Covalent Immobilization of Enzymes Using Commercially Available CDI-Activated Agarose

Carbonyldiimidazole (CDI) activated supports for enzyme immobilization are commercially available. The urethane linkage that is formed when a protein is bound to these supports is about 20-fold more stable than the N-substituted isourea linkage formed during protein immobilization on cyanogen bromide-activated matrices (1 ). Complete characterization of the immobilized preparation requires the development of new methodology or the modification of existing methodology to measure the amount of protein bound to, and the enzymatic activity residing in, the immobilized preparation. In this chapter we present detailed descriptions of the immobilization of two enzymes, lipoxygenase (2 ,3 ) and lactamase, on CDI-activated agarose, and procedures for characterization of the immobilized enzymes.