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

Autori
Rainer, Daniela ; Kratzer, Regina ; Müller, Mario ; Slavica, Anita ; Schiller, Margaretha ; Nidetzky, Bernd

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
ProStab2009, 8th International Conference on Protein Stabilisation, Programme and Abstract Book / - Graz : Research Centre Applied Biocatalysis (A-B), Graz, 2009, 82-82

Skup
8th International Conference on Protein Stabilisation – ProStab2009

Mjesto i datum
Graz, Austrija, 14.04.2009. - 17.04.2009

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

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

Sažetak
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.

Izvorni jezik
Engleski

Znanstvena područja
Biotehnologija

POVEZANOST RADA