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 enzym es against hydrogen peroxide. (a) Multipoint covalent immobilization. Throu gh this technique, enzyme rigidity would be greatly increased and hence, an y conformational change on the enzyme structure involved before or after ox idation with hydrogen peroxide becomes greatly prevented. (b) Oriented immo bilization on supports having large internal surfaces. The immobilization o f 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 grea tly restricted. (c) Additional chemical modification of immobilized enzyme derivatives with polymers. By adding thick barriers surrounding the whole e nzyme molecule, the effective concentration of hydrogen peroxide in the pro ximity of the most sensitive residues may be strongly reduced. Multipoint c ovalently immobilized D-amino acid oxidase (DAAO) from Rhodotorula gracilis on glyoxyl-agarose is Ii-fold more stable than native enzyme against the d eleterious effect of hydrogen peroxide. On the other hand, DAAO from Trigon opsis variabilis was not stabilized by rigidification but it could be highl y stabilized by an adequate combination of the best orientation on the supp ort plus an additional modification with poly-aldehyde polymers. (C) 1999 E lsevier Science B.V. All rights reserved.