Covalent Binding Method
An enzyme can be covalently bound to' support materials by different methods. The enzyme forms a covalent link with active groups of support material either (i) through the reactive groups on side chains of its amino acids like lysine, arginine, histidine, tyrosine, cysteine, serine, aspartic acid and glutamic acid or (ii) with terminal amino and carboxyl groups of the polypeptide chains. Some examples of covalent linkage between the support and the enzyme are as follows.

(b) Support with -COOH group
Such supports including carboxymethyl cellulose can be activated via azide derivative or acyl isourea formation. The reaction again involves –NH₂ group of lysine. However, other amino acids such as tyrosine, cysteine and serine are also implicated.

Immobilization of Enzymes, Using Supporths Having-COOH, with Hydrazine (NH₂ - NH₂) or with - NH₂ Groups Through Covalent Linkage Involving (A) Azide Derivative

Immobilization of Enzymes, Using Supporths Having-COOH, with Hydrazine (NH₂ - NH₂) or with - NH₂ Groups Through Covalent Linkage Involving (B) Acyl Isourea

Glutaraldehyde is also used sometime to activate the support containing –NH₂ group. The reaction involves a Schiffs - base formation between amino group of support and amino group of one of the amino acids of protein.

Immobilization of Enzymes Using Supports Having - NH₂ Group Involving (B) Activation with Glutaraldehyde

Number of bonds between support and the enzyme molecule during covalent linkage is variable. There are, for example, 17 bonds per papain molecule and 8 for subtilopeptidase immobilized on semi aldehyde starch derivative. Papain bound to porous glass is linked through three azo links per enzyme molecule.