根癌土壤杆菌SCUEC1菌株尼古丁降解代谢中agnE基因的克隆与功能鉴定  

Cloning and charaterization of the agnE gene for nicotine degradation in Agrobacterium tumefaciens strain SCUEC1

作　　者：皮婷 姚家成 黄粤 夏珍珍 梅枫 何冬兰[1] 程国军[1] 刘涛[1] 李晓华[1] Ting Pi;Jiacheng Yao;Yue Huang;Zhenzhen Xia;Feng Mei;Donglan He;Guojun Cheng;Tao Liu;Xiaohua Li(Hubei Provincial Engineering and Technology Research Center for Resources and Utilization of Microbiology, College of Life Sciences, South-Central University for Nationalities, Wuhan 430074, Hubei Province, Chin)

机构地区：[1]中南民族大学生命科学学院,微生物资源与利用湖北省工程技术研究中心,湖北武汉430074

出　　处：《微生物学报》2018年第5期907-914,共8页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31070087,30570046); 湖北省自然科学基金(2011CDA079,2008CDB076); 中央科研基本业务费(CZW16005,YCZW15104)

摘　　要：【目的】本研究对尼古丁降解菌根癌土壤杆菌SCUEC1菌株中agnE基因进行克隆表达,并对agnE基因表达蛋白进行功能鉴定。【方法】通过PCR扩增获得agn 全长基因(1029 bp),构建重组质粒pET28a-agnE,转化大肠杆菌BL21(DE3)菌株进行异源表达,采用SDS-PAGE检测重组蛋白。以2,5-二羟基吡啶作为反应底物,在检测波长320 nm下测定反应液吸光度值,进一步研究温度、pH值和金属离子对AgnE蛋白酶活性的影响。【结果】克隆得到基因agnE,构建了p ET28a-agnE重组质粒并进行了表达,AgnE蛋白分子量为42.0 kDa,表达蛋白以包涵体形式存在于细胞中。酶促反应0、5、10、20 min时反应液吸光度分别为0.6170、0.2273、0.0907、0.0667。在pH 8.0、温度20°C下,AgnE蛋白具有较高的2,5-二羟基吡啶双加氧酶活性,且Fe2+对酶活性具有明显的促进作用。【结论】明确了Agn E蛋白具有2,5-二羟基吡啶双加氧酶活性。[Objective] We cloned and expressed the agn E gene involved in nicotine-degrading in Agrobacterium tumefaciens and characterized the Agn E protein. [Methods] The agn E gene fragment（1029 bp） was amplified with PCR. Recombinant plasmid p ET28 a-agn E was constructed and transformed into Escherichia coli BL21（DE3） for heterologous expression, and the expressed protein was detected with SDS-PAGE. The enzyme activity of Agn E was determined spectrophotometrically by monitoring the decrease of 2,5-dihydroxy pyridine at 320 nm. The effect of temperature, p H and metal ion on enzyme activity was investigated. [Results] The agn E gene was cloned and the recombinant plasmid p ET28 a-agn E was expressed. The molecular weight of the recombinant protein was 42.0 k Da, and the protein was expressed in the form of inclusion bodies in cells. The Agn E protein had high enzyme activity at p H 8.0 and and 20 °C. In addition, Fe^（2＋） showed significant promoting effect on enzyme activity. [Conclusion] The activity of Agn E protein with 2,5-dihydroxy pyridine dioxygenase was clarified.

关 键 词：根癌土壤杆菌SCUEC1菌株 尼古丁降解代谢 agnE基因功能 2 5-二羟基吡啶 

分 类 号：Q78[生物学—分子生物学]