CLONING, SEQUENCING AND EXPRESSION IN E-COLI OF A D-AMINO-ACID OXIDASE CDNA FROM RHODOTORULA-GRACILIS ACTIVE ON CEPHALOSPORIN-C

We have cloned the cDNA coding for the Rhodotorula gracilis D-amino ac id oxidase (DAAO), an enzyme that performs with high catalytic efficie ncy biotechnologically relevant bioconversions, by PCR amplification. The first strand cDNA was synthesised from the total mRNA fraction iso lated from R. gracilis cells grown under DAAO-inducing conditions. The R. gracilis DAAO cDNA consists of 1104 bp encoding a protein of 368 a mino acids. The insertion of the cDNA. into the pKK223-3 plasmid allow ed the expression of recombinant DAAO in Escherichia coli as a wholly soluble and catalytically active holoenzyme (approximate to 0.5 U mg(- 1) protein) with a fermentation yield, in terms of DAAO units, of 800 U l(-1). This level of expression allowed the purification, in homogen eous form and high yield (50%), of the recombinant enzyme which showed a high catalytic activity on cephalosporin C as substrate. The nucleo tide sequence reported in this paper will appear in the GenBank nucleo tide sequence databases under accession number U60066. (C) 1997 Elsevi er Science B.V.