Abstract
Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases (DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic D-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus D-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His6-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg(-1) protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 degrees C, respectively. The half-life of His6-tagged DHP was 25 days at 50 degrees C. The enzyme activity was stimulated by Co2+ and Mn2+ ions. His6-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies (kcat/Km) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s(-1) mM(-1), respectively.
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