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

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。

Abstract:

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.