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標題: Bacillus sp. TS-23 dihydropyrimidinase 基因的選殖與酵素性質分析
Cloing of the dihydropyrimidinase gene from Bacillus sp. TS-23 and characterization of enzyme properties
作者: 許偉意
Hsu, Wei-Yi
關鍵字: dihydropyrimidinase;醯亞胺水解酵素
出版社: 分子生物學研究所
本研究嘗試從嗜熱性Bacillus sp. 選殖耐熱的D-hydantoinase 基因。比對目前已經發表的 D-hydantoinase基因的片段,設計兩條degenerate primers,以六株嗜熱性Bacillus spp.的染色體當模板,利用聚合酉每連鎖反應(Polymerase Chain Reaction),分別得到 6條 330 bp 左右的DNA片段,經 DNA 定序證實為 D-hydantoinase 基因片段。利用這些片段當作探針,從Bacillus sp. TS-23 genomic library中篩選到一個純株 (clone),攜帶質體 pBK-CMV-5,能在Escherichia coli 菌體表現D-hydantoinase活性。經過 DNA 定序發現: pBK-CMV-5 含有 3.8 kb的嵌入片段,包含三個基因可能分別為: dihydropyrimidine dehydrogenase、dihydropyrimidinase及 transcription regulator。根據前人研究推測,前兩個酵素可能參與 pyrimidine 的降解,而 transcription regulator的角色不明。
Dihydropyrimidinase 基因全長為 1,422 bp,可轉譯出472個胺基酸殘基,與 Bacillus stearothermophilus NS1122A的dihydropyrimidinase 相似性達 92 %,而與人類的 dihydropyrimidinase 相似性為 45% 。將Bacillus sp. TS-23 dihydropyrimidinase 基因選殖入表現載體 pQE-30 中大量表現,純化回收酵素並經過透析,發現此酵素在 Co2+ 離子存在時,活性會提高十四倍;Mn2+ 存在時,活性提高五倍。此酵素活性不受 EDTA影響,但 2,6-dipicolinic acid 會完全抑制酵素活性。酵素最適反應溫度及最適 pH 值分別為 60℃ 及 8.0 。在含 dihydropyrimidinase的緩衝液 (50mM Tris-HCl, pH 8.0) 中添加 500 μM 的 Co2+ 離子,於 50℃ 保溫 30 天,酵素仍保有 22% 活性,其半衰期約為 25 天。 Bacillus sp. TS-23 dihydropyrimidinase 對於 dihydrouracil 的比活性為3.46 U/mg,對於D,L-methylthioethylhydantoin、 D,L-p-hydroxyphenylhydantoin (D,L-p-HPH) 及 hydantoin 的比活性分別為1.69 U/mg、0.62 U/mg 及 0.02 U/mg,對 1-n-butylhydantoin 及 1-methylhydantoin 則不具催化能力。酵素動力學分析結果發現, dihydropyrimidinase 對 dihydrouracil 及hydantoin 的催化效率 (Kcat/Km),分別為15.6 min-1mM-1 及 2 ×10-3 min-1mM-1。

Two degenerate primers were designed from the alignment of the highly conserved amino acid sequences of bacterial D-hydantoinase genes. DNA fragments were amplified by polymerase chain reaction from chromosomal DNA of six thermophilic Bacillus spp. using degenerate primers and confirmed to be a part of D-hydantoinase genes. Using these DNA fragments as probe, Escherichia coli XLOLR (pBK-CMV-5) exhibited D-hydantoinase activity was isolated from Bacillus sp. TS-23 genomic library by plaqe hybridization. A 3.8 kb insert of pBK-CMV-5 containing three putative genes encoding dihydropyrimidine dehydrogenase, dihydropyrimidinase and transcription regulator was found. The former two genes were proposed to be involved in the pyrimidine degradation, whereas the role of the transcription regulator is unclear.
The ORF of dihydropyrimidinase gene consisted of 1,422 bp and deduced to contain 472 amino acids with a molecular mass of 52 kDa. The amino acid sequence showed 92% identity with dihydropyrimidinase of Bacillus stearothermophilus NS1122A. The dihydropyrimidinase gene was cloned into pQE30 and overexpressed in E. coli NovaBlue. His- tagged dihydropyrimidinase was purified by immobilized metal affinity chromatography and dialyzed. The enzyme activity was enhanced by Co2+ and Mn2+ ions. The optimal pH and temperature for the catalytic activity were 8.0 and 60℃, respectively. The thermostability of purified dihydropyrimidinase was determined in 50mM Tris-HCl (pH 8.0) containing 500μM CoCl2 and incubated at 50℃. The results showed that the half-life of the enzyme is 25 days and 22% activity still retained after 30 days incubated at 50℃. This dihydropyrimidinase was most active toward dihydrouracil, while less active in the catalysis of D,L-methylthioethylhydantoin, D,L-p-hydroxyphenylhydantoin (D,L-p-HPH) and hydantoin. No catalytic activity was found with 1-methylhydantoin and 1-n-butylhydantoin as substrates. The Kcat/Km of the dihydropyrimidinase for dihydrouracil and hydantoin was 15.6 min-1mM-1 and 2×10-3 min-1mM-1,respectively.
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