耐辐射奇异球菌R12海藻糖合成酶基因的克隆与表达

Cloning and Expression of treS Gene from Deinococcus Wulumuqiensis R12

耐辐射奇异球菌(Deinococcus wulumuqiensis)R12在环境胁迫下具有合成海藻糖的能力。采用PCR方法从耐辐射奇异球菌R12基因组中分离得到分子量约1 700bp的海藻糖合成酶基因,为验证其功能而在大肠杆菌中进行了表达,经IPTG诱导表达出约66kDa的目的蛋白。经酶活检测发现,目的蛋白能够催化麦芽糖一步合成海藻糖。以30%麦芽糖为底物反应1h,进行海藻糖转化实验,结果表明,重组海藻糖合成酶的最适反应温度为35℃、最适反应pH值为8.0,转化率达67%,具有较高的应用价值。

Deinococcus wulumuqiensis R12 has the ability to produce trehalose under the extreme conditions.1 700 bp DNA fragment encoding trehalose synthase was cloned from D.wulumuqiensis R12 by PCR method.Then it was expressed in E.coli to verify its function.By adding IPTG to induce the recombinant plasmid,a novel target protein band of 66 kDa was detected by SDS-PAGE analysis.After determination of the enzymatic activity,it was found that this trehalose synthase could catalyze the reversible interconversion of maltose and trehalose.When using 30% maltose as substrate,reacting for 1h,it was found that the optimum reaction temperature and pH value for recombinant trehalose synthase was 35 ℃,8.0,respectively,the conversion rate reached 67%.