一种新型糖磷酸异构酶在大肠杆菌中的重组表达

Recombination and expression of a novel phosphosugar isomerase in E.coli

摘要: 核糖-5-磷酸异构酶（RpiB）是新型功能性稀有糖D-阿洛糖生物合成过程中的重要酶。克隆Thermotoga lettingae TMO来源的RpiB基因,以pET-22b（+）为载体,E.coli BL21（DE3）为宿主细胞,构建了基因重组菌。诱导剂IPTG可诱导目的蛋白表达;经金属亲和层析纯化得到电泳纯的重组蛋白,SDS-PAGE显示17ku处出现显著的特征蛋白质条带。活性检测结果表明,该重组RpiB具有较高的产D-阿洛糖生物活性,静息细胞和纯酶反应3h后转化率分别为19%和23%。

Abstract: Ribose-5-phosphate isomerase B ( RpiB) is a main enzyme for biotransformation D-psicose to D-allose, a novel rare sugar.Ribose-5-phosphate isomerase B from Thermotoga lettingae TMO was cloned and expressed in Escherichia coli BL21 ( DE3) .The genetically engineered bacterium was induced by IPTG, then the recombinant RpiB was purified to electrophoretical homogeneity with affinity chromatography. The recombinant RpiB was analyzed by SDS- PAGE, and approximately 17ku target protein was observed on the SDS- PAGE. The activity assay of recombinant enzyme showed a relatively high enzyme activity of D- allose- producing. Conversion rates after 3h of resting cells and pure enzyme reaction were 19% and 23%, respectively.