holds the enzyme molecules, restricting their vibration (movement), so fewer hydrogen and ionic bonds break; the tertiary structure and active site keep their shape, so fewer enzyme molecules are denatured. (Also accept: the matrix may slow heat transfer to the enzyme.)

Bead size and surface area (moderate)

A student made two batches of alginate beads containing the same total mass of enzyme: batch A with beads $3\ \text{mm}$ in diameter and batch B with beads $6\ \text{mm}$ in diameter.

(a) Calculate the surface area to volume ratio of one bead of each size. (Surface area of a sphere $= 4\pi r^2$ ; volume $= \tfrac{4}{3}\pi r^3$ .) (b) Predict which batch would produce product faster and explain why.

Solution

(a) Bead A: $r = 1.5\ \text{mm}$ . Surface area $= 4\pi(1.5)^2 = 28.3\ \text{mm}^2$ ; volume $= \tfrac{4}{3}\pi(1.5)^3 = 14.1\ \text{mm}^3$ . Ratio $= 28.3 / 14.1 = 2.0 : 1$ (or $2.0\ \text{mm}^{-1}$ ).

Bead B: $r = 3.0\ \text{mm}$ . Surface area $= 4\pi(3.0)^2 = 113.1\ \text{mm}^2$ ; volume $= \tfrac{4}{3}\pi(3.0)^3 = 113.1\ \text{mm}^3$ . Ratio $= 1.0 : 1$ .

(For a sphere, the ratio simplifies to $3/r$ : $3/1.5 = 2.0$ and $3/3.0 = 1.0$ .)

(b) Batch A (small beads). The same total volume of gel has a larger total surface area (twice as large), so substrate can diffuse into the beads faster; the diffusion distance to enzymes at the centre of each bead is shorter (1.5 mm rather than 3.0 mm). More substrate reaches active sites per unit time, so more ES complexes form and products diffuse out faster.

See Surface area to volume ratio for the same idea applied to cells and organisms.

Flow rate through a column (moderate)

Milk was passed through a column of immobilised lactase at different flow rates. The glucose concentration in the milk leaving the column was measured.

Flow rate / cm³ min⁻¹	2	4	6	8	10
Glucose concentration / mmol dm⁻³	28	20	14	10	8

Describe and explain the effect of flow rate on the glucose concentration.

Solution

Description: as flow rate increases, the glucose concentration in the collected milk decreases, from $28$ to $8\ \text{mmol dm}^{-3}$ between 2 and 10 cm³ min⁻¹; the decrease is greatest at low flow rates.

Explanation: at a higher flow rate, each portion of milk spends less time in contact with the beads. Less lactose has time to diffuse into the beads and bind to the active sites of lactase, so fewer enzyme–substrate complexes form and less lactose is hydrolysed into glucose and galactose before the milk leaves the column.

A manufacturer must balance a slow flow rate (more complete hydrolysis) against a faster flow rate (more milk processed per hour).

Exam-hard: planning the comparison (6 marks)

Plan an investigation to compare the activity of sucrase immobilised in alginate beads with the activity of the same quantity of sucrase free in solution.

Solution

Prepare beads: mix e.g. $2\ \text{cm}^3$ sucrase solution with $2\ \text{cm}^3$ sodium alginate; drip into calcium chloride; leave to harden; rinse with distilled water.
Prepare free enzyme: mix $2\ \text{cm}^3$ of the same sucrase solution with $2\ \text{cm}^3$ distilled water (so the amount of enzyme is the same).
Place the beads in one beaker and the free enzyme in another, each with the same volume and concentration of sucrose solution (e.g. $20\ \text{cm}^3$ of 2%), both at the same temperature in a water bath and at the same pH (buffer).
At fixed intervals (e.g. every minute for 10 minutes) remove a small sample from each and test for glucose with a glucose test strip (or carry out a semi-quantitative Benedict's test with fixed volumes and heating time).
Record the glucose concentration (or time until glucose is first detected) and calculate the rate.
Controls: beads made with alginate but no enzyme (or with boiled enzyme) in sucrose solution, to show that glucose is produced by the enzyme and not by the beads or by spontaneous hydrolysis.
Repeat at least three times and calculate means.

An extension compares activity after heating both forms to a range of temperatures, or measures the activity of the same beads used again in several successive batches to show reuse.

Practical skills

Free versus immobilised enzyme: points the practical paper asks about

Why rinse the beads? To remove free enzyme from the bead surface, which would otherwise give a falsely high rate for the immobilised enzyme, and to remove calcium chloride.
Why use glucose test strips with lactase? Lactose is a reducing sugar, so Benedict's solution gives a positive result whether or not lactose has been hydrolysed. Glucose test strips detect glucose specifically.
Variables to standardise: amount (volume and concentration) of enzyme in each form; volume and concentration of substrate; temperature; pH; bead size (same syringe, same height); number of beads; flow rate (for a column).
Sources of error: beads of unequal size; enzyme leaking from beads; colour matching on test strips is subjective (use a colour chart and the same observer, or a colorimeter with Benedict's); sampling at long intervals.
Expected result: the free enzyme usually produces product faster at first; the immobilised enzyme gives enzyme-free product and can be reused, and keeps more activity after heating.

Watch out

Immobilised enzymes are not faster. In most comparisons the free enzyme has a higher initial rate, because substrate must diffuse into the beads. The advantages are reuse, purity of product and stability.
"Immobilised enzymes do not denature" is wrong. They denature at higher temperatures (or more slowly) than free enzymes.
Do not confuse sodium alginate (soluble, mixed with the enzyme) with calcium alginate (insoluble gel formed in calcium chloride).
With lactase, Benedict's solution cannot show that lactose has been hydrolysed. Use glucose test strips.

Exam tip

"State two advantages" questions: give two different advantages (e.g. reuse; product not contaminated). "Cheaper and saves money" counts as one point at most.
For "explain the difference in rate" between free and immobilised enzyme, the key ideas are diffusion into the bead and fewer accessible active sites.
For stability, link to bonds and active site shape, exactly as for denaturation: the matrix holds the enzyme in shape.
In planning answers, make the comparison fair by stating that the same quantity of enzyme is used in both forms, and include a control of beads without enzyme.

Summary

An immobilised enzyme is trapped in or fixed to an inert insoluble matrix, e.g. calcium alginate beads.
Beads are made by dripping enzyme mixed with sodium alginate into calcium chloride; rinse before use.
Beads are packed in a column; substrate flows through; slower flow gives more conversion.
Free enzymes usually work faster at first; immobilised enzymes are more stable to temperature and pH.
Advantages: reuse; product not contaminated with enzyme; greater stability over a wider temperature and pH range; continuous processing; easy separation and control.
Smaller beads have a larger surface area to volume ratio and shorter diffusion distances, so they give a faster rate.
Glucose test strips detect glucose specifically: needed for lactase because lactose is reducing.

Practice questions

Question

Define the term immobilised enzyme.
Describe how an enzyme can be immobilised in alginate beads. (4 marks)
State three advantages of using immobilised enzymes in industry.
Suggest why the initial rate of reaction with immobilised enzyme is usually lower than with the same quantity of free enzyme. (2 marks)
Explain why glucose test strips, rather than Benedict's solution, are used to test milk that has passed through a column of immobilised lactase.
Explain why the beads are rinsed with distilled water before use.
A column of immobilised sucrase converted 85% of the sucrose passing through it when new. After 20 uses, it converted 68%. Calculate the percentage decrease in its activity and suggest two reasons for the decrease.
Calculate the surface area to volume ratio of an alginate bead of diameter $4\ \text{mm}$ , and compare it with a bead of diameter $2\ \text{mm}$ .
A company is choosing between free and immobilised glucose isomerase to convert glucose to fructose at $60\ ^\circ\text{C}$ . Use your knowledge of immobilisation to explain which they should choose. (4 marks)
A student found that a column of lactase beads produced milk with a glucose concentration of $24\ \text{mmol dm}^{-3}$ at a flow rate of $3\ \text{cm}^3\ \text{min}^{-1}$ at $25\ ^\circ\text{C}$ . Predict, with reasons, the effect on glucose concentration of (a) raising the temperature to $40\ ^\circ\text{C}$ and (b) using beads of twice the diameter but the same total mass of enzyme. (5 marks)

Answers

An enzyme that is fixed to, or trapped within, an inert insoluble material (matrix), so that it can be separated from the reaction mixture and reused.
Mix enzyme solution with sodium alginate solution; draw into a syringe; add drop by drop to calcium chloride solution; calcium ions cross-link alginate to form insoluble calcium alginate beads that trap the enzyme; leave to harden, then strain and rinse with distilled water.
Any three: enzyme can be reused; product not contaminated with enzyme / no purification needed; more stable to high temperatures / wider pH range; continuous process possible; easy to separate enzyme from product / easy to control.
Substrate must diffuse into the bead (and product out) before/after reaction, which takes time; some active sites are inside the matrix or blocked by it, so fewer active sites are accessible; fewer ES complexes form per unit time.
Lactose (the substrate) is a reducing sugar, so it gives a positive Benedict's result whether or not it has been hydrolysed; glucose test strips detect only glucose, a product of the reaction.
To remove any enzyme on the surface of the beads (not immobilised), which would otherwise be washed into the product and give a misleadingly high rate; and to remove excess calcium chloride.
Decrease $= \dfrac{85 - 68}{85} \times 100 = 20\%$ . Reasons: some enzyme leaks out of the beads; some enzyme molecules are gradually denatured (e.g. by temperature or pH); beads may be damaged or broken down; beads/column partly blocked by deposits, reducing contact with substrate.
Ratio $= 3/r$ : for $d = 4\ \text{mm}$ , $r = 2\ \text{mm}$ , ratio $= 1.5 : 1$ ; for $d = 2\ \text{mm}$ , $r = 1\ \text{mm}$ , ratio $= 3.0 : 1$ . The smaller bead has twice the ratio. (Check for $d = 4$ : area $= 50.3\ \text{mm}^2$ , volume $= 33.5\ \text{mm}^3$ .)
Immobilised: at $60\ ^\circ\text{C}$ the free enzyme is likely to denature (hydrogen/ionic bonds break, active site changes shape); the immobilised enzyme is more thermostable because the matrix holds the enzyme in shape; it can also be reused and gives a product not contaminated with enzyme; a continuous process is possible; the higher temperature also gives a faster rate (more kinetic energy) without denaturing.
(a) Glucose concentration increases (as long as $40\ ^\circ\text{C}$ is at or below the optimum and the beads protect against denaturation): more kinetic energy, faster diffusion into beads and more frequent collisions with active sites, more ES complexes, more lactose hydrolysed in the same contact time. (b) Glucose concentration decreases: larger beads have a smaller surface area to volume ratio (half) and longer diffusion distances, so lactose reaches fewer active sites in the time available and less is hydrolysed.

What immobilisation means

Making alginate beads

Using immobilised enzymes in a column

Comparing immobilised and free enzymes

Uses

Practice questions

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