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

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agnE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pET28a-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 ℃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AgnE蛋白具有2,5-二羟基吡啶双加氧酶活性.%[Objective] We cloned and expressed the agnE gene involved in nicotine-degrading in Agrobacterium tumefaciens and characterized the AgnE protein. [Methods] The agnE gene fragment (1029 bp) was amplified with PCR. Recombinant plasmid pET28a-agnE 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 AgnE was determined spectrophotometrically by monitoring the decrease of 2,5-dihydroxy pyridine at 320 nm. The effect of temperature, pH and metal ion on enzyme activity was investigated. [Results] The agnE gene was cloned and the recombinant plasmid pET28a-agnE was expressed. The molecular weight of the recombinant protein was 42.0 kDa, and the protein was expressed in the form of inclusion bodies in cells. The AgnE protein had high enzyme activity at pH 8.0 and and 20 °C. In addition, Fe2+ showed significant promoting effect on enzyme activity. [Conclusion] The activity of AgnE protein with 2,5-dihydroxy pyridine dioxygenase was clarified.