构建植物乳杆菌(Lactobacillus sp. LMY-20)中亚硝酸盐还原酶(nitrite reductase,NiR)的重组大肠杆菌、纯化重组蛋白并对其进行酶学性质分析。将人工合成的密码子优化的亚硝酸盐还原酶基因(nir)亚克隆至载体pET28a(+),构建重组表达载体pET28a(+)-nir并转化到E. coli BL21(DE3)中实现表达。包涵体复性,利用镍柱亲和层析纯化重组亚硝酸盐还原酶。成功构建产亚硝酸盐还原酶的重组大肠杆菌并纯化了重组亚硝酸盐还原酶,纯化后经变性聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)分析在45 kDa附近出现明显条带。Na2S2O4-MV法测得酶活为2 332.72 U/L,最适pH值为6.5,最适作用温度为37 ℃,在4~70 ℃下孵育40 min后可保持超过85%的活力。该研究实现植物乳杆菌来源的NiR在大肠杆菌中的重组表达、纯化及其酶学性质分析,重组NiR具有较好的温度适应性和稳定性,为其在农业、食品及医药等领域应用奠定基础。
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.
[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.
