中华根瘤菌NP1亚硝酸盐还原酶基因的表达和酶学性质研究

Expression of Nitrite Reductase Gene of Sinorhizobium sp. NP1 and the Enzymatic Properties

[目的]研究中华根瘤菌NP1(Sinorhizobium sp.NP1)中亚硝酸盐还原酶基因(nir)的原核表达情况以及粗酶液的化学性质。[方法]构建该基因的原核表达载体p ET32a-nir,然后对重组蛋白质进行SDS-PAGE和Western blotting分析获得NIR酶在原核生物中的表达情况。通过测定粗酶液对亚硝酸盐降解情况研究相关酶学性质。[结果]SDS-PAGE检测结果表明,可溶性融合表达产物约为57 ku,其中亚硝酸盐还原酶大小约40 ku,与酶分子量的预估值一致;Western blotting证实该蛋白可与根据亚硝酸盐还原酶合成的多克隆抗体发生强阳性反应;酶学性质分析表明,亚硝酸盐还原酶活力约为247.15 U/m L,最适pH 6.5,最适反应温度37℃,该酶对Na Cl的耐受性为0.3 mol/L。[结论]亚硝酸盐还原酶蛋白重组质粒能在大肠杆菌系统中高效表达,研究其酶学性质为NO_2^-生物清除提供了技术保障。

[Objective] To study the prokaryotic expression and enzymatic properties of nitrite reductase gene of Sinorhizobium sp.NP1.[Method] Prokaryotic expression vector pET32a-nir was constructed.Then,Western blotting and SDS-PAGE analysis were performed to study its expression in prokaryote.The enzyme properties were studied by measuring the degradation of nitrite.[Result] The result of SDS-PAGE showed that the fusion expression product was about 57 ku,including 40 ku target protein,which was consistent with the molecular weight of the enzyme.Western blotting confirmed that the protein had a strong positive reaction with the polyclonal antibody which was synthesized by NIR.Enzymatic properties analysis showed that the activity of the enzyme was 247.15 U/mL,the optimum reaction pH value was 6.5,the optimum reaction temperature was 37 ℃,the tolerance to NaCl was 0.3 mol/L.[Conclusion] The recombinant plasmid of NIR could be efficiently expressed in E.coli,and the study of enzymatic properties provides a technical support for the biological removal of NO2-.