請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/21670
標題: Cloning and expression of the D-hydantoinase from Bacillus circulans and characterization of the enzyme
Bacillus circulans D-hydantoinase 基因的選殖、表現及酵素特性
作者: 高肇鴻
Kao, Chao-Hung
關鍵字: D-hydantoinase
海因酉每
N-carbamoyl-D-amino acid amidohydrolase
Cloning
Expression
水解酉每
選殖
表現
出版社: 分子生物學研究所
摘要: 比對已知的 D-hydantoinase 基因的高保留區域,根據其DNA 序列設計引子,以 Bacillus circulans CCRC 12254 的染色體作為模版,利用聚合酉每鏈鎖反應,可以合成一 DNA 片段。以此片段作為探針 (probe),於B. circulans 的染色體基因庫 (genomic library),以溶菌斑雜交法 (plaque hybridization) 篩選出一個在 E. coli 菌體內可表現 D-hydantoinase 活性的純株 (clone) pBK-CMV-6。經由 DNA 序列分析,顯示 pBK-CMV-6 含有 5.0 kb 的嵌入片段,並發現含有三個很接近的 open reading frame (ORF),分別為 dihydropyrimidine dehydrogenase、D-hydantoinase (dihydropyrimidinase)及 β-ureidopropionase。這三個酵素可能參與 pyrimidine 的降解,且此三個基因的轉錄方向相同,形成基因叢集 (cluster)。 D-hydantoinase 基因 ORF 全長為1,386 bp,可轉譯出含 461 個胺基酸殘基的蛋白質、其分子量為 51 kDa。其胺基酸殘基序列與其他的 D-hydantoinase 相同性達 45~70% 之間。將 D-hydantoinase 基因選殖入表現載體 pQE-30 中,並使其在 E. coli 中表現,隨後,純化 D-hydantoinase 酵素,探討其酵素特性,發現Mn2+、Co2+ 或 Ni2+ 離子存在時,活性會提高,而Zn2+ 離子則會抑制酵素活性。D-hydantoinase 對 H2O2 的氧化作用有很高的抗性,且不受 NH4+的回饋抑制。酵素的最適反應pH 及溫度分別為 8.5 及 60℃。在D-hydantoinase 酵素中添加 500 μM Mn2+ 離子,於 50℃ 中保溫 30 天,酵素仍保有 60% 的活性。B. circulans的 D-hydantoinase 對於 dihydro-uracil、Hydantoin及 D,L-p-hydroxyphenylhydantoin (D,L-p-HPH) 的催化活性較高,對於 D,L-methylthioethylhydantoine 較低;而對於 1-methyl-hydantoin、1-p-butylhydantoin則不具催化能力。以酵素動力學分析 D-hydantoinase 對 dihydrouracil 及 D,L-p-HPH 的催化效率,其 Kcat/Km 分別為 122.2 min-1mM-1 及 26.3 min-1mM-1。
Two primers were designed from the conserved sequences of D-hydantoinase genes. A DNA fragment was amplified from Bacillus circulans CCRC 12254 chromosomal DNA using polymerase chain reaction. Using the PCR amplified DNA fragment as the probe, an Escherichia coli XLOLR (pBK-CMV-6) clone that exhibited D-hydantoinase activity was selected from the B. circulans genomic library with plaque hybridization. Plasmid pBK-CMV-6 contains a 5.0 kb insert from B. circulans genomic DNA. DNA sequencing of the insert DNA revealed three closely spaced open reading frames predicted to encode dihydropyrimidine dehydrogenase, D-hydantoinase (dihydropyrimidinase) and β-ureidopropionase. These three genes were transcribed in the same orientation and proposed to be involved in the pyrimidine degradation. The ORF of D-hydantoinase gene consisted of 1,386 bp and was predicted to encode a peptide of 461 amino acids, with a calculated molecular mass of about 51 kDa. The amino acid sequence showed 45~70% identity with other D-hydantoinases. The D-hydantoinase gene was cloned into pQE-30 and expressed in E. coli NovaBlue. D-hydantoinase was purified by metal affinity chromatography. The enzyme activity was enhanced by Mn2+, Co2+ or Ni2+ ions and inhibited by Zn2+ ion. The enzyme was resistant to H2O2 oxidation and NH4+ ion inhibition. The optimal pH and temperature for the catalytic activity were 8.5 and 60℃, respectively. After incubation at 50℃ for 30 days, about 60% of enzyme activity was still retained in a reaction mixture containing 500 μM Mn2+ ion. The D-hydantoinase from B. circulans was most active in the catalysis of dihydrouracil, hydantoin and D,L-p-hydroxyphenyl-hydantoin, while less active in the catalysis of D,L-methylthioethylhydantoin. No catalytic activity was exhibited with 1-methyl-hydantoin and 1-p-butylhydantoin as substrates. The Kcat/Km of the D-hydantoinase for dihydrouracil and D,L-p-hydroxyphenylhydantoin was 122.2 min-1mM-1 and 26.3 min-1mM-1, respectively.
URI: http://hdl.handle.net/11455/21670
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