Thermostable and highly specific l-aspartate oxidase from Thermococcus litoralis DSM 5473: cloning, overexpression, and enzymological properties

Autor: Tadao Oikawa, Tsubasa Washio
Rok vydání: 2017
Předmět:
Zdroj: Extremophiles. 22:59-71
ISSN: 1433-4909
1431-0651
DOI: 10.1007/s00792-017-0977-4
Popis: We successfully expressed the l-aspartate oxidase homolog gene (accession no: OCC_06611) of Thermococcus litoralis DSM 5473 in the soluble fraction of Escherichia coli BL21 (DE3) using a pET21b vector with 6X His tag at its C-terminus. The gene product (Tl-LASPO) showed l-aspartate oxidase activity in the presence of FAD in vitro, and this report is the first that details an l-aspartate oxidase derived from a Thermococcus species. The homologs of Tl-LASPO existed mainly in archaea, especially in the genus of Thermococcus, Pyrococcus, Sulfolobus, and Halobacteria. The quaternary structure of Tl-LASPO was homotrimeric with a subunit molecular mass of 52 kDa. The enzyme activity of Tl-LASPO increased with temperature up to 70 °C. Tl-LASPO was active from pH 6.0 to 9.0, and its highest activity was at pH 8.0. Tl-LASPO was stable at 80 °C for 1 h. The highest k cat/K m value was observed in assays at 70 °C. Tl-LASPO was highly specific for l-aspartic acid. Tl-LASPO utilized fumaric acid, 2,6-dichlorophenolindophenol, and ferricyanide in addition to FAD as a cofactor under anaerobic conditions. The absorption spectrum of holo-Tl-LASPO exhibited maxima at 380 and 450 nm. The FAD dissociation constant, K d, of the FAD-Tl-LASPO complex was determined to be 5.9 × 10−9 M.
Databáze: OpenAIRE
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