Cloning, sequencing and expression of D-amino acid oxidase gene

The D-amino acid oxidase gene(daao) was cloned from the total DNA of Trigonopsis vairabilis by intron-deleting PCR amplification and TA cloning methods. The daao gene consists of 1071bp encoding a protein of 357 amino acids. Comparing its nucleotide and amino acid sequences with DAAOs from other sources, considerable homologies were observed. The insertion of the daao gene into the high-expression vector pET-28b allowed the expression of recombinant DAAO in E. coli as a soluble and catalytically active enzyme with a fermentation yield, in terms of DAAO units, of 802u/L.