Porcine D-amino acid oxidase: Production of the biologically active enzyme in Escherichia coli

Abstract

DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.

Triple fusion of d-amino acid oxidase from Trigonopsis variabilis with polyhistidine and Vitreoscilla hemoglobin

“…Of different sources (Pollegioni et al 2004), the DAAOs from porcine kidney (pKDAAO; Ciccarelli et al 1989), Rhodotorula gracilis (RgDAAO; Alonso et al 1998), and Trigonopsis variabilis (TvDAAO; Gonzalez et al 1997;Pollegioni et al 2004) are more interesting in kinds of applications. High-level expression of RgDAAO and TvDAAO (28% homology identity) in Escherichia coli (Hwang et al 2000;Lin et al 2000) is specially emphasized due to its important industrial applications, especially the enzymatic conversion of Cephalosporin C to glutaryl 7-ACA, the intermediate for cephalosporin antibiotics backbone of 7-ACA.…”