海栖热袍菌耐高温β-半乳糖苷酶基因的克隆和表达

Cloning and Expression of a β-galactosidase Gene from Thermotoga maritima

摘要:

本文主要研究了在大肠杆菌中克隆和表达海栖热袍菌(Thermotoga maritima) MSB8的一个b-半乳糖苷酶基因 (TM_0310)。以海栖热袍菌基因组DNA(GenBank登录号AE000512)为模板, 设计特异性引物带有NcoI-HindIII酶切位点, 克隆得到的该基因全序列为2019 bp, 编码672个氨基酸, 分子量为78.972 kD。该基因编码的蛋白质属于42族, 根据氨基酸同源性分析, 该b-半乳糖苷酶与Thermotoga petrophila RKU-1来源的假想的b-半乳糖苷酶(GenBank登录号ABQ46628.1)以及Thermotoga sp. RQ2来源的b-半乳糖苷酶(GenBank登录号EDQ29256.1)同源性最高, 均为95％。重组转化子经诱导酶比活可达2.08 U/mg蛋白。粗酶液经过80℃热处理10 min, 酶活残留70％以上, 表明该酶有很好的耐热性, 在高温工业环境中有良好应用前景。

Abstract:

The cloning and expression of a β-galactosidase gene (TM_0310) from Thermotoga maritima MSB8 was studied. The gene consists of 2019 bp, and the translated protein encodes 672 amino acids and its molecular mass is approximately 78.972 kD. The homology analysis of the deduced amino acid sequences showed that the enzyme shared 95% identity with a putative β-galactosidase from Thermotoga petrophila RKU-1 and a β-galactosidase from Thermotoga sp. RQ2. The galactosidase activity was up to 2.08 U/mg after the recombinant E. coli BL21 was induced by IPTG. The crude lysate remained about 70% activity after treated at 80℃ for 10 min, indicating that the recombinant enzyme is thermostable and may be used at high temperatures.