【作者】 李莉莉；

【导师】 刘建；

【作者基本信息】 长春理工大学 ， 生物医学工程， 2010， 硕士

【摘要】 为了获得能用于D-氨基酸工业化生产的D-氨基酰化酶（D-aminoacylase DAA）,我们拟从Microbacterium natoriense菌属中分离出DAA,并对其特性进行了分析。通过多步层析分离技术从TNJL143-2菌株中纯化了DAA,应用SDS-PAGE和层析分离技术分析了DAA的纯度、分子量大小和聚体数;利用酶促反应与TLC（薄层层析）及DAO（D-氨基酸氧化酶）技术检测了DAA的催化底物的特异性、最适酶促反应温度和pH值、以及耐受热、酸、碱、高浓度底物以及金属离子和EDTA的能力;通过蛋白质序列分析仪分析了DAA的氨基酸序列;应用巢式PCR技术从菌体DNA中钓取了目的基因,并推定了蛋白开放阅读框架（ORF）;对比了TNJL143-2菌株DAA与其它菌株DAA的同源性。结果显示获得的DAA为单聚体,其分子量为56kDa,对于N-乙酰-D-氨基酸底物具有高活性。催化反应的相对活性为46.4 U/mg、最适pH值为7.0、最适温度为50℃、以及在上述条件下反应1小时后仍具有85%残存酶活性。相对耐受Ca2⁺、Mn²⁺、Co²⁺和Cd²⁺和EDTA。该DAA的氨基酸序列与来自Alcaligenes xylosoxydans A-6、Alcalygenesfacelis DA1和paradoxus Iso1的DAA分别具有为25%、24%和26%的同源性。表明从Microbacterium natoriense TNJL143-2菌株中分离的DAA具有独特的结构和功能特性,以及潜在的应用价值。更多还原

【Abstract】 A D-aminoacylase (DAA) was found in a novel Microbacterium natoriense TNJL143-2 strain that isolated from soil in Japan miyagi natrori. The enzyme was purified by multistep chromatography. The native molecular mass was 56kDa, which agree with predicted molecular mass 56 kDa and the enzyme appered to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 46.4 U/mg was finally obtained. The enzyme had an optimal pH and temperature of 7.0 and 50℃, respectively, after 1 hour heat treatment at 45℃at pH 7.0 85% activity remained. The DAA had higher hydrolyzing activity aganst N-acetyl-D-amino acid. After N-terminal and internal peptide sequending by LC/MS/MS, and designing the degenerate primers according to the N-terminal and internal peptide sequence. The gene was cloned and sequenced. The gene consisted of a 1485-bp ORF encoding a polypeptide of 495 amino acid. The M. natorines DAA showed a low amino acid similarity to Alcaligenes xylosoxydans A-6 (25%), Alcalygenes facelis DA1 (24%), and paradoxus Isol (26%).更多还原