STABILIZATION OF MICROCOCCAL ENDONUCLEASE BY IMMOBILIZATION ON AGAROSE GELS HIGHLY ACTIVATED WITH CNBR

The attachment of enzymes, through their amino groups, to CNBr activated agarose gels has been tested as an immobilization stabilization system. By using this system, the development of a strategy to immobilize enzymes through multipoint covalent attachment has been studied. We have prepared different staphylococcal nuclease-Agarose derivatives by using Sepharose 2B gels previously activated with CNBr. Activity and stability of the derivatives obtained were very dependent on the degree of activation of the support. The most stable derivatives, prepared with the most activated supports, were 700 fold more stable than the soluble enzyme in irreversible thermal inactivation experiments, at 40-degrees-C. In contrast, a significant loss of catalytic activity (k(cat) decreases down to 40%) was associated with the increase in stability. Colorimetric titration of amine groups in the stabilized derivatives suggested that enzyme-support multipoint attachment was the main reason for the observed stabilizing effect.