Immobilised Enzymes (OCR A-Level Biology A): Revision Notes

Immobilised Enzymes

Introduction to enzyme immobilisation

Enzymes used in laboratory work and many industrial applications are typically in solution. While soluble enzymes are relatively inexpensive to produce, they present a practical challenge: after a single use, recovering them for reuse becomes costly. This economic limitation prompted scientists to develop methods for enzyme immobilisation.

Enzymes for industrial use are extracted from microorganisms cultured in laboratory conditions. When microorganisms secrete enzymes into their growth medium, extraction is straightforward. However, intracellular enzymes (those that remain inside cells) require the microorganisms to be broken open, making them more expensive to obtain.

What are immobilised enzymes?

This technique allows enzymes to remain in one location while substrate molecules pass through, undergo catalysis, and products are collected separately from the enzyme.

Methods of immobilisation

Enzymes can be immobilised using several approaches:

Attachment to inert support materials:

• Glass beads
• Resin beads
• Collagen fibres

Enzymes attach to these surfaces through chemical bonds whilst keeping active sites accessible to substrate molecules.

Encapsulation methods:

• Trapping in polyacrylamide microcapsules
• Encapsulation in calcium alginate beads
• Containment within partially permeable membranes

Industrial applications of immobilised enzymes

Immobilised enzymes play important roles across various industries:

Enzyme	Microbial source	Industrial application
Glucose isomerase	Genetically modified Streptomyces	Converts glucose to fructose in high-fructose corn syrup manufacture
Penicillin acylase	Escherichia coli	Production of semisynthetic penicillins
Lactase	Yeasts and mould fungi (e.g. Aspergillus spp.)	Hydrolysis of lactose to glucose and galactose
Aminoacylase	Saccharomyces cerevisiae, Bacillus, Streptomyces, Aspergillus oryzae	Production of pure L-amino acids for animal feeds
Glucoamylase	Aspergillus spp.	Conversion of dextrins (short-chain carbohydrates) to glucose
Nitrile hydratase	Rhodococcus	Conversion of acrylonitrile to acrylamide for plastics manufacture

Medical applications:

Immobilised enzymes are used in diagnostic test strips, including those for detecting glucose in urine and blood glucose monitors used by people with diabetes.

Food industry applications:

Immobilised lactase breaks down lactose in milk products for people with lactose intolerance, who cannot digest lactose and experience symptoms such as diarrhoea when they consume it.

Case study: high-fructose corn syrup production

Commercial advantages:

Fructose is sweeter than sucrose, meaning smaller quantities achieve the same sweetness level. Since corn starch is less expensive than sucrose as a raw material, HFCS provides a cost-effective alternative for food manufacturers.

Advantages of immobilised enzymes

Immobilised enzymes offer several benefits compared to soluble enzymes:

Economic advantages:

• Enzymes can be reused multiple times before replacement
• No contamination of the final product with enzyme protein
• Lower long-term costs despite higher initial setup expenses

Process advantages:

• Enable continuous processes rather than batch production
• Products can be collected continuously without stopping production
• Simplified product purification (no enzyme separation needed)

Stability advantages:

• Greater tolerance of temperature extremes
• Wider functional pH range
• Longer operational lifetime

Practical advantages:

• Easy to pack into columns for continuous flow systems
• Simple filtration and separation from products
• Straightforward recovery and replacement

Practical procedures with immobilised enzymes

Using immobilised enzymes:

Once packed into a column, substrate solution flows over the beads. The substrate molecules diffuse through the gel matrix to reach enzyme active sites. After catalysis, products diffuse out and collect at the column base.

Testing the product:

For immobilised sucrase breaking down sucrose:

• Test for glucose using glucose test strips (positive result confirms enzyme activity)
• Test for protein using biuret solution (negative result confirms no enzyme leakage)

Temperature effects on immobilised versus soluble enzymes

When comparing immobilised and soluble forms of the same enzyme, temperature affects them differently.

Experimental observations with lactase:

Research comparing immobilised lactase (in calcium alginate beads) with soluble lactase across temperatures from $15°\text{C}$ to $55°\text{C}$ reveals:

Key findings:

Soluble lactase:

• Shows optimal activity at 40°C
• Maintains high activity at $45°\text{C}$ (above $90\%$ of maximum)
• Activity drops sharply at $50°\text{C}$ and $55°\text{C}$ due to denaturation
• Shows the typical enzyme-temperature relationship

Immobilised lactase:

• Also shows optimal activity at 45°C
• Maintains higher activity at 50°C compared to soluble form
• Still shows substantial activity at $50°\text{C}$ (approximately $70\%$ of maximum)
• More resistant to denaturation at high temperatures

Practical significance:

The enhanced thermal stability of immobilised enzymes allows industrial processes to operate at higher temperatures, which can increase reaction rates without complete enzyme denaturation.

Remember!