纳豆芽孢杆菌谷氨酸脱氢酶基因的克隆表达及其生物信息学分析

发布时间：2018-07-20 15:27

【摘要】：克隆表达Bacillus subtilis natto HSF 1410谷氨酸脱氢酶(GDH),测定其酶活性,并阐述其体内功能。将gdhA基因克隆入pET28a质粒在E.coli BL21(DE3)中表达,用生物信息学方法探究其蛋白结构、结构域特点。经测定Bacillus subtilis natto GDH同时具有NADP~+和NAD~+依赖活性,酶活比活力分别为3.140U/mg蛋白和0.251 4U/mg蛋白。生物信息学分析结果表明gdhA基因包含一个长度为1 275bp的开放阅读框,编码424个氨基酸,蛋白分子量为47.05kDa,理论等电点为5.58,不含跨膜区域,二级结构以α-螺旋和无规则卷曲为主。其三级结构表明,该蛋白在45—174位氨基酸残基形成二聚化结构域,191—424位氨基酸残基形成NAD(P)结合位域。B.subtilis natto HSF 1410与E.coli中gdhA的二聚化结构域、NAD(P)结合位域的氨基酸相似度分别为29.23%、29.27%,显著高于B.subtilis 168中gdhA的氨基酸相似度;与B.subtilis 168中gdhA蛋白空间结构相比,B.subtilis natto HSF 1410与E.coli中gdhA在空间结构上更为相似,在体内主要负责促α-酮戊二酸转化成谷氨酸。
[Abstract]:Bacillus subtilis natto HSF 1410 glutamate dehydrogenase (GDH) was cloned and expressed. GdhA gene was cloned into pET28a plasmid and expressed in E. coli BL21 (DE3). The protein structure and domain characteristics were investigated by bioinformatics. It was determined that Bacillus subtilis natto GDH had NADP- and NAD- dependent activities at the same time. The specific activity of the enzyme was 3.140 U / mg protein and 0.251 4 U / mg protein, respectively. Bioinformatics analysis showed that the gdhA gene contained an open reading frame with a length of 1 275bp, encoding 424 amino acids, with a molecular weight of 47.05 kDa, a theoretical isoelectric point of 5.58, and no transmembrane region. The secondary structure was mainly 伪 -helix and irregular curl. Its tertiary structure shows that, The amino acid similarity of natto (P) binding domain of gdhA and that of dimer domain of gdhA in E. coli was 29.23 and 29.27, respectively, and the amino acid binding domain of the protein was formed in the domain of amino acid at the 45174 amino acid residues at position 191-424. The amino acid similarity of the binding domain. B. subtilis natto HSF 1410 was 29.23 and 29.27, respectively, and the amino acid similarity of the binding domain of gdhA was 29.23 and 29.27, respectively. The amino acid similarity of gdhA in B. subtilis 168 was higher than that in B. subtilis 168. Compared with B.subtilis natto HSF 1410 and E. coli, the spatial structure of gdhA protein is more similar to that of B.subtilis 168, and it is mainly responsible for the conversion of 伪 -ketoglutaric acid to glutamic acid in vivo.
【作者单位】：陕西师范大学食品工程与营养科学学院;陕西省科学院酶工程研究所;
【基金】：陕西省科学技术研究发展计划(2015KTCQ02-07) 陕西省科学院科技计划(2011k-13)
【分类号】：Q936;Q811.4

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