| D-乳酸高产菌菊糖芽胞乳杆菌Y2-8磷酸果糖激酶基因在大肠杆菌中的克隆和表达 |
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| 引用本文： | 郑璐,;柏中中,;许婷婷,;何冰芳.D-乳酸高产菌菊糖芽胞乳杆菌Y2-8磷酸果糖激酶基因在大肠杆菌中的克隆和表达[J].生物加工过程,2014(4):37-42. |
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| 作者姓名： | 郑璐 ;柏中中 ;许婷婷 ;何冰芳 |
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| 作者单位： | [1] 南京工业大学 生物与制药工程学院,南京211800; [2] 南京中医药大学 基础医学院,南京210046 |
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| 基金项目： | 国家重点基础研究发展计划（973计划）（2011CB707400）;江苏省普通高校研究生科研创新计划（CXZZ11_0358） |
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| 摘 要： | 以D-乳酸高产菌菊糖芽胞乳杆菌Y2-8基因组DNA为模板,通过PCR扩增得到960 bp的磷酸果糖激酶基因(pfk)。氨基酸序列比对分析表明,该磷酸果糖激酶(PFK)与其他乳酸菌PFK具有保守的底物结合位点,但是其变构效应物结合位点存在差异。将pfk基因克隆到表达载体pSE380上,获得重组菌E-pSE-pfk。进一步通过诱导条件的优化,重组菌的PFK比酶活达到4.89 U/mg,是优化前的4.79倍。采用低温诱导策略有助于实现菊糖芽胞乳杆菌pfk基因在大肠杆菌中可溶性表达。
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| 关 键 词： | D-乳酸 大肠杆菌 菊糖芽胞乳杆菌 表达 优化 磷酸果糖激酶 |
Cloning and expression of phosphofructokinase gene from Sporolactobacillus inulinus in Escherichia coli |
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| Institution: | ZHENG Lu, BAI Zhongzhong ,XU Tingting ,HE Bingfang ( 1. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211800, China; 2. College of Pre-clinical Medicine, Nanjing University of Chinese Medicine, Nanjing 210046, China) |
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| Abstract: | The gene encoding phosphofructokinase （ pfk ） amplified by PCR using Sporolactobacillus inulinus Y2-8 genomic DNA as a template was 960 bp.Alignments of the amino acid sequences revealed that compared with phosphofructokinase （ PFK） from other representative lactic acid bacteria,PFK from S.inulinus Y2-8 had strictly conserved substrate binding sites,but different allosteric sites.The pfk gene was cloned into the vector pSE380,producing recombinant strain E-pSE-pfk.After optimization,the PFK specific activity of recombinant strain reached 4.89 U/mg, thus it was 4.79-fold higher than that of primary expression condition.The result showed that low-temperature induction was helpful to the soluble expression of PFK from S.inulinus in E.coli. |
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| Keywords: | D-lactic acid Escherichia coli Sporolactobacillus inulinus expression optimization phosphofructokinase |
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