A thermostable L-aspartate oxidase: a new tool for biotechnological applications.
L-Amino acid oxidases (LAAOs) are homodimeric flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Unlike the D-selective counterpart, the biotechnological application of LAAOs has not been thoroughly advanced because of the difficulties in their expression as recombinant protein in prokaryotic hosts. In this work, L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, specific for L-aspartate and L-asparagine only) was efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. This recombinant flavoenzyme shows the classical properties of FAD-containing oxidases. Indeed, StLASPO 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. This latter property significantly distinguishes StLASPO from the E. coli counterpart. StLASPO represents an appropriate novel biocatalyst for the production of D-aspartate and a well-suited protein scaffold to evolve a LAAO activity by protein engineering.
Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, Via J.H. Dunant 3, Varese, Italy.
Davide
D
Bifulco
Loredano
L
Pollegioni
Davide
D
Tessaro
Stefano
S
Servi
Gianluca
G
Molla
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D003067:Coenzymes / Q000378:metabolism; D026603:D-Aspartic Acid / Q000378:metabolism; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D062907:Oxaloacetic Acid / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D003067:Coenzymes; D019641:DNA, Archaeal; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D062907:Oxaloacetic Acid; D026603:D-Aspartic Acid; D000594:Amino Acid Oxidoreductases; C031955:L-aspartate oxidase, E coli