Cloning, sequencing and expression in E. coli of a d-amino acid oxidase cDNA from Rhodotorula gracilis active on cephalosporin C

Abstract

We have cloned the cDNA coding for the Rhodotorula gracilis d-amino acid oxidase (DAAO), an enzyme that performs with high catalytic efficiency biotechnologically relevant bioconversions, by PCR amplification. The first strand cDNA was synthesised from the total mRNA fraction isolated from R. gracilis cells grown under DAAO-inducing conditions. The R. gracilis DAAO cDNA consists of 1104 bp encoding a protein of 368 amino acids. The insertion of the cDNA into the pKK223-3 plasmid allowed the expression of recombinant DAAO in Escherichia coli as a wholly soluble and catalytically active holoenzyme (≈0.5 U mg −1 protein) with a fermentation yield, in terms of DAAO units, of 800 U 1 −1. This level of expression allowed the purification, in homogeneous form and high yield (50%), of the recombinant enzyme which showed a high catalytic activity on cephalosporin C as substrate. The nucleotide sequence reported in this paper will appear in the GenBank nucleotide sequence databases under accession number U60066.