摘要
该研究通过聚合酶链反应(PCR)方法从木糖氧化产碱菌(Achromobacter xylosoxidans DL-1)基因组DNA中成功克隆含铜亚硝酸还原酶基因。PCR测序表明该基因全长1083个核苷酸,编码360个氨基酸,预测其理论分子质量约38.924 k Da,等电点(p I)4.83,命名为Cu NiR(Genbank登录号KX674378)。结构域分析该蛋白编码区包含信号肽,1个铜离子结合位点,1个氧化还原酶催化域。将Cu NiR基因构建到p ET22b载体,并转化至大肠杆菌(Escherichia coli,E.coli)BL21(DE3)中诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测显示目的蛋白可溶表达。利用镍柱亲和层析纯化重组蛋白,酶活检测显示比活力为123.82 U/mg,为后期含铜亚硝酸还原酶(Cu NiR)的生化性质表征奠定理论基础。
In this study,a novel copper nitrite reductase gene was cloned from Achromobacter xylosoxidans DL-1 by using a PCR protocol.The gene contained a 1083 bp open reading frame encoding a 360 amino acid protein with an estimated molecular mass of 38.924 k Da and isoelectronic point（ p I） of 4.83,named Cu NiR（ Genebank no.KX674378）.Based on domains analysis,Cu NiR had a signal peptide,a copper binding site classified as Cu-oxidase-3,and one nitrite reductase catalytic domain（ Cuoxidase）. A recombinant plasmid,p ET22b-Cu NiR was constructed and transformed into E.coli BL21（ DE3）. The cells were induced by addition of IPTG. Cu NiR had been successfully expressed by the analysis of sodium dodecyl sulphate-polyacrylamide gel electrophoresis（ SDS-PAGE）. The recombinant protein was purified by Ni-NTA resin,and the specific activity was 123.82 U/mg,which lay the theoretical basis for biochemical characterization of the copper nitrite reductase.
出处
《食品工业科技》
CAS
CSCD
北大核心
2017年第14期101-105,共5页
Science and Technology of Food Industry
基金
国家自然科学基金项目(31500039)
