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

Investigating Enzyme Activity: Immobilised in Alginate vs. Free in Solution

Learning Objective

To compare the catalytic activity of an enzyme immobilised in calcium‑alginate beads with the same enzyme dissolved freely in solution, and to explain the practical and industrial advantages of using immobilised enzymes.

Background Theory

1. Active Site, Specificity & Models of Action

  • Active site: a defined region of the enzyme where substrate molecules bind and are converted into product. It confers substrate specificity; only substrates that fit the site can be catalysed.

  • Lock‑and‑key model: the substrate and active site have complementary shapes, like a key fitting a lock.

  • Induced‑fit model: binding of the substrate induces a conformational change in the enzyme, improving the fit and lowering the activation energy.

2. Factors Affecting Enzyme Activity (Cambridge Syllabus Topic 3 – Enzymes)

FactorHow it influences the rateTypical optimum / effect
Temperature (°C)Increases kinetic energy → more collisions; above the optimum denaturation destroys the active site.Bell‑shaped curve; optimum often 30‑40 °C for many enzymes.
pHAlters ionisation of active‑site residues; extreme pH disrupts hydrogen‑bonding and ionic interactions.Bell‑shaped curve; optimum near pH 7 for many cytosolic enzymes.
Substrate concentration, [S]Follows Michaelis–Menten kinetics:

v = (Vmax [S]) / (Km + [S])

Low [S] → rate ∝ [S]; high [S] → rate approaches Vmax.

Rate rises until the enzyme becomes saturated.
Enzyme concentration, [E]Rate is directly proportional to [E] when substrate is in excess; otherwise the reaction is substrate‑limited.Linear increase in initial rate with enzyme amount under saturating substrate.
Inhibitors

  • Competitive: inhibitor resembles substrate and binds the active site. Increases Km (lower apparent affinity); Vmax unchanged.

  • Non‑competitive: inhibitor binds elsewhere, altering enzyme conformation. Decreases Vmax; Km unchanged.

Both are reversible.

Effect observed by comparing rates before and after adding the inhibitor.
Ionic strength & cofactors

  • High salt can shield electrostatic interactions, reducing activity.

  • Many enzymes require metal ions (e.g., Mg²⁺, Zn²⁺) or organic cofactors (e.g., NAD⁺) for catalysis.

Include appropriate controls to confirm that observed rate changes are due to the factor under study.

3. Determining Vmax and Km

  • Measure initial rates (v) at several substrate concentrations.

  • Plot the data:

    • Non‑linear fit: fit the Michaelis–Menten equation directly (software or spreadsheet).

    • Lineweaver‑Burk (double‑reciprocal) plot: plot 1/v against 1/[S]. The y‑intercept = 1/Vmax, the x‑intercept = –1/Km.

  • Compare the parameters for the immobilised and free enzyme to assess the effect of diffusion and matrix interactions.

4. Immobilisation of Enzymes

  • Enzyme molecules are trapped within a solid matrix (e.g., calcium‑alginate beads) while retaining catalytic activity.

  • Key matrix properties that influence observed rates:

    • Porosity & bead size: control diffusion of substrate into, and product out of, the bead.

    • Diffusion limitation: reduces the apparent initial rate compared with a free enzyme.

  • Effective diffusion coefficient (D) can be estimated by preparing beads of different radii and plotting initial rate versus 1/r² (Fick’s law approximation).

Experimental Design

The model enzyme is catalase, but any soluble enzyme that gives a measurable change (colour, gas evolution, absorbance) may be used.

StepImmobilised in AlginateFree in Solution
1. Prepare enzymeMix catalase (2 U mL⁻¹) with 2 % (w/v) sodium alginate. Drop the mixture into 0.1 M CaCl₂ to form beads (≈3 mm diameter). Rinse beads with phosphate buffer (pH 7.0).Dissolve the same total enzyme units in phosphate buffer (pH 7.0).
2. Standardise enzyme amountCount beads and calculate total units (e.g., 10 beads ≈ 20 U). Record bead volume for later normalisation.Transfer an equivalent volume of enzyme solution containing the same 20 U.
3. Set‑up reactionPlace beads in a cuvette containing 5 mL 0.03 % H₂O₂ (substrate). Start timer.Add enzyme solution to identical H₂O₂ solution in a separate cuvette.
4. Measure activityMonitor decrease in absorbance at 240 nm (O₂ evolution) every 15 s for 2 min. Alternatively, collect evolved O₂ with a gas syringe.Same measurement protocol.
5. Controls & repeats

  • Blank (no enzyme)

  • Competitive inhibitor test (add 1 mM NaN₃)

  • Co‑factor control (add 5 mM MgCl₂ if the enzyme requires Mg²⁺)

  • Ionic‑strength control (add 0.5 M NaCl to test high‑salt effect)

  • Repeat each condition ≥3 times

Same set of controls.
6. Data analysis

  • Calculate initial rate (ΔA min⁻¹) from the linear portion of the curve.

  • Convert ΔA to µmol O₂ min⁻¹ using Beer‑Lambert law (ε₂₄₀ ≈ 1.0 × 10⁴ M⁻¹ cm⁻¹).

  • Plot rate versus [S] and fit the Michaelis–Menten equation (non‑linear) to obtain Km and Vmax.

  • Construct a Lineweaver‑Burk plot (1/v vs. 1/[S]) to verify the parameters.

  • If bead size is varied (2 mm, 3 mm, 4 mm), plot initial rate against 1/r² to estimate the effective diffusion coefficient (D) for H₂O₂ in alginate.

Identical analysis.

Expected Results and Interpretation

  1. Initial rates: Immobilised catalase usually shows a lower initial rate because substrate must diffuse through the alginate matrix. The reduction is larger for bigger beads.

  2. Michaelis–Menten parameters:

    • Km may increase slightly for the immobilised enzyme, reflecting an apparent decrease in affinity caused by diffusion resistance.

    • Vmax (expressed per unit of enzyme) is often comparable, indicating that the catalytic centre itself is unchanged.

  3. Thermal & pH stability: Immobilised enzyme retains a higher proportion of activity at temperatures above the free‑enzyme optimum and at pH values farther from the optimum, showing the protective effect of the alginate matrix.

  4. Inhibition test: Adding a competitive inhibitor reduces the rate of both forms; removal of the inhibitor restores activity, confirming reversibility.

  5. Diffusion analysis (optional): A linear relationship between initial rate and 1/r² confirms that diffusion through the bead limits the observed rate and allows calculation of D.

Advantages of Immobilised Enzymes

  • Reusability: Beads can be recovered (by filtration or decanting) and reused in multiple cycles, reducing cost.

  • Easy separation: No need for downstream purification of product; the solid matrix can be removed simply.

  • Enhanced stability: Immobilisation protects against thermal denaturation, extreme pH, and proteolytic degradation.

  • Continuous processing: Suitable for packed‑bed reactors, allowing substrate to flow continuously over immobilised enzyme.

  • Controlled micro‑environment: The matrix can be engineered (e.g., by adding buffering agents) to maintain optimal local pH or ionic strength.

  • Reduced product inhibition: In some systems the matrix limits accumulation of product near the active site.

Schematic of substrate diffusion into an alginate bead containing immobilised enzyme compared with free enzyme in solution

Figure: Substrate diffuses into the alginate bead (left) where the enzyme is immobilised, whereas in the free‑enzyme system (right) substrate and enzyme are already in the same phase.