Molecular cloning and expression of uricase gene from Arthrobacter globiformis in Escherichia coli and characterization of the gene product.
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 pUOD1, 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 pUOD1 produced 20-fold higher uricase than the original Arthrobacter strain, even without an inducer.
Suzuki
Koji
K
Department of Chemistry and Materials Engineering, Ibaraki National College of Technology, 866 Nakane, Hitachinaka, Ibaraki 312-8508, Japan. ksuzuki@chem.ibaraki-ct.ac.jp
Sakasegawa
Shin-Ichi
S
Misaki
Hideo
H
Sugiyama
Masanori
M