03.10 Immobilising Enzymes
Overview of Immobilised Enzymes
- Immobilised Enzymes:
- Enzymes are fixed in place, often in alginate beads or similar matrices, which prevents them from diffusing freely in a solution.
- Immobilisation allows reusability, reducing costs, especially in commercial applications like medicine, food processing, and industrial production.
Example: Immobilised Lactase for Lactose-Free Products
- Lactase and Alginate Beads:
- Lactase: An enzyme that breaks down lactose (milk sugar) into glucose and galactose.
- Application:
- Milk is passed through a column containing lactase-immobilised alginate beads.
- Lactase hydrolyses lactose, producing lactose-free milk for lactose-intolerant consumers.
- Advantages:
- No enzyme contamination in milk, ensuring a clean product.
- Enzyme reusability: Lactase can be reused multiple times, reducing costs.
Advantages of Immobilised Enzymes
Cost Efficiency:
- Enzymes can be retrieved and reused, minimizing the need for constant replenishment.
Purity of Product:
- No enzyme residue in the final product, preventing contamination.
Increased Stability:
- Greater tolerance to changes in temperature and pH compared to enzymes in solution.
- Immobilised enzymes are partly protected by the matrix (e.g., alginate beads) which holds them.
- Reduced exposure of enzyme structure to environmental changes decreases risk of denaturation.
Experimental Investigations
Comparing Temperature Stability:
- Objective: Determine the temperature at which lactase denatures within 10 minutes.
- i) Free in Solution:
- Expose a lactase solution to incrementally higher temperatures for 10 minutes, checking enzyme activity after each interval.
- ii) Immobilised in Alginate Beads:
- Repeat procedure using lactase immobilised in alginate beads, comparing temperatures at which activity ceases.
- i) Free in Solution:
Time to Denature at 90°C:
- Objective: Determine how long lactase remains active at 90°C.
- i) Free in Solution:
- Maintain lactase solution at 90°C, testing activity at regular intervals to find denaturation time.
- ii) Immobilised in Alginate Beads:
- Use immobilised lactase, testing activity periodically until denaturation is observed.
- i) Free in Solution:
Determining Optimum pH:
- Objective: Find the pH at which lactase works most efficiently.
- i) Free in Solution:
- Test lactase activity in solutions of varying pH levels, identifying pH with maximum activity.
- ii) Immobilised in Alginate Beads:
- Repeat with immobilised lactase, comparing optimum pH to free enzyme.
- i) Free in Solution:
Practise Questions
Question 1
What are immobilised enzymes, and how are they typically prepared for industrial use? (4 marks)
Mark Scheme:
- Immobilised enzymes are enzymes that are fixed in place, preventing them from freely diffusing in solution. (1 mark)
- They are often embedded in matrices like alginate beads or bound to solid supports. (1 mark)
- Immobilisation allows the enzymes to be reused multiple times, reducing costs. (1 mark)
- Example: Lactase immobilised in alginate beads is used to hydrolyse lactose in milk. (1 mark)
Question 2
Describe how immobilised lactase is used to produce lactose-free milk and its advantages over free lactase. (6 marks)
Mark Scheme:
- Lactase is immobilised in alginate beads. (1 mark)
- Milk is passed through a column containing these beads, where lactase hydrolyses lactose into glucose and galactose. (1 mark)
- Advantages over free lactase:
- Immobilised lactase can be reused, reducing costs. (1 mark)
- It prevents enzyme contamination in the milk, ensuring a clean product. (1 mark)
- Immobilised lactase is more stable, tolerating temperature and pH fluctuations better than free enzymes. (1 mark)
- The process is efficient and allows continuous production of lactose-free milk. (1 mark)
Question 3
Explain the experimental setup to compare the temperature stability of free and immobilised lactase. (6 marks)
Mark Scheme:
- Prepare two setups:
- Free lactase in solution.
- Immobilised lactase in alginate beads. (1 mark)
- Expose both enzyme forms to incrementally higher temperatures (e.g., 30°C, 40°C, etc.) for 10 minutes each. (1 mark)
- After each interval, test enzyme activity by measuring the rate of lactose hydrolysis. (1 mark)
- Record the temperature at which enzyme activity ceases for both setups. (1 mark)
- Compare results: Immobilised lactase is expected to remain active at higher temperatures than free lactase. (1 mark)
- Control variables: Substrate concentration, reaction volume, and exposure time for consistent comparison. (1 mark)
Question 4
What are the advantages of using immobilised enzymes in industrial processes? (6 marks)
Mark Scheme:
- Reusability: Immobilised enzymes can be recovered and reused, reducing operational costs. (1 mark)
- Product purity: Immobilised enzymes do not contaminate the final product, ensuring a clean outcome. (1 mark)
- Enhanced stability: Immobilised enzymes have greater tolerance to changes in temperature and pH. (1 mark)
- Reduced denaturation risk: The matrix provides partial protection against environmental fluctuations. (1 mark)
- Continuous processes: Immobilised enzymes are suitable for continuous production systems, improving efficiency. (1 mark)
- Example: Lactase immobilised in alginate beads for lactose-free milk production. (1 mark)
Question 5
Why do immobilised enzymes show greater stability compared to free enzymes? (4 marks)
Mark Scheme:
- Immobilised enzymes are protected by the matrix (e.g., alginate beads) that holds them in place. (1 mark)
- This reduces exposure of the enzyme structure to environmental changes such as temperature or pH fluctuations. (1 mark)
- The matrix helps maintain the enzyme’s 3D structure, decreasing the likelihood of denaturation. (1 mark)
- Immobilised enzymes tolerate more extreme conditions than free enzymes, making them more durable in industrial applications. (1 mark)
Question 6
Outline how to determine the optimum pH for free and immobilised lactase. (6 marks)
Mark Scheme:
- Prepare solutions of varying pH levels (e.g., pH 4, 5, 6, etc.) using buffers. (1 mark)
- Add free lactase to each pH solution and measure enzyme activity by monitoring lactose hydrolysis. (1 mark)
- Repeat the experiment using immobilised lactase in alginate beads. (1 mark)
- Record the reaction rates for each pH level and identify the pH with the highest activity. (1 mark)
- Compare results: The optimum pH for immobilised lactase may differ slightly from that of free lactase due to matrix effects. (1 mark)
- Control variables: Substrate concentration, reaction time, and temperature to ensure fair comparisons. (1 mark)
Question 7
Compare the time to denature at 90°C for free and immobilised lactase. Why might immobilised lactase denature more slowly? (5 marks)
Mark Scheme:
- At 90°C, maintain both free lactase and immobilised lactase at constant exposure, periodically testing enzyme activity. (1 mark)
- Record the time at which enzyme activity ceases for both forms. (1 mark)
- Immobilised lactase is expected to remain active for a longer duration than free lactase. (1 mark)
- Reason: Immobilisation in the alginate matrix provides partial protection against heat, reducing the risk of denaturation. (1 mark)
- The matrix stabilises the enzyme’s structure, allowing it to withstand higher temperatures for longer. (1 mark)
Question 8
What experimental controls are essential when comparing free and immobilised enzyme activity? (4 marks)
Mark Scheme:
- Use the same substrate concentration and volume in all experiments. (1 mark)
- Maintain constant temperature and pH across setups. (1 mark)
- Use identical exposure times for both free and immobilised enzymes. (1 mark)
- Ensure consistent methods for measuring enzyme activity (e.g., rate of lactose hydrolysis). (1 mark)
Question 9
Explain why immobilised lactase is preferred for industrial lactose hydrolysis. (5 marks)
Mark Scheme:
- Immobilised lactase can be reused, reducing the need for frequent enzyme replacement. (1 mark)
- It does not contaminate the final product, ensuring clean, lactose-free milk. (1 mark)
- Immobilised lactase has increased stability, tolerating temperature and pH changes better than free lactase. (1 mark)
- Continuous processing is possible with immobilised lactase, improving production efficiency. (1 mark)
- The cost-effectiveness and durability of immobilised lactase make it ideal for large-scale industrial use. (1 mark)
Question 10
Summarise the key advantages of immobilised enzymes over free enzymes in industrial processes. (5 marks)
Mark Scheme:
- Reusability: Immobilised enzymes can be recovered and reused, lowering costs. (1 mark)
- Product purity: No contamination of final products with enzyme residue. (1 mark)
- Enhanced stability: Greater tolerance to environmental changes, reducing denaturation risk. (1 mark)
- Efficiency: Suitable for continuous processes, improving industrial productivity. (1 mark)
- Durability: Increased lifespan due to protection provided by the immobilisation matrix. (1 mark)