| 大黄欧文菌中蔗糖异构酶基因的克隆表达及应用 |
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| 引用本文： | 李莎,任贲,林璐,徐虹,蔡恒.大黄欧文菌中蔗糖异构酶基因的克隆表达及应用[J].南京工业大学学报(自然科学版),2011,33(1):84-89. |
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| 作者姓名： | 李莎 任贲 林璐 徐虹 蔡恒 |
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| 作者单位： | 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 |
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| 基金项目： | 国家科技支撑计划，国家自然科学基金资助项目，江苏省自然科学基金资助项目，江苏省高校自然科学研究计划 |
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| 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%。
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| 关 键 词： | 蔗糖异构酶 大黄欧文菌 克隆表达 条件优化 |
Cloning expression and application of sucrose isomerase from Erwinia rhapontici |
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| LI Sha,REN Ben,LIN Lu,XU Hong,CAI Heng.Cloning expression and application of sucrose isomerase from Erwinia rhapontici[J].Journal of Nanjing University of Technology,2011,33(1):84-89. |
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| Authors: | LI Sha REN Ben LIN Lu XU Hong CAI Heng |
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| Institution: | (State Key Laboratory of Materials-Oriented Chemical Engineering,College of Food Science and Light Industry,Nanjing University of Technology,Nanjing 210009,China) |
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| Abstract: | 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. |
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| Keywords: | sucrose isomerase Erwinia rhapontici clonging expression optimization |
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