Immobilization-stabilization of thermolysin onto activated agarose gels

A method for immobilization-stabilization of thermolysin onto activated agarose gels is reported based on the formation of covalent bonds between the enzyme and the support. All derivatives prepared retained 100% of the enzymatic activity and they show higher stability than free thermolysin. The effect of different variables concerning the strength of the enzyme-support attachment on the stability of the immobilized thermolysin derivatives has been established under different inactivation conditions: presence of a water miscible solvent (DMF); stirred biphasic systems, 1,2-dichloroethane/acetate buffer; acid conditions (pH = 3) as well as in the absence of calcium ions. The possible reactivation of the derivatives inactivated by the loss of calcium ions was also studied.