地衣芽孢杆菌谷氨酸消旋酶基因克隆、原核表达及生物信息学分析

Cloning, prokaryotic expression and bioinformatics analysis of glutamic acid racemase gene of Bacillus licheniformis

为了进一步分析谷氨酸消旋酶的结构和生物信息功能,试验以γ-PGA生产菌株地衣芽孢杆菌(Bacillus licheniformis)DY136为材料,PCR扩增其谷氨酸消旋酶基因(racE),纯化racE基因进行原核表达,并运用多种在线生物信息学分析软件对该基因编码蛋白质的理化性质、亲/疏水性、信号肽、结构域等进行分析。结果表明:PCR扩增racE基因条带大小为819 bp,对racE基因进行大肠杆菌原核表达,通过构建表达载体和优化表达条件,成功表达了大小为30.15 ku的重组融合蛋白。racE蛋白编码272个氨基酸,氨基酸序列与NCBI中公布的地衣芽孢孢杆菌racE蛋白(GenBank登录号为KAA0845975.1)氨基酸同源性为99.62%。racE蛋白理化性质稳定,是亲水蛋白,无跨膜结构域,不含有信号肽,α-螺旋是其主要的二级结构,主要定位于细胞质中(73.90%)。说明racE基因保守且相对稳定,在谷氨酸的合成中发挥重要作用。

In order to further analyze the structure and bioinformatics function of glutamate racemase, the experiment used γ-PGA-producing strain Bacillus licheniformis DY136 as the material;the glutamic acid racemase gene racE was amplified by PCR, the target gene was purified for prokaryotic expression, and a variety of online bioinformatics analysis softwares were used to analyze its physicochemical properties, hydrophilicity/hydrophobicity, signal peptide, structural domain, etc. The results showed that the size of the PCR-amplified racE gene was 819 bp. Prokaryotic expression of the racE gene was carried out in E. coli;by constructing an expression vector and optimizing expression conditions, a recombinant fusion protein with a size of 30.15 ku was successfully expressed. racE protein encoding 272 amino acids. The amino acid sequence was 99.62% homologous to the Bacillus licheniformis racE protein published in NCBI(GenBank registration number KAA0845975.1). racE protein was stable in physicochemical properties, which was a hydrophilic protein without transmembrane domain and signal peptide. α-helix was its main secondary structure, which was mainly located in the cytoplasm(73.90%). The results suggested that the racE gene was conserved and relatively stable, and played an important role in the synthesis of glutamate.