亚硝酸还原酶基因克隆、表达与纯化

魏计东; 张庆芳; 窦少华; 于爽; 迟乃玉; 王晓辉

doi:10.13386/j.issn1002-0306.2017.14.020

亚硝酸还原酶基因克隆、表达与纯化

魏计东^1,2,
张庆芳^1,2,
窦少华^1,2,
于爽^1,2,
迟乃玉^1,2,
王晓辉^1,2

1. 大连大学生命科学与技术学院
2. 辽宁省海洋微生物工程技术研究中心

基金项目:

国家自然科学基金项目(31500039)；

详细信息

作者简介:
魏计东 (1991-) , 男, 硕士研究生, 研究方向:微生物与酶工程, E-mail:weijidongsk@163.com。;

王晓辉 (1981-) , 女, 博士, 副教授, 研究方向:微生物与酶工程, E-mail:wangxiaohui@dlu.edu.cn。;

中图分类号: Q78
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出版历程
- 收稿日期: 2016-12-13

Cloning, expression and purification of copper nitrite reductase

School of Life Science and Biotechnology, Dalian University
Liaoning Technology of Marine Microbiological Engineering Research Center

摘要

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。
- 木糖氧化产碱菌DL-1 /
- 含铜亚硝酸还原酶(Cu NiR) /
- 克隆 /
- 表达 /
- 纯化
Abstract: 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.
- Alcaligenes xylosoxidans DL-1 /
- copper nitrite reductase /
- cloning /
- expression /
- purification

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参考文献(20)

[1]	Orellana L H, Rodriguez-R L M, Higgins S, et al.Detecting nitrous oxide reductase (Nos Z) genes in soil metagenomes:method development and implications for the nitrogen cycle[J].Mbio, 2014, 5 (3) :191-194.
[2]	Allen J W, Barker P D, Daltrop O, et al.Why isn’t‘standard’heme good enough for c-type and d1-type cytochromes[J].Dalton Transactions, 2005, 21 (21) :3410-3418.
[3]	Sharma S, Szele Z, Schilling R, et al.Influence of freeze-thaw stress on the structure and function of microbial communities and denitrifying populations in soil[J].Journal of Experimental Medicine, 2006, 72 (3) :2148-2154.
[4]	Antonyuk S V, Strange R W, Sawers G, et al.Atomic resolution structures of resting-state, substrate-and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism[J].Proceedings of the National Academy of Sciences, 2005, 102 (34) :41-46.
[5]	Hallin S, Lindgren P E.PCR Detection of genes encoding nitrite reductase in denitrifying bacteria PCR detection of genes encoding nitrite reductase in denitrifying bacteria[J].Applied and Environmental Microbiology, 1999, 65 (4) :1652-1657.
[6]	Tsuneda S, Miyauchi R, Ohno T, et al.Characterization of denitrifying polyphosphate-accumulating organisms in activated sludge based on nitrite reductase gene[J].Journal of Bioscience and Bioengineering, 2005, 99 (4) :403-408.
[7]	Yamaguchi K, Deligeer, Nakamura N, et al.Isolation and characterization of two distinct azurins from Alcaligenes xylosoxidans subsp.xylosoxidans NCIB11015 or GIFU1051[J].Chemistry Letters, 1995, 89 (5) :353-354.
[8]	Moura I, Moura J J.Structural aspects of denitrifying enzymes[J].Current Opinion in Chemical Biology, 2001, 5 (2) :168-175.
[9]	Bykov D, Neese F.Six-electron reduction of nitrite to ammonia by cytochrome c nitrite reductase:insights from density functional theory studies[J].Inorganic Chemistry, 2015, 54 (19) :3-16.
[10]	Suzuki M, Hirai T, Arai H, et al.Purification, characterization, and gene cloning of thermophilic cytochrome cd1nitrite reductase from Hydrogenobacter thermophilus TK-6[J].Journal of Bioscience&Bioengineering, 2006, 101 (5) :391-397.
[11]	Kataoka K, Furusawa H, Takagi K, et al.Functional analysis of conserved aspartate and histidine residues located around the type 2 copper site of copper-containing nitrite reductase[J].Journal of Biochemistry, 2000, 127 (2) :345-348.
[12]	李美玉, 张庆芳, 王晓辉.几丁质结合蛋白基因克隆、表达与纯化[J].中国酿造, 2015 (11) :41-46.
[13]	Rosa M.Purification and characterisation of a possible assimilatory nitrite reductase from the halophile archaeon Haloferax mediterranei[J].FEMS Microbiology Letters, 2001, 196 (2) :113-118.
[14]	Treusch A H, Leininger S, Kletzin A, et al.Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling[J].Environmental Microbiology, 2005, 7 (12) :85-95.
[15]	Ai-Jia J I, Ning X B.Construction and expression of Prokaryotic expression Vector p ET28a-EGFP[J].Journal of Microbiology, 2011, 31 (4) :69-73.
[16]	Yuan Z G, Zhang J P, Chu Y W, et al.Expression of target gene in eukaryotic cells driven by prokaryotic T7 promoter and its RNA polymerase][J].Chinese Journal of Biotechnology, 2005, 21 (2) :182-186.
[17]	杜永成, 王玉波, 范文婷, 等.不同氮素水平对甜菜硝酸还原酶和亚硝酸还原酶活性的影响[J].植物营养与肥料学报, 2012, 18 (3) :717-723.
[18]	Rajasekhar V K, Oelmüller R.Regulation of induction of nitrate reductase and nitrite reductase in higher plants[J].Physiologia Plantarum, 2006, 71 (4) :517-521.
[19]	Prudêncio M, Eady R R, Sawers G.The Blue CopperContaining Nitrite Reductase from Alcaligenes xylosoxidans:Cloning of the nir A Gene and Characterization, of the Recombinant Enzyme[J].Journal of Bacteriology, 1999, 181 (8) :2323-2329.
[20]	Ho W H, Ooi B L.Cytoplasmic expression of the Achromobacter xylosoxidans blue copper nitrite reductase in Escherichia coli and characterisation of the recombinant protein[J].Protein Expression&Purification, 2003, 32 (2) :288-292.