Enzyme Activity: Immobilised v Free
Diffusion Limitations: In immobilised systems, the substrate must travel from the bulk liquid to the enzyme site, which can slow the observed reaction rate compared to free enzymes where collision is immediate.
Microenvironment Effects: The local pH or charge near the support surface may differ from the bulk solution, causing a shift in the enzyme's optimal operating conditions.
Thermal Stability: Immobilisation often restricts the enzyme's conformational flexibility, making it more resistant to unfolding (denaturation) at higher temperatures or extreme pH levels.
Kinetic Parameters: The apparent Michaelis constant () often increases upon immobilisation because a higher bulk substrate concentration is required to overcome diffusion resistance and saturate the enzyme.
| Feature | Free Enzymes | Immobilised Enzymes |
|---|---|---|
| Recovery | Difficult; usually lost after one use | Easy; filtered or settled out |
| Product Purity | Low; enzyme contaminates product | High; enzyme remains separate |
| Stability | Sensitive to heat and pH changes | Enhanced; rigid structure protects active site |
| Reaction Mode | Batch processing only | Batch or Continuous Flow |
| Initial Activity | High (no diffusion barriers) | Lower (diffusion and masking effects) |
Analyze the Graph: When comparing temperature curves, look for the 'peak' shift; immobilised enzymes often show a broader peak or a peak shifted toward higher temperatures, indicating better thermal tolerance.
Identify the Method: If a question mentions 'beads' or 'membranes', it is likely discussing entrapment; if it mentions 'chemical bonds', it is discussing covalent attachment.
Economic Justification: Always link the use of immobilised enzymes to cost-effectiveness in industry, specifically mentioning the ability to reuse the catalyst and reduce purification costs.
Check for Changes: Remember that an 'apparent' increase does not necessarily mean the enzyme itself has changed, but rather that the physical setup makes it harder for the substrate to reach the enzyme.
Activity vs. Stability: Students often confuse these; immobilisation usually decreases the immediate rate of reaction (activity) but increases the duration the enzyme remains functional (stability).
Universal Application: Not all enzymes are suitable for immobilisation; if the active site is blocked by the attachment method, the enzyme will become completely inactive.
Substrate Size: Large substrate molecules (like proteins) may be physically unable to enter the pores of an entrapment matrix, rendering the immobilised enzyme useless for that specific reaction.