engleski

Immobilization of D-amino acid oxidase and two oxidation-resistant variants thereof

D-Amino acid oxidase from the yeast Trigonopsis variabilis (TvDAO) has a long-standing history in industrial biocatalysis. The enzyme was successfully applied in the conversion of cephalosporin C to 7-aminocephalosporanic acid and has now found viable alternative uses in the preparation of chiral amino acids. TvDAO shows absolute enantioselectivity for a-amino acids in R-configuration. The specificity with respect to the structure of the amino acid side chain is however extremely relaxed, allowing a diversity of substrates to be converted. Although TvDAO is a reasonably robust catalyst, even in the presence of the oxidants present in the process (O2, H2O2), interest has been high in developing a more stable form of the enzyme that can be reused easily. Immobilization on a solid carrier was often the preferred choice. Dissociation of the cofactor FAD can limit the stability of immobilized TvDAO [1]. We have found in earlier work [1] that Cys108 of TvDAO can undergo irreversible oxidation into a stable Cys sulfinic acid. This oxidative modification leads to partial loss of enzyme activity and stability. A more rapid overall release of the cofactor FAD from oxidized compared to native enzyme is responsible for the decrease in stability [2]. Site-directed mutagenesis of Cys108 into the non-oxidized residues Ser and Asp were used in an effort to mimic the native and oxidized form of TvDAO. We present results of a characterization of C108S and C108D variants with respect to activity and stability in solution. The two mutants were immobilized on epoxy-activated Sepabeads and the stabilities of carrier-bound forms of wild-type [3] and mutated TvDAO were analyzed.

D-amino Acid Oxidase; Two Oxidation-resistant Variants of the enzyme; Immobilisation

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