investigate the difference in activity between an enzyme immobilised in alginate and the same enzyme free in solution, and state the advantages of using immobilised enzymes

Published by Patrick Mutisya · 14 days ago

Cambridge A-Level Biology 9700 – Factors that affect enzyme action

Factors that affect enzyme action

Objective

To investigate the difference in activity between an enzyme immobilised in alginate beads and the same enzyme free in solution, and to state the advantages of using immobilised enzymes.

Background

Enzymes are biological catalysts that accelerate chemical reactions by lowering the activation energy. Their activity is influenced by a range of physical and chemical factors. Understanding these factors is essential for interpreting experimental results and for the practical application of enzymes in industry and research.

  • Temperature: Increases kinetic energy, raising reaction rates up to an optimum. Beyond the optimum, denaturation reduces activity.

  • pH: Affects ionisation of active‑site residues. Each enzyme has an optimum pH at which activity is maximal.

  • Substrate concentration ([S]): Influences the rate according to the Michaelis‑Menten relationship:

    \$v = \frac{V{\max}[S]}{Km + [S]}\$

  • Enzyme concentration: Rate is directly proportional to enzyme amount when substrate is abundant.

  • Inhibitors: Competitive, non‑competitive or uncompetitive inhibitors alter \$V{\max}\$ and/or \$Km\$.

  • Ionic strength and cofactors: Can stabilise the enzyme structure or be required for activity.

Experimental Design

The experiment compares the catalytic activity of a chosen enzyme (e.g., catalase) in two forms:

StepImmobilised in AlginateFree in Solution
1. Preparation of enzymeEnzyme mixed with sodium alginate (2 % w/v) and dropped into CaCl₂ solution to form beads.Enzyme dissolved directly in buffer.
2. Standardisation of enzyme amountCalculate enzyme units per bead based on volume and concentration.Adjust volume to contain the same total units as in the beads.
3. Reaction set‑upBeads added to substrate solution (e.g., H₂O₂) in a cuvette.Enzyme solution added to identical substrate solution.
4. Measurement of activityMonitor decrease in absorbance at 240 nm (O₂ evolution) over time.Same monitoring method.
5. RepetitionRepeat at least three times for each condition.Repeat the same number of trials.
6. Data analysisCalculate initial reaction rate (ΔA/min) and compare with free enzyme.Same calculations.

Expected Results and Interpretation

Typical observations may include:

  1. Lower initial rates for the immobilised enzyme due to diffusion limitations of substrate into the alginate matrix.

  2. Similar \$V_{\max}\$ values after correcting for diffusion, indicating that the catalytic centre remains unchanged.

  3. Greater thermal stability of the immobilised enzyme, reflected by a higher temperature at which activity declines.

Data can be plotted as reaction rate versus substrate concentration for both forms. Fitting the Michaelis‑Menten equation will yield \$Km\$ and \$V{\max}\$ values, allowing quantitative comparison.

Advantages of Immobilised Enzymes

  • Reusability: Enzymes can be recovered from the reaction mixture and used in multiple cycles, reducing cost.

  • Ease of separation: No need for downstream purification; beads can be filtered or decanted.

  • Enhanced stability: Immobilisation often protects enzymes from denaturation by heat, pH extremes, or proteolysis.

  • Continuous operation: Immobilised enzymes are ideal for packed‑bed reactors and flow‑through systems.

  • Controlled reaction environment: The matrix can be engineered to create optimal micro‑environments (e.g., local pH).

Suggested diagram: Schematic of an alginate bead containing immobilised enzyme compared with a free enzyme solution, showing substrate diffusion into the bead.