重组鱼腥藻脂肪氧合酶基因的克隆表达、分离纯化及活性分析
Cloning and expression of lipoxygenase gene from Anabaena sp. PCC 7120 and purification, characterization of the recombinatant enzyme
投稿时间:2011-09-20  
DOI:  
中文关键词:重组脂肪氧合酶,克隆表达,定点突变,分离纯化,活性分析
英文关键词:recombinant lipoxygenase, clone and expression, site directed, purification, characterization
基金项目:南京农业大学青年科技创新基金 (No. Y201069),南京农业大学基本科研业务费专项基金 (No. KYZ200910), 国家自然科学基金 (No. 31071605)资助。
作者单位E-mail
张充 南京农业大学食品科技学院 酶工程研究室江苏 南京 210095  
周孝伟 南京农业大学食品科技学院 酶工程研究室江苏 南京 210095  
吕凤霞 南京农业大学食品科技学院 酶工程研究室江苏 南京 210095  
别小妹 南京农业大学食品科技学院 酶工程研究室江苏 南京 210095  
陶婷婷 南京农业大学食品科技学院 酶工程研究室江苏 南京 210095  
应琦 南京农业大学食品科技学院 酶工程研究室江苏 南京 210095  
陆兆新 南京农业大学食品科技学院 酶工程研究室江苏 南京 210095 fmb@njau.edu.cn 
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中文摘要:
      克隆鱼腥藻PCC7120基因组中脂肪氧合酶 (ana-LOX) 基因,对该功能基因进行了定点突变研究,确定了ana-LOX的最短功能基因长度,构建原核重组表达载体,对重组ana-LOX进行了分离纯化和性质研究。从GenBank中搜索到鱼腥藻PCC7120基因组中含有LOX基因,通过序列分析和比对,发现LOX功能基因位于双功能酶AOS (单加氧酶) -LOX的C端,通过定点突变研究,证实了ana-LOX活性中心位点为His197、His202、His369、Asn373和Ile455。通过逐步缩短基因长度的策略,获得ana-LOX基因的最短功能基因长度为1 254 bp。构建的表达载体pET-32a/ana-LOX转化入BL21 (DE3) 宿主内,在低温16 ℃条件下的诱导表达,重组脂肪氧合酶活力可达6 750 U/mL。表达产物通过Ni-NTA亲和柱进行分离纯化,比活达到11.4×104 U/mg蛋白,酶活回收率为60.89%。重组ana-LOX最适反应温度45 ℃,最适反应pH 6.0,在常温下具有较好的稳定性,金属离子Fe2+、Mg2+、Ca2+对该酶存在明显的激活作用,而Fe3+和Cu2+对该酶有强烈的抑制作用。重组ana-LOX能够改善面团的显微结构。该研究获得了高效表达重组ana-LOX,为实现其在食品加工中的应用提供了参考。
英文摘要:
      We cloned the lipoxygenase gene (ana-LOX) from Anabaena sp. PCC 7120 and expressed it in Escherichia coli BL21 (DE3) pLysS. We determined the active site of the recombinant ana-LOX through site-directed gene mutagenesis and obtained the shortest length of the functional gene. Meanwhile, we studied the properties of recombinant ana-LOX after purification. The C-terminal of the Aos (allene oxide synthase)-LOX fusion gene in Anabaena sp. PCC 7120 genome was found belonging to LOXs family by bioinformatics analysis. Further results of site-directed gene mutagenesis confirmed that the active sites of ana-LOX were His197, His202, His369, Asn373and Ile455. The shortest length of functional gene was identified to be 1 254 bp based on the strategy of shortening the gene length gradually. The highest activity of recombinant ana-LOX of 6 750 U/mL could be achieved when constructed to pET-32a vector and expressed at low temperature 16 °C. We purified the enzyme by Ni-NTA chelating affinity chromatography, with 60.89% yield and specific activity of 11.4×104 U/mg. The optimum reaction temperature and pH for ana-LOX were 45 oC and 6.0, respectively. Furthermore, the obtained ana-LOX was stable at room temperature. The effect of metal ions on ana-LOX was determined also. Fe2+, Mg2+, Ca2+ could markedly promote the activity of this enzyme whereas Fe3+ and Cu2+ had a strong inhibitory effect on it. Finally, the ana-LOX could improve the microscopical structure of dough. The results of this study will provide a basis for future improvements and food industrial applications of ana-LOX.
张充,周孝伟,吕凤霞,别小妹,陶婷婷,应琦,陆兆新.重组鱼腥藻脂肪氧合酶基因的克隆表达、分离纯化及活性分析[J].生物工程学报,2012,28(4):440~456
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