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 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 (approximately 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 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 nucleotide sequence databases under accession number.
Pollegioni
L
L
Department of Structural and Functional Biology, University of Milano, Varese, Italy.
Molla
G
G
Campaner
S
S
Martegani
E
E
Pilone
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002511:Cephalosporins / Q000378:metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003605:D-Amino-Acid Oxidase / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D012248:Rhodotorula / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D002511:Cephalosporins; D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; C025163:cephalosporin C; D003605:D-Amino-Acid Oxidase