Immobilised Enzymes

Enzyme immobilisation is a technique which prevents the free movement of enzymes within a reaction mixture containing a substrate.

Enzymes are immobilised by trapping them within, or binding them to, a material that is then packed into a reaction column.

Reaction columns are used in chemical immobilisation to provide a vessel in which the material to which the enzyme is bound can be packed.

The diagram below shows a typical reaction column containing immobilised enzymes:

Enzymes can be immobilised either chemically or physically:

Chemically binding enzymes with chemical bonds to the surface of a solid material.
Physically trapping enzymes involves allowing the substrate to diffuse through a material that binds the enzymes within a matrix or keeps the enzymes separate by acting as a partially permeable membrane.

The two types of chemical immobilisation are adsorption and covalent bonding:

Adsorption binds enzyme molecules to the surface of an inert supporting material (e.g. clay, glass beads) by weak hydrogen bonds, hydrophobic interactions, or ionic bonds.
Covalent bonding binds enzyme molecules to a support by strong covalent bonds.

The table below outlines some of the advantages and disadvantages of the different types of chemical immobilisation:

Method	Advantage(s)	Disadvantage(s)
Adsorption	Enzymes can be reused, lowering costs. The active site is exposed to the reaction mixture containing the substrate.	The enzyme-surface bonding is weak, so the enzyme may detach and leak into the mixture.
Covalent bonding	Enzymes can be reused, lowering costs. Strong covalent bonds prevent enzymes from detaching and leaking into the reaction mixture.	Expensive to covalently bind enzymes to the surface. The enzyme-surface bonding is strong, so the enzyme’s active site may be distorted, reducing the rate of reaction.

The two types of physical immobilisation are entrapment and membrane separation:

Entrapment holds enzymes inside a matrix or gel (e.g. calcium alginate beads) that they cannot leave, but the substrate and product molecules can diffuse in and out of the matrix or gel.
Membrane separation holds enzymes on one side of a partially permeable membrane, which separates them from the reaction mixture containing the substrate.

It is useful to know that adsorption is more commonly classified as a form of physical immobilisation.

The table below outlines the advantages and disadvantages of the different types of physical immobilisation:

Method	Advantage(s)	Disadvantage(s)
Entrapment	Enzyme active site is unaffected and fully active. Enzymes can be reused, lowering costs.	The need for diffusion limits the rate of reaction.
Membrane separation	Enzyme active site is unaffected and fully active. Enzymes can be reused, lowering costs.	Need for diffusion limits the rate of reaction. The substrate and product must be small enough to diffuse through the partially permeable membrane, limiting its use to certain reactions.