摘要
本文拟克隆新阿波罗栖热袍菌(Thermotoga neapolitana DSM 4359)的一种α-葡萄糖苷酶基因(TNAG),其GenBank注册号为lcl27401。首先以T.neapolitana DSM 4359基因组DNA为模板,通过PCR扩增目的基因并完成T克隆,在NCBI数据库中比对结果表明:目的基因与Thermotoqa sp.RQ2等氨基酸序列相似度高于99%。再将目的基因连接至表达载体pET-32a(+),并导入大肠杆菌Rosetta中用IPTG诱导表达。SDS-PAGE显示表达蛋白的大小约为72KDa。热纯化后酶液测活反应的最适温度为80℃,最适pH在5.0左右。
To produce a kind of Thermotoga neapolitana DSM 4359 α-glucosidase（TNAG） in large quantity which GenBank registration number was lcl27401,the gene was cloned and expressed in Escherichia coli.First,the genome DNA of T.neapolitana DSM 4359 as a template and through the PCR amplification target gene and complete the T cloning,the results of database contrast in NCBI indicated： the similarity of amino acid sequences of target gene and Thermotoqa sp.RQ2 was more than 99%.Then the target gene was connected to expression vector pET-32a（＋） and E.coli Rosetta was imported with isopropyl β-D-1-thiogalactopyranoside（IPTG） induction expression.A obvious protein band of about 72 KDa was displayed by sodium dodecyl sulfate polyaclamide gel electrophoresis（SDS-PAGE）.After heat treaments,the enzyme＇s optimal temperature and pH were 80℃ and about 5.0.
出处
《食品与发酵科技》
CAS
2011年第6期58-62,共5页
Food and Fermentation Science & Technology
基金
吉林省科技厅自然科学基金(20090553)
