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

Cloning,expression and bioinformatics analysis of glutamate dehydrogenase cDNA fromBacillus subtilis natto

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。

The gdhA gene of B. subtilis natto was cloned into plasmid pET28a and expressed in E. coli BL21 （DE3）, and explored the protein structure, structure domains features using bioinfor- matics methods. This GDH has both NADP＋-dependent and NAD＋-dependent enzymatic activi- ties, which were 3. 140 U/mg protein and 0. 251 4 U/mg protein, respectively. The results of in- formatics analysis showed that the coding region sequence contains a complete ORF （1 275 bp） which encoding 424 amino acids. The molecular weight was 47. 05 kDa and theoretical pI was 5.58. The gdhA protein had no transmembrane regions. Its secondary structure was composed of a-helix and random coil. And its tertiary structure showed that the protein formed a dimerization domain and a NAD （P） binding domain in the 45 174 and 191-424 amino acid residues,respec- tively. The amino acid similarities of dimerization domains and NAD （P） binding domains of B. subtilis natto HSF 1410 and E. coli gdhA protein were 29.23% and 29.27% ,respectively. These were s gdhA, baeteri lgn B. ifieantly higher than gdhA amino subtilis natto HSF 1410 and E. co a is mainly responsible for promoting acids of B. subtilis 168. Compared to B. subtilis 168 li gdhA are more similar in the space structure, the α- ketone glutaric acid into glutamic acid.