The big idea: To study an enzyme you change one factor, keep everything else the same, and measure how fast the reaction goes.
The factor you deliberately change is the independent variable (for example temperature, pH or substrate concentration).
What you measure — usually the rate of reaction — is the dependent variable.
Every other factor that could affect the rate must be held constant; these are the controlled variables. Without them the comparison is not fair.
- Independent variable
- The one factor you deliberately change (e.g. temperature, pH, or substrate concentration).
- Dependent variable
- What you measure to see the effect — for an enzyme experiment this is usually the rate of reaction.
- Controlled variable
- A factor kept constant in every run so it does not affect the result and the comparison stays fair.
- Rate of reaction
- How fast substrate is used up or product is made — for example the amount of product formed per minute.
- Fair test
- An experiment in which only the independent variable changes, so any change in the result is due to that one factor.
How do you actually measure the rate?: You can't see an enzyme working, so you measure something that changes as the reaction happens, then time it.
Common choices: the volume of gas released (e.g. oxygen bubbles), the colour change of an indicator, the time for a substrate to disappear, or the amount of product formed.
Whatever you choose, it must reliably reflect how much the enzyme has done — this is the idea of a valid measure (proxy) of enzyme activity.
Why controls matter most: If you compare two runs at different temperatures and at different pH, you can't tell which factor caused the change.
Controlling every other variable is what makes the result trustworthy — this is the single most common thing examiners ask you to discuss in a data question.
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In industry the enzyme is often not left floating in the mixture. Instead it is immobilized — attached to, or trapped on, a solid support such as tiny gel beads.
The substrate flows past the fixed enzyme, the reaction happens, and the product flows on — but the enzyme stays behind.
- Immobilized enzyme
- An enzyme attached to or trapped on a solid support (for example gel beads) so it stays in one place instead of mixing freely with the substrate.
- Free enzyme
- An enzyme dissolved and mixed freely in solution with its substrate.
- Solid support
- The material the enzyme is fixed to or trapped in — commonly small beads of gel through which the substrate flows.
Why immobilize an enzyme?: Immobilizing an enzyme gives several practical advantages:
Reuse — the enzyme stays put, so it can be used over and over instead of thrown away (cheaper).
Pure product — the enzyme doesn't end up mixed into the product, so the product is not contaminated.
Greater stability — the support helps the enzyme tolerate a wider range of temperature and pH before it denatures.
Easy to stop and control — you can simply remove the beads to halt the reaction.
| Feature | Free enzyme (in solution) | Immobilized enzyme (fixed to a surface) |
|---|---|---|
| Where the enzyme is | Dissolved freely, mixed in with the substrate | Attached to / trapped on a solid support (e.g. tiny gel beads) |
| Recovering the enzyme afterwards | Hard — it is mixed into the product | Easy — the beads are simply removed or the product runs past them |
| Reusing the enzyme | Usually used once, then lost | Can be used again and again (cost-effective) |
| Contaminating the product | The enzyme ends up in the product | The product stays enzyme-free / purer |
| Stability | Denatures more easily with heat or pH change | More stable — the support helps it tolerate a wider range of conditions |
A real application to remember: The classic example is lactase.
Lactase is immobilized on beads, and milk is passed over them. The lactase breaks the lactose in the milk into glucose and galactose.
The result is lactose-free milk for people who are lactose intolerant — and because the lactase is immobilized, none of it ends up in the milk and the beads can be reused.
| Immobilized enzyme | What it does | Application |
|---|---|---|
| Lactase | Breaks lactose into glucose and galactose | Making lactose-free milk for people who are lactose intolerant |
| Protease | Breaks proteins into amino acids | Removing protein stains / making protein-digested foods |
| Glucose isomerase | Converts glucose into sweeter fructose | Producing high-fructose syrup for the food industry |
Free enzyme
- Dissolved and mixed with the substrate
- Hard to recover — ends up in the product
- Usually used once, then lost
- Denatures more easily with heat / pH change
Immobilized enzyme
- Fixed to a solid support (e.g. beads)
- Easy to recover — the product stays pure
- Can be reused many times (cheaper)
- More stable over a wider range of conditions
A memory hook: Immobilized = 'not moving'. The enzyme is stuck in place, so you keep it, reuse it, and get a clean product.
Think beads of lactase + milk → lactose-free milk.
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How this is tested: Paper 1B loves a data question on an enzyme investigation. A 2-mark Discuss question can ask which variables must be controlled so a comparison (for example free versus immobilized enzyme) is fair — name them and say why.
A 3-mark Justify question can ask why a chosen measurement (such as the amount of product released) is a valid measure of enzyme activity.
Paper 3 often wants a 1-mark State: name one application of immobilized enzymes (e.g. lactase for lactose-free milk), or Outline how a practical method (low temperature, acid, wrapping) slows enzyme action.
IB-style question — variables to control in an enzyme investigation
A student compares how fast a free enzyme and an immobilized enzyme break down the same substrate. Discuss the variables that must be controlled so the comparison is valid. [2]
How to score both marks
- Name the variables to keep constant. Temperature, pH, substrate concentration, the amount (mass) of enzyme used, and the time the reaction is allowed to run must all be kept the same for both the free and the immobilized enzyme.
- Say why this matters. Each of these factors changes enzyme rate on its own, so if any of them differed between the two runs you could not tell whether a difference in rate was caused by immobilization or by the uncontrolled factor — keeping them constant makes the test fair. (Award 1 mark for naming two or more controlled variables, 1 mark for explaining that this keeps the comparison fair / valid.)
Final answer
Keep temperature, pH, substrate concentration, amount of enzyme and reaction time the same for both — otherwise any difference in rate might be due to one of those factors rather than to immobilization, so the comparison would not be fair.
Why this scores full marks: The answer names the controlled variables and explains the purpose of controlling them (a fair, valid comparison).
A 'discuss' worth 2 marks needs both halves — listing variables alone, with no reason, usually scores only 1.
| Variable | Why it changes enzyme rate | How to control it |
|---|---|---|
| Temperature | Higher temperature speeds the reaction up to the optimum, then denatures the enzyme | Use a water bath at one fixed temperature for every run |
| pH | Too acidic or too alkaline denatures the enzyme and slows the rate | Use the same buffer / pH in every run |
| Substrate concentration | More substrate gives a faster rate until the enzyme is saturated | Use the same starting substrate concentration each time |
| Amount of enzyme | More enzyme gives more active sites, so a faster rate | Use the same mass / volume of enzyme (or the same number of beads) |
| Time / reaction period | A longer time lets more product build up | Measure for the same length of time in every run |