大黄欧文菌中蔗糖异构酶基因的克隆表达及应用

摘要

The pal Ⅰ gene encoding sucrose isomerase(Slase) was amplified by PCR with the genomic DNA of Erwinia rhapontici NX-5 as the template, and the recombinant cloning vector pUC18- pal Ⅰ was constructed with inserting the pal Ⅰ into pUC18. After the DNA sequence was determined, the pal Ⅰ was subcloned into expression vector pET-22b(+) to construct the recombinant expression vector pET-22b-palⅠ , and the gene pal Ⅰ was expressed in recombinant E. coli BL21 ( DE3 ) for the protein with a single band about 66 000 on SDS-PAGE. After Slase was purified by Ni-NTA affinity chromatography, its specified activity was about 40 U/mg. The optimization of conversion conditions showed that 85％ of sucrose solution (50％) could be converted into isomaltulose by recombinant cells.%以大黄欧文菌 (Erwinia rhapontici)NX-5基因组DNA为模板, PCR扩增得到编码蔗糖异构酶(SIase)的基因 palⅠ, 构建克隆载体pUC18-palⅠ.经测序正确后, 将palⅠ亚克隆至表达载体pET-22b( + )上,并在E.coli BL21 (DE3)中成功表达相对分子质量约为66 000的可溶性蛋白.通过Ni-NTA柱对表达产物进行纯化, 纯酶的比活为40U/mg.转化条件研究表明:重组菌能够高效转化质量分数为50%的蔗糖溶液,转化液中异麦芽酮糖得率为85%.