Immobilised Enzymes

Immobilised enzymes are enzymes that have been physically or chemically attached to an inert support so that they are held in place during a reaction. This allows reuse of expensive enzymes, simplifies product purification and improves enzyme stability. OCR A-Level Biology A specification 6.2.1 (i) requires you to explain the methods of enzyme immobilisation, name examples of industrial use and evaluate the advantages and disadvantages compared with free enzymes.

Key Definitions:

• Immobilised enzyme — an enzyme attached to or entrapped within an insoluble support, allowing it to be reused.
• Adsorption — binding to a surface by weak forces (hydrogen bonds, hydrophobic interactions, van der Waals).
• Covalent bonding — formation of strong chemical bonds between enzyme and support.
• Entrapment — trapping the enzyme inside a gel or fibre without direct chemical bonding.
• Membrane separation — keeping enzyme on one side of a selective membrane through which substrate and product can pass.

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Why Immobilise Enzymes?

Enzymes are powerful biological catalysts used in industry to make products ranging from high-fructose corn syrup to penicillin antibiotics. Free enzymes have problems:

• They contaminate the product and must be removed.
• They cannot easily be recovered for reuse.
• They may be denatured by high temperatures or extreme pH.
• They can be expensive, especially human or designer enzymes.

Immobilising the enzyme solves these problems: the enzyme stays on its support while substrate flows through, product leaves the reactor enzyme-free, and the same batch of enzyme can be used for weeks or months.

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Methods of Immobilisation

1. Adsorption

The enzyme is allowed to bind to the surface of an inert support such as clay, porous carbon, glass beads or a resin. Binding forces are weak (hydrogen bonds, hydrophobic interactions, van der Waals).

Advantages: simple, cheap, preserves enzyme activity well because the active site is usually unaffected. Disadvantages: enzyme can leak off the support ("leaching") with changes in pH, temperature or ionic strength.

2. Covalent Bonding

The enzyme is chemically linked to the support (e.g. cellulose, sepharose, polyacrylamide) by strong covalent bonds. Cross-linking agents such as glutaraldehyde are often used.

Advantages: strong attachment, no leaching. Disadvantages: expensive; the chemical modification may partially denature the enzyme or block the active site.

3. Entrapment

The enzyme is trapped inside a gel (e.g. calcium alginate beads, silica gel) or hollow fibres. The gel is porous enough for substrate and product to diffuse in and out, but the enzyme molecules are too large to escape.

Advantages: the enzyme is not chemically modified, so its activity is preserved; simple to prepare. Disadvantages: diffusion of substrate and product through the gel is slow, limiting reaction rate; small enzymes can sometimes leak.

4. Membrane Separation

The enzyme is kept in solution on one side of a semi-permeable membrane. Substrate diffuses in and product diffuses out; the enzyme cannot cross the membrane.

Advantages: enzyme remains in solution, so activity is unaffected. Disadvantages: membranes can clog; equipment is more complex.

Method	Attachment	Activity preserved	Leaching risk	Cost
Adsorption	Weak forces	High	High	Low
Covalent	Chemical bond	Medium	Very low	High
Entrapment	Gel matrix	High	Low	Low
Membrane	Physical barrier	Very high	None	High

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Industrial Examples

Glucose Isomerase — High-Fructose Corn Syrup

Glucose isomerase converts glucose to fructose. Because fructose is much sweeter than glucose, fructose syrups allow less sugar to be used in food and drink, cutting costs and calories. The enzyme is immobilised (usually by adsorption on to a resin) in packed-bed reactors, through which glucose syrup flows. The resulting high-fructose corn syrup (HFCS) is used in soft drinks, breakfast cereals and processed foods worldwide. Global production exceeds 10 million tonnes per year. Immobilisation is essential because free enzyme would contaminate the syrup and cost too much to replace.

Penicillin Acylase — Semi-Synthetic Antibiotics

Penicillin acylase (penicillin amidase) removes the side chain from penicillin G, producing 6-aminopenicillanic acid (6-APA). Chemists then attach different side chains to make semi-synthetic penicillins such as amoxicillin, ampicillin and oxacillin — crucial in the fight against antibiotic-resistant bacteria. The enzyme is immobilised by covalent bonding or entrapment in large packed-bed reactors, and used continuously to produce tonnes of 6-APA each year.

Lactase — Lactose-Free Milk