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首页> 外文期刊>Journal of bacteriology >Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production
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Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

机译：嗜热古细菌激烈热球菌丙氨酸氨基转移酶的纯化，鉴定及其在丙氨酸生产中的作用

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Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosusby multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6.5 to 7.8 and at a temperature of over 95°C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in theP. furiosus genome database. The gene was expressed inEscherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. Thek cat/Km values for alanine and pyruvate formation were 41 and 33 s?1mM?1, respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aatgene. In addition, both the aat and gdh(encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdhgenes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.
机译：通过多步色谱法从嗜热古细菌 Pyrococcus furiosus 的细胞提取物中纯化出丙氨酸氨基转移酶（AlaAT）。经凝胶过滤估计，该酶的表观分子量为93.5 kDa，由十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和基因序列推导的两个相同的46 kDa亚基组成。与来自真核来源的AlaAT相比，AlaAT表现出更广泛的底物特异性，并以丙氨酸，谷氨酸和天冬氨酸（以2-氧戊二酸酯或丙酮酸为氨基受体）表现出显着的活性。发现最佳活性在pH范围6.5至7.8和温度超过95°C的情况下。测定了纯化的AlaAT的N末端氨基酸序列，并使得能够鉴定 P中编码AlaAT的基因（ aat ）。 furiosus 基因组数据库。该基因在大肠埃希菌中表达，并纯化了重组酶。重组体的pH和温度依赖性，分子量和动力学参数与来自 P的天然酶的pH和温度依赖性没有区别。愤怒。丙氨酸和丙酮酸形成的 k cat / K m 值为41和33 s ？1 mM ？1 分别表明该酶不偏向于丙酮酸或丙氨酸的形成。 Northern分析确定了 aat 基因的一个1.2kb转录物。此外， aat 和 gdh （编码谷氨酸脱氢酶）的转录本似乎在转录水平上均受调控，因为这两种基因的表达都是在细胞被诱导时被诱导的。生长在丙酮酸上。发现 aat 和 gdh 基因的协调控制与这些酶协同作用以在 P中形成电子库的行为非常吻合。愤怒。

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