大黄欧文菌中蔗糖异构酶基因的克隆表达及应用  

Cloning expression and application of sucrose isomerase from Erwinia rhapontici

作　　者：李莎[1] 任贲[1] 林璐[1] 徐虹[1] 蔡恒[1] 

机构地区：[1]南京工业大学食品与轻工学院,材料化学工程国家重点实验室,江苏南京210009

出　　处：《南京工业大学学报（自然科学版）》2011年第1期84-89,共6页Journal of Nanjing Tech University(Natural Science Edition)

基　　金：国家科技支撑计划(2008BAI63B07);国家自然科学基金资助项目(20906050);江苏省自然科学基金资助项目(BK2009357);江苏省高校自然科学研究计划(08KJB530004)

摘　　要：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.coliBL21(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40 U/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%。The palⅠ gene encoding sucrose isomerase（SIase） was amplified by PCR with the genomic DNA of Erwinia rhapontici NX-5 as the template,and the recombinant cloning vector pUC18-palⅠwas constructed with inserting the palⅠ into pUC18.After the DNA sequence was determined,the palⅠwas subcloned into expression vector pET-22b（＋） to construct the recombinant expression vector pET-22b-palⅠ,and the gene palⅠ was expressed in recombinant E.coli BL21（DE3） for the protein with a single band about 66 000 on SDS-PAGE.After SIase 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.

关 键 词：蔗糖异构酶 大黄欧文菌 克隆表达 条件优化 

分 类 号：O611.62[理学—无机化学]