%0 Journal Article
%T Cloning and expression of lipoxygenase gene from Anabaena sp. PCC 7120 and purification, characterization of the recombinatant enzyme<br>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
%A Zhang Chong
%A Zhou Xiaowei
%A Lü Fengxia
%A Bie Xiaomei
%A Tao Tingting
%A Ying Qi
%A Lu Zhaoxin
%A <br>张充
%A 周孝伟
%A 吕凤霞
%A 别小妹
%A 陶婷婷
%A 应琦
%A 陆兆新
%J 生物工程学报
%D 2012
%I 
%X 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.
%K recombinant lipoxygenase
%K clone and expression
%K site directed
%K purification
%K characterization<br>重组脂肪氧合酶
%K 克隆表达
%K 定点突变
%K 分离纯化
%K 活性分析
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=878F493A8FA2CF127858822A78D4DD36&yid=99E9153A83D4CB11&vid=D3E34374A0D77D7F&iid=E158A972A605785F&sid=78976D931AD1540F&eid=522844664D9E629A&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=0&reference_num=0