Stabilization of enzymes (D-amino acid oxidase) against hydrogen peroxide via immobilization and post-immobilization techniques

Three different approaches are proposed to increase the resistance of enzymes against hydrogen peroxide. (a) Multipoint covalent immobilization. Through this technique, enzyme rigidity would be greatly increased and hence, any conformational change on the enzyme structure involved before or after oxidation with hydrogen peroxide becomes greatly prevented. (b) Oriented immobilization on supports having large internal surfaces. The immobilization of enzymes, through different areas of their surface on solid supports with internal morphology composed by large surfaces, promotes a certain masking of the enzyme areas that are very close to the support surface. In this way, the accessibility of hydrogen peroxide to such protein areas becomes greatly restricted. (c) Additional chemical modification of immobilized enzyme derivatives with polymers. By adding thick barriers surrounding the whole enzyme molecule, the effective concentration of hydrogen peroxide in the proximity of the most sensitive residues may be strongly reduced. Multipoint covalently immobilized D-amino acid oxidase (DAAO) from Rhodotorula gracilis on glyoxyl-agarose is Ii-fold more stable than native enzyme against the deleterious effect of hydrogen peroxide. On the other hand, DAAO from Trigonopsis variabilis was not stabilized by rigidification but it could be highly stabilized by an adequate combination of the best orientation on the support plus an additional modification with poly-aldehyde polymers. (C) 1999 Elsevier Science B.V. All rights reserved.