title: The stabilizing effects of immobilization in D-amino acid oxidase from Trigonopsis variabilis.
abstract: BACKGROUND
  Immobilization of Trigonopsis variabilis D-amino acid oxidase (TvDAO) on solid support is the key to a reasonably stable performance of this enzyme in the industrial process for the conversion of cephalosporin C as well as in other biocatalytic applications.
  
  
  RESULTS
  To provide a mechanistic basis for the stabilization of the carrier-bound oxidase we analyzed the stabilizing effects of immobilization in TvDAO exposed to the stress of elevated temperature and operational conditions. Two different strategies of immobilization were used: multi-point covalent binding to epoxy-activated Sepabeads EC-EP; and non-covalent oriented immobilization of the enzyme through affinity of its N-terminal Strep-tag to Strep-Tactin coated on insoluble particles. At 50 degrees C, the oriented immobilizate was not stabilized as compared to the free enzyme. The structure of TvDAO was stabilized via covalent attachment to Sepabeads EC-EP but concomitantly, binding of the FAD cofactor was weakened. FAD release from the enzyme into solution markedly reduced the positive effect of immobilization on the overall stability of TvDAO. Under conditions of substrate conversion in a bubble-aerated stirred tank reactor, both immobilization techniques as well as the addition of the surfactant Pluronic F-68 stabilized TvDAO by protecting the enzyme from the deleterious effect of gas-liquid interfaces. Immobilization of TvDAO on Sepabeads EC-EP however stabilized the enzyme beyond this effect and led to a biocatalyst that could be re-used in multiple cycles of substrate conversion.
  
  
  CONCLUSIONS
  Multi-point covalent attachment of TvDAO on an isoluble porous carrier provides stabilization against the denaturing effects of high temperature and exposure to a gas-liquid interface. Improvement of binding of the FAD cofactor, probably by using methods of protein engineering, would further enhance the stability of the immobilized enzyme.
authors.affiliation: Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria. iskandar.dib@gmx.at
authors.forename: Iskandar
authors.initials: I
authors.lastname: Dib
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D002384:Catalysis; D002511:Cephalosporins / Q000737:chemistry; D003605:D-Amino-Acid Oxidase / Q000737:chemistry*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D020442:Poloxamer / Q000737:chemistry; D015202:Protein Engineering; D004718:Saccharomycetales / Q000201:enzymology*; D013696:Temperature
chemical_list: D002511:Cephalosporins; D004800:Enzymes, Immobilized; D020442:Poloxamer; C025163:cephalosporin C; D003605:D-Amino-Acid Oxidase
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