Molecular Cloning and Expression of Uricase Gene from Arthrobacter globiformis in Escherichia coli and Characterization of the Gene Product(GENETICS, MOLECULAR BIOLOGY, AND GENE ENGINEERING)

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Arthrobacter globiformis FERM BP-360 produces uricase (urate oxidase ; EC 1.7.3.3) intracellularly. A genomic library of the bacterium, prepared in the plasmid vector PUC118, was screened with probes based on the amino acid sequence of the purified uricase. We found that a chimeric plasmid in the library, designated pUODl, carries a 2.0-kb DNA insert from the Arthrobacter DNA that hybridizes with the probe. The DNA insert contains an ORF consisting of 302 amino acids with a calculated molecular mass of 33,858. The protein translated from the ORF displays the highest identity (67%) to uricase from a bacterium, Cellulomonas flavigena. X-ray fluorescence analysis showed that the Arthrobacter uricase contains copper ion. However, we found that the catalytic activity of uricase is inhibited by the excessive addition of copper ion. Although the production of A. Globiformis uricase is induced by the addition of uric acid to the culture medium, Escherichia coli harboring pUODl produced 20-fold higher uricase than the original Arthrobacter strain, even without an inducer.
社団法人日本生物工学会の論文
2004-09-25