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

作　　者：张充[1] 周孝伟[1] 吕凤霞[1] 别小妹[1] 陶婷婷[1] 应琦[1] 陆兆新[1] 

机构地区：[1]南京农业大学食品科技学院酶工程研究室,江苏南京210095

出　　处：《生物工程学报》2012年第4期440-456,共17页Chinese Journal of Biotechnology

基　　金：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~~

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11.4×104U/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。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℃. We purified the enzyme by Ni-NTA chelating affinity chromatography, with 60.89% yield and specific activity of 11.4×10^4 U/mg. The optimum reaction temperature and pH for ana-LOX were 45℃ 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.

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 

分 类 号：Q943.2[生物学—植物学]