This study constructed a recombinant Escherichia coli that expressed nitrite reductase (NiR) from Lactobacillus sp. LMY-20, and the enzymatic properties of the NiR were investigated. A synthetic codon-optimized Lactobacillus plantarum nitrite reductase gene (nir) was cloned into vector pET28a(+) and expressed in Escherichia coli BL21(DE3). The recombinant NiR was purified by nickel column affinity chromatography with a molecular weight of 45 kDa on SDS-PAGE. Moreover, its optimal reaction pH and temperature were 6.5 and 37 ℃, respectively. Besides, more than 85% of the original enzyme activity was preserved after 40 min incubation at 4-70 ℃. In conclusion, the recombinant NiR has good temperature adaptability and stability, which lays a foundation for its applications in the fields of agriculture, food, and medicine.
ZHANG Qingfang
,
LI Meiyu
,
WANG Xiaohui
,
HU Shansong
,
YU Shuang
,
CHI Naiyu
. Expression, purification and characterization of Lactobacillus plantarum nitrite reductase in Escherichia coli[J]. Food and Fermentation Industries, 2019
, 45(12)
: 28
-34
.
DOI: 10.13995/j.cnki.11-1802/ts.019986
[1] 丁少南.植物乳杆菌中亚硝酸还原酶的研究[D].上海:上海师范大学, 2013.
[2] LINTULUOTO M, LINTULUOTO J M. DFT study on nitrite reduction mechanism in copper-containing nitrite reductase[J].Biochemistry,2016,55(1):210-223.
[3] EZZINE M, GHORBEL M H. Physiological and biochemical responses resulting from nitrite accumulation in tomato(Lycopersicon esculentum Mill.cv.Ibiza F1) [J].Journal of Plant Physiology,2006,163(10):1 032-1 039.
[4] SOLOMON E I. Spectroscopic methods in bioinorganic chemistry:Blue to green to red copper sites[J].Inorganic Chemistry,2006,42(20): 8 012-8 025.
[5] ADMAN E T,GODDEN J W, TURLEY S. The structure of copper-nitrite reductase from Achromobacter cycloclastes at five pH values,with NO-2 bound and with type Ⅱ copper depleted[J]. J Biol Chem,1995,270(46):27 458-27 474.
[6] TOCHEVA E I, ROSELL F I, MAUK A G, et al. Side-on copper-nitrosyl coordination by nitrite reductase [J].Science,2004,304(5 672):867-870.
[7] TIKHONOVA T V, SLUTSKY A, ANTIPOV A N, et al.Molecular and catalytic properties of a novel cytochrome c nitrite reductase from nitrate-reducing haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens[J].Biochim Biophys Acta,2006,1 764(4):715-723.
[8] HORRELL S, KEKILLI D, STRANGE R W, et al.Recent structural insights into the function of copper nitrite reductases[J].Metallomics,2017,9(11):1 470-1 482.
[9] ZERBINO D R, BIRNEY E. Algorithms for de novo short read assembly using de Bruijn graphs[J].Genome Res,2008,18(5):821-829.
[10] AZIZ R K, BARTELS D, BEST A A, et al. The RAST Server: Rapid annotations using subsystems technology[J].BMC Genomics,2008,9(1):75.
[11] 赵东岳,林莉莉,温福利.结核分枝杆菌Rv3194c蛋白的表达、纯化及活性鉴定[J].微生物学报,2016,56(12):1 847-1 855.
[12] 赵云,朱蓓霖,汪正华,等.麦芽四糖淀粉酶基因优化表达及酶学性质分析[J].中国生物工程杂志,2013,33(5):100-106.
[13] 蔡婀娜,贺淹才,刘治江,等.重组产几丁质酶C工程菌包涵体的复性[J].河南师范大学学报(自然科学版),2011,39(1):137-141.
[14] 李美玉,曹洪玉,张庆芳,等.几丁质结合蛋白基因克隆、表达与纯化[J].中国酿造,2015,34(11):41-46.
[15] GAO H, LI C, RAMESH B, et al.Cloning,purification and characterization of novel Cu-containing nitrite reductase from the Bacillus firmus GY-49[J].World J Microbiol Biotechnol, 2017,34(1):10.
[16] 陈思敏,罗彤晖,费永涛,等.蜡样芽孢杆菌Bacillus cereus LJ01中亚硝酸盐还原酶的基因克隆、表达和纯化[J].食品科学,2018,39(6):69-74.
[17] 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].Environ Microbiol,2005,7(12):1 985-1 995.
[18] TERPE K. Overview of bacterial expression systems for heterologous protein production:from molecular and biochemical fundamentals to commercial systems[J].Appl Microbiol Biot,2006,72(2):211-222.
[19] 黄佳明,姜宁,张爱忠.基因工程菌生产抗菌肽的研究进展[J].微生物学通报,2019,46(3): 654-659.
[20] 罗惠霞,李敏王,玉炯.包涵体蛋白复性的几种方法[J].生物技术通报,2007(5):96-98.
[21] 何庆,刘帅,周海霞.人胱抑素C大肠杆菌表达载体构建及包涵体复性研究[J].惠州学院学报,2018, 6: 29-31;38.
[22] 袁志刚, 张进平,储以微,等. 原核表达系统T7 RNA聚合酶/启动子在真核细胞中表达目的基因的实验研究[J]. 生物工程学报,2005,21(2):182-186.
[23] YANG Y Q,WANG H,LIANG M L,et al. Construction and expression of prokaryotic expression vectors fused with genes of Magnaporthe oryzae effector proteins and mCherry[J].Genetics and Molecular Research,2015,14(3):10 827-10 836.
[24] 季爱加,宁喜斌.原核表达载体pET28a-EGFP的构建与表达[J].微生物学杂志,2011,34(1):69-73.
[25] 李爽.水稻土厌氧硝酸盐还原耦合亚铁氧化与砷氧化机制[D].广州:中国科学院大学,2018.
[26] NAKANO S,TAKAHASHI M,SAKAMOTO A,et al.The reductive reaction mechanism of tobacco nitrite reductase derived from a combination of crystal structures and ultraviolet-visible microspectroscopy[J]. Proteins,2012,80(8):2 035-2 045.
