Immobilised lactase column losing activity
A small dairy uses a packed-bed column of lactase immobilised on calcium alginate beads to make lactose-free milk. The column contains 2.0 g of immobilised enzyme, is run at 40 °C and pH 6.5, and is fed milk containing 50 g lactose dm−3 at 5.0 cm3 min−1. On day 1 the outflow contains 7.5 g lactose dm−3. After 30 days of continuous use the outflow contains 31 g lactose dm−3. The operator proposes three explanations: enzyme has leaked from the beads; the active sites are blocked by product; or the enzyme has changed shape. A sample of outflow concentrated 20-fold shows no detectable protein. Adding extra lactose does not restore the rate. Storing a spare batch of beads in 0.50 mol dm−3 NaCl at 4 °C for two weeks is also proposed, although the supplier recommends 0.05 mol dm−3 phosphate buffer at pH 6.5 and 4 °C.
1. Calculate the percentage of lactose hydrolysed on day 1 and on day 30. Using the evidence, decide which of the operator's three explanations is best supported. Justify your answer by referring to each explanation.
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Calculation
- Day 1: lactose hydrolysed = (50 - 7.5) / 50 × 100 = 85.0%.
- Day 30: (50 - 31) / 50 × 100 = 38.0%.
Which explanation?
- Enzyme leakage would put enzyme protein into the outflow. A 20-fold concentrate with no detectable protein makes substantial leakage unlikely, although it does not prove zero loss. If leakage were the main cause, the fall in activity should be matched by protein loss.
- Product inhibition is less likely because the products are continuously washed out of a packed-bed column, and adding more substrate did not restore activity. Competitive product inhibition would be partly relieved by extra substrate; the failure to restore activity argues against a simple competitive effect. Non-competitive product inhibition cannot be excluded completely, but it is not the best fit.
- Change in enzyme shape (denaturation) fits the slow loss over 30 days at 40 °C: activity falls without protein release and is not reversed by substrate. A second possibility is fouling of the alginate beads or microbial growth, which would reduce substrate access by mass-transfer limitation rather than change the active site.
Best supported: denaturation or inactivation of the immobilised enzyme, with bead fouling as a competing explanation. To distinguish them, assay the beads directly for residual activity and check whether the column pressure has risen.
2. Evaluate the proposal to store beads in 0.50 mol dm−3 NaCl at 4 °C instead of the supplier's buffer. Explain the likely effects on the immobilised enzyme.
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0.50 mol dm−3 NaCl is about ten times the ionic strength of the recommended 0.05 mol dm−3 phosphate buffer. High ionic strength screens ionic attractions and can weaken hydrophobic interactions, so the enzyme may partly unfold; NaCl can also draw water out of the alginate beads osmotically and shrink the pores. Storage at 4 °C slows thermal denaturation, but it does not protect against ionic or osmotic damage. The buffer at pH 6.5 keeps the enzyme's ionisable groups in their normal protonation state and maintains a stable microenvironment. The NaCl batch should not be assumed equivalent: measure residual activity of beads stored by both methods, and use the buffer unless the NaCl batch retains activity.
3. The dairy wants to restore about 85% hydrolysis without buying new enzyme. Suggest two operational changes and explain the trade-off for each.
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Any two sensible changes with trade-offs:
- Lower the flow rate, for example from 5.0 to 2.0 cm3 min−1. Residence time rises, so each milk portion contacts enzyme longer and hydrolysis increases. Trade-off: throughput falls, so the dairy needs more columns or longer operating hours to process the same volume.
- Add a second column in series or increase bed length. This increases total residence time without reducing flow and can restore conversion. Trade-off: higher back-pressure, greater risk of clogging, and extra pumping cost.
- Reduce temperature to 30-35 °C. This slows denaturation and may prolong useful life. Trade-off: reaction rate falls, roughly halving for a 10 °C drop, so a longer residence time is needed.
- Use smaller beads or a higher surface-area support. This improves substrate access. Trade-off: smaller beads increase pressure drop and are harder to retain.
First check whether the column is fouled and whether pH has drifted; replacing or cleaning beads addresses fouling rather than enzyme inactivation.