L-Amino acid oxidases (LAAOs, EC 1.4.3.2) catalyze the stereospecific oxidative deamination of L-amino acids to alpha-ketoacids, ammonia and hydrogen peroxide. However LAAOs have not been widely employed in biotechnological applications because of the difficulties in their expression as recombinant proteins in prokaryotic hosts. In this work L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, E.C.1.4.3.16) has been efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. Recombinant StLASPO shows the classical properties of FAD-containing oxidases. Indeed, it shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80 °C) and high temperature optimum, stable activity in a broad range of pH (7.0–10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. Studies of immobilization on different supports have been also carried out in order to allow the recyclability of the enzyme for potential preparative purposes. The best immobilization yield was achieved using an epoxidic resin, high ionic strength and a temperature > or equal to 24 °C. Because of the very interesting properties of this enzyme, we propose StLASPO as an attractive tool for biotechnological applications: it could be amenable of scale-up and innovative industrial processes for the efficient resolution of D,L-aspartate mixtures. Indeed, it seems a well-suited protein scaffold to evolve a LAAO activity with broader substrate specificity by protein engineering, as previously performed for D-amino acid oxidase.

Studies on Racemic Resolution of D,L-aspartate by StLASPO

D'ARRIGO, PAOLA;TESSARO, DAVIDE;SERVI, STEFANO;CERIOLI, LORENZO;FIORATI, ANDREA;
2013-01-01

Abstract

L-Amino acid oxidases (LAAOs, EC 1.4.3.2) catalyze the stereospecific oxidative deamination of L-amino acids to alpha-ketoacids, ammonia and hydrogen peroxide. However LAAOs have not been widely employed in biotechnological applications because of the difficulties in their expression as recombinant proteins in prokaryotic hosts. In this work L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, E.C.1.4.3.16) has been efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. Recombinant StLASPO shows the classical properties of FAD-containing oxidases. Indeed, it shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80 °C) and high temperature optimum, stable activity in a broad range of pH (7.0–10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. Studies of immobilization on different supports have been also carried out in order to allow the recyclability of the enzyme for potential preparative purposes. The best immobilization yield was achieved using an epoxidic resin, high ionic strength and a temperature > or equal to 24 °C. Because of the very interesting properties of this enzyme, we propose StLASPO as an attractive tool for biotechnological applications: it could be amenable of scale-up and innovative industrial processes for the efficient resolution of D,L-aspartate mixtures. Indeed, it seems a well-suited protein scaffold to evolve a LAAO activity with broader substrate specificity by protein engineering, as previously performed for D-amino acid oxidase.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11311/762853
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