Aromatic hydrocarbonization reaction is a widely used method in enzyme immobilization technology. It involves alkylating and aromatizing polymers with halogen-substituted aromatic rings or heterocycles, followed by reactions with amino, phenol, or sulfhydryl groups on enzyme molecules under alkaline conditions.

Aromatic hydrocarbons, commonly referred to as aromatics, are fundamental components of aromatic compounds. These compounds exhibit chemical properties distinct from aliphatic compounds, such as resistance to oxidation and a tendency for substitution rather than addition reactions. Despite their name, many aromatic compounds do not possess pleasant odors. Aromaticity refers to the unique stability of their unsaturated cyclic structures, characterized by resistance to addition reactions and a preference for substitution.

Coupling Reactions in Enzyme Immobilization

1. Diazotization Reaction

This method involves:

• Treating carriers with aromatic amino groups using NaNO₂ and dilute HCl to form diazonium salts.
• Coupling diazonium salts with enzymes under neutral to alkaline conditions (pH 8–9).

Common carriers:

• Polysaccharides with aromatic amino derivatives (e.g., cellulose, dextran).
• Amino acid copolymers (e.g., L-Leu and p-nitrogen-DL-Phe copolymers).
• Polyacrylamide derivatives (e.g., Enzacry, Bio-Gel).
• Phenylacetamide resin and aminosilane derivatives of porous glass.

2. Aromatic Hydrocarbonization Reaction

This reaction combines halogen-substituted aromatic rings or heterocycles with polymers containing haloacetyl groups. Common carriers include haloacetyl derivatives (e.g., chloroacetylcellulose, bromoacetylcellulose).

3. Glutaraldehyde Reaction

Glutaraldehyde, a bifunctional aldehyde, reacts with polymers containing primary amino groups to generate aldehyde-functionalized polymers. These polymers immobilize enzymes by forming irreversible bonds.

Common carriers:

• Aminoethyl cellulose, DEAE-cellulose.
• Amino derivatives of agarose and chitin.
• Aminosilane derivatives of porous glass.

4. Thiol-Disulfide Exchange Reaction

Carriers with sulfhydryl (-SH) or disulfide groups react with thiol groups on enzyme molecules. For instance, carriers treated with 2,2'-dipyridine disulfide can exchange sulfhydryl groups with enzymes under acidic conditions.

5. Four-Component Condensation Reaction

This reaction involves carboxylic acids, amines, aldehydes, and isocyanic acids to form N-substituted amides. Using suitable reaction conditions and carriers, enzymes can be coupled efficiently.

6. Carbonic Acid Group Reaction with CNBr

Under alkaline conditions, carriers with hydroxyl groups react with cyanogen bromide (CNBr) to form active imine carbonate groups, enabling enzyme immobilization through coupling with amino groups.

7. Acid Chlorination Reaction

Carriers containing carboxyl groups are treated with thionyl chloride to form acid chloride derivatives, which then couple with enzyme amino groups to produce immobilized enzymes.