Clostridium cellulolyticum D-塔格糖3-差向异构酶基因的克隆表达及酶活性

摘要

D-塔格糖3-差向异构酶是生物法生产新型功能性因子D-阿洛酮糖最为有效的酶.作者克隆到一种新型的D塔格糖3-差向异构酶基因,来源于微生物Clostridium cellulolyticum H10.以pET-22b(+)为载体质粒,E.coli BL21(DE3)为宿主细胞,构建了基因重组菌,IPTG可诱导目的蛋白质的过量表达;经亲和层析纯化的重组蛋白质样品进行SDS-PAGE分析,在约31 000处出现显著的特征蛋白质条带;活性检测结果表明:该重组酶具有较高的转化活性.%D-tagatose 3-epimerase (DTE) is the most effective enzyme for the biological production of D-psicose, a novel functional factor. In this study, a novel gene encoding DTE from Clostridiurn cellulolyticurn H10 was cloned, pET-22b (+) was for the plasmid, E. coli BL21 (DE3) acted as host cells, then the recombinant strains were constructed. The target protein was over-expressed by IPTG induction; the recombinant DTE purified to electrophoretical homogeneity with affinity chromatography was analyzed by SDS-PAGE, showing approximately 31 000 characteristic protein. The result of activity detection revealed that the recombined enzyme had a higher transforming activity.