engleski

Influence of sulfhydryl-specific modification on stability of D-amino acid oxydase from Trigonopsis variabilis

The flavoprotein D-amino acid oxydase (DAO ; EC 1.4.3.3) catalyzes the oxydation of D-amino acids to their corresponding imino acids that hydrolyze spontaneously to the alpha-keto acids and ammonia. Reoxidation of the flavin cofactor by molecular oxygen is accompanied by the formation of H2O2. DAO displays a broad substrate specificity, making it an interesting and widely used biocatalyst. The most important present application of immobilized DAO is in the conversion of cephalosporin C into glutaryl-7-aminocephalosporanic acid (Gl-7-ACA) for the large-scale production of several commercial cephalosporins. Other relevant applications include the detection of alpha-D-amino acids in chemical and biological samples, and the production of chiral intermediates fro drugs. Immobilized DAO provides a good example of a biocatalyst which stability is not fully satisfactory for the industrial process and can be influenced by different factors. Many efforts have thus been made to extend the half-life of DAO under the operational conditions. We have investigated the stability of soluble DAO from the yeast Trigonopsis variabilis (TvDAO) using a direct oxygen-independent assay to determine enzyme activity. TvDAO is shown to follow a complex kinetic mechanism of dentauration and displays significantly increased stability when antioxidant, thiol-protecting reagent, and an excess of cofactor (FAD) are added to the reaction mixture. To elucidate the mechanism underlying the inactivation, a technical grade preparationof TvDAO was fractionated into two main enzymatically active components, F1 and F2, which were characterized. F1 represents TvDAO component with lower overall polarity, higher specific activity, and it follows a two-step mechanism of inactivation. In F1 all six cysteine residues and in F2 five cysteines are reduced and available for reaction with Ellmans reagent in the folded state of the protein, and for alkylation by iodoacetamide in denatured preparation. Tandem mass spectometry revealed that oxidation of Cys-108 distinguishes F2 from F1, whereas other cysteine residues remained unmodified. Coupling of PEG-maleimide to protein thiol groups was used for preparing well defined, active and more stable conjugates.

D-amino acid oxydase; Trigonopsis variabilis; oxygen-independent assay; two enzymatically active components; Cys-108

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