Abstract
Myrothecium verrucaria bilirubin oxidase (EC 1.3.3.5) is an enzyme catalyzing the oxidation of bilirubin to biliverdin and other substrates. We have purified bilirubin oxidase from the medium of M. verrucaria and determined its partial amino acid sequence and isolated cDNA fragment amplified by polymerase chain reaction using oligonucleotide primers designed on the basis of the partial amino acid sequence. The gene for bilirubin oxidase has been cloned from a genomic library using the cDNA fragment as a probe. The gene encodes a precursor of bilirubin oxidase consisting of 572 amino acid residues, which comprises the prepro-region of 38 amino acid residues and the mature enzyme of 534 amino acid residues containing one cysteine. Five introns were found within the coding region. Sequence comparison of bilirubin oxidase with other blue copper proteins (laccase, ascorbate oxidase, human ceruloplasmin, plastocyanin, and azurin) revealed the presence of four domains corresponding to potential copper ligands. We have expressed this bilirubin oxidase gene in Saccharomyces cerevisiae under the repressible acid phosphatase promotor and found an active recombinant bilirubin oxidase, establishing the functional identity of the gene.
Highlights
From the $Tsukuba Research Laboratories,A m a m Pharmaceutical Co., Ltd., 22 Miyukigmka, Tsukubq &ragi 305,Japan, TDepartment of Chemistry, College of Science and Engineering, AoyamaGakuin University, Setagaya-ku,Tokyo 157,Japan, and the Department of $$Applied Microbial Techmbgy, TheKumanwto Znstitute of Techmbgy, Kumanwto 860, Japan
This enzyme has been expected to be used in cloned from a genomic library using the cDNA fragment amplified by polymerase chain reaction (PCR)’ as a probe
This result indicates that the nucleotide bilirubin oxidase appear to be related to the binding of the eequence ofcDNA amplified by PCR is correct
Summary
Dept. of Chemistry, Faculty of Science, Tokyo medium (potato extract containing 1%glucose) for 3 days [1]. To design the PCR primers on the basis of the amino acid sequence for cDNA amplification, S-carboxymethylated enzyme and its fragments were prepared. The digest was runon 1% agarose gel and the 1.6-kbp XhoI-EcoRV fragment, which contains the sequence encoding a-prepro-bilirubin oxidase fusion protein, and the phosphoglycerate kinase terminator was isolated. This fragment was subcloned between the XhoI and PvuII sites located at the downstream side of the repressible acid phosphatase (PH05) protrifluoroacetic acid in 80% acetonitrile)in solution A (0.1% (v/v) moter of the yeast-E. coli shuttle expression vector pAM82 yielding trifluoroacetic acid in H20) a t a flow rate of 0.5 ml/min
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