The enzyme can be immobilized through several methods such as:
a. INTERACTION WITH THE CARRIER

• Adsorption
• Ionic interaction

b. CHEMICAL BINDING

• Cross-linking
• Covalent binding with functional group

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a. Interaction with the carrier

The enzyme is adsorbed on the carrier at suitable conditions (e.g. pH, salt content) for the interaction with the functional groups distributed on the carrier surface. The interaction is reversible and the enzyme can be de-adsorbed changing the conditions. In some cases the adsorption can be quite strong (e.g. hydrophobic interaction)

The reversible interaction can take place through:

• Hydrophobic adsorption: Hydrophobic adsorbents e.g. Relizyme™ BU403 or OD403 and SEPABEADS™ EC-BU or EC-OD;
• Ionic interaction: Amino functionalised carriers e.g. Relizyme™ EA403 or HA403 and SEPABEADS™ EC-EA or EC-HA;

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b. Chemical binding

The reversible immobilisation can become irreversible by means of a cross-linking step: the enzyme is adsorbed onto the carrier and then crosslinked by using, for example, glutaraldehyde. The crosslinked enzyme molecules cover the carrier like a net:

The elective method to obtain an irreversible immobilization is the covalent binding between the enzyme and the functional groups of the carrier. This immobilization can be performed using carriers with oxyrane groups (e.g. Relizyme™ EP403 or HFA403 and SEPABEADS™ EC-EP or EC-HFA) or with an amino functionalised (e.g. Relizyme™ EA403 or HA403 and SEPABEADS™ EC-EA or EC-HA). In case of amino groups is necessary a pre-activation step with a bifunctional coupling agent, such as glutaraldheyde. Another nethod is the activation of the hydroxyl functional groups of ReliZyme™ HG403 and SEPABEADS™ EC-HG.

Several factors related to the micro-environment surrounding the immobilized enzyme have effects on the efficiency of the immobilized biocatalyst, as it is schematically resumed in this table:

Matrix features	Effect	Effect on catalytic efficiency
Functional group type and surface polarity	Enzyme orientation	Active sites availability
Porosity	Mass transfer	Turnover rate
Functional group type and surface polarity	Reagent or product accumulation	Inhibition phenomena
Spacer arm length	Mass transfer; system flexibility	Stability aand active sites accessibility