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

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