Ensifer meliloti 1021烟酰胺酶的酶学特性及3-氰基吡啶调控机理的研究

doi:10.13560/j.cnki.biotech.bull.1985.2019-0027

生物技术通报 ›› 2019, Vol. 35 ›› Issue (8): 51-58.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0027

Ensifer meliloti 1021烟酰胺酶的酶学特性及3-氰基吡啶调控机理的研究

郭静静¹, 郭磊磊², 赵云岫², 戴亦军²

1. 南京师范大学中北学院,南京 210023;
2. 南京师范大学生命科学学院江苏省微生物与功能基因组学重点实验室江苏省微生物资源产业化工程技术研究中心,南京 210023

收稿日期:2019-01-08 出版日期:2019-08-26 发布日期:2019-08-05
作者简介:郭静静,女,硕士研究生,研究方向：微生物转化;E-mail：guojjhz@163.com
基金资助:
国家自然科学基金面上项目（31570104）

Research on the NAMase of Ensifer meliloti 1021 and Regulation Mechanism of 3-Cyanopyridine

GUO Jing-jing¹, GUO Lei-lei², ZHAO Yun-xiu², DAI Yi-jun²

1. Nanjing Normal University Zhongbei College,Nanjing 210023;
2. Jiangsu Key Laboratory for Microbes and Functional Genomics,Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources,College of Life Science,Nanjing Normal University,Nanjing 210023

Received:2019-01-08 Published:2019-08-26 Online:2019-08-05

摘要/Abstract

摘要： 克隆和异源表达固氮菌Ensifer meliloti 1021烟酰胺酶（NAMase）,研究其酶学特性,并探究3-氰基吡啶（3-CP）对该酶的调控机制。PCR扩增获得E. meliloti 1021烟酰胺酶基因（nam）序列,构建含nam的重组质粒并导入 Escherichia coli Rosetta;（DE3）中异源表达,用Ni-NTA亲和层析纯化蛋白,探究温度、pH、有机溶剂及金属离子对NAMase活性的影响,并对烟酰胺酶进行了固定化研究。结果显示,克隆得到长636 bp的nam,编码的蛋白分子量为22.6 kD,等电点（PI）为5.5。NAMase的最适pH为7.0,在30-50℃温度范围内孵育2 h酶活维持在97.1%以上,最适温度在30-70℃。Ag²⁺和异戊醇对NAMase的活性抑制率最大。在E. meliloti 1021野生菌株及NAMase过表达菌株转化烟酰胺的过程中3-CP会抑制烟酸的产量,这种效应并非底物抑制作用。

关键词: 草木樨剑菌, 静息转化, 烟酰胺酶, 酶学特性

Abstract: This work aims to clone and express the NAMase of Ensifer meliloti 1021,and to investigate its enzymatic properties and explore the enzymatic mechanisms regulated by 3-CP. PCR was used to amplify the gene nam of E. meliloti 1021 and the constructed recombinant nam-containing plasmid was imported to Escherichia coli Rosetta（DE3）cells for heterologous expression. Ni-NTA affinity chromatography was employed to purify the protein. The effects of temperature,pH,organic solvents and metal ions on NAMase activity were explored,and the immobilization of NAMase was further investigated. The results showed that the full-length of nam was 636 bp,encoded a 22.6 kD protein with PI of 5.5. The optimal pH for NAMase was 7,it retained over 97.1% of its initial activity after incubation at 30-50℃ for 2 h,and the optimal temperature ranged in 30-70℃. Ag²⁺and isoamyl alcohol demonstrated the highest inhibitory effect on NAMase activity. 3-CP inhibited nicotinic acid production during the transformation of nicotinamide by E. meliloti 1021 and over-expressed NAMase in E. coli Rosetta（DE3）,and this was not a substrate inhibition.

Key words: Ensifer meliloti 1021, resting conversion, NAMase, enzymatic properties

郭静静, 郭磊磊, 赵云岫, 戴亦军. Ensifer meliloti 1021烟酰胺酶的酶学特性及3-氰基吡啶调控机理的研究[J]. 生物技术通报, 2019, 35(8): 51-58.

GUO Jing-jing, GUO Lei-lei, ZHAO Yun-xiu, DAI Yi-jun. Research on the NAMase of Ensifer meliloti 1021 and Regulation Mechanism of 3-Cyanopyridine[J]. Biotechnology Bulletin, 2019, 35(8): 51-58.

参考文献

[1] Vaughan PA, Knowles CJ, Cheetham PSJ.Conversion of 3-cyanopyridine to nicotinic acid by Nocardia rhodochrous LL100-21[J]. Enzyme & Microbial Technology, 1989, 11(12):815-823.
[2] Kim SH, Oriel P.Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp. BR449 into Escherichia coli[J]. Enzyme and Microbial Technology, 2000, 27:492-501.
[3] Alfani F, Cantarella M, Spera A.et al.Operational stability of Brevibacterium imperalis CBS489-74 nitrile hydratase[J]. Journal of Molecular Catalysis(B:Enzymatic), 2001, 11(4/6):687-697.
[4] Park HJ, Park H, Rutger JF, et al.Characterization of nitrile hydrolyzing enzymes produced from Rhodococcus erythropolis[J]. Korean Society for Microbiology and Biotechnology, 2006, 34(3):204-210.
[5] Prasad S, Bhalla TC.Nitrile hydratases(NHases):At the interface of academia and industry[J]. Biotechnology Advances, 2010, 28(6):725-741.
[6] Pace HC, Brenner C.The nitrilase superfamily:classification, structure and function. Genome Biology, 2001, 2(1):1-9.
[7] Anderson RM, Bitterman KJ, Wood JG, et al.Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae[J]. Nature, 2003, 423(6936):181-185.
[8] French JB, Cen Y, Sauve AA, et al.High-resolution crystal structures of Streptococcus pneumoniae nicotinamidase with trapped intermediates provide insights into the catalytic mechanism and inhibition by aldehydes[J]. Biochemistry, 2010, 49(40):8803-8812.
[9] Rongvaux A, Shea RJ, Mulks MH, et al.Pre-B-cell colony-enhancing factor, whose expression is up-regulated in activated lymphocytes, is a nicotinamide phosphoribosyl transferase, a cytosolic enzyme involved in NAD biosynthesis[J]. European Of Immunology, 2002, 11(32), 3225-3234.
[10] Belenky P, Bogan KL, Brenner C.NAD+ metabolism in health and disease[J]. Trends in Biochemical Sciences, 2007, 32(1):12-19.
[11] Smith BC, Anderson MA, Hoadley KA, et al.Structural and kinetic isotope effect studies of nicotinamidase(Pnc1)from Saccharomyces cerevisiae[J]. Biochemistry, 2012, 51(1):243-256.
[12] Balan V, Miller GS, Kaplun L, et al.Life span extension and neuronal cell protection by drosophila nicotinamidase[J]. Journal of Biological Chemistry, 2008, 283(41):27810-27819.
[13] Rogina B, Helfand SL, Ames BN.Sir₂ mediates longevity in the fly through a pathway related to calorie restriction[J]. Proc Natl Acad Sci USA, 2004, 101(45):15998-16003.
[14] Ge F, Zhou LY, Wang Y, et al.Hydrolysis of the neonicotinoid insecticide thiacloprid by the N₂-fixing bacterium Ensifer meliloti CGMCC 7333[J]. International Biodeterioration & Biodegradation, 2014, 93:10-17.
[15] Mathew CD, Nagasawa T, Kobayashi M, et al.Nitrilase-catalyzed production of nicotinic acid from 3-cyanopyridine in Rhodococcus rhodochrous J1[J]. Applied & Environmental Microbiology, 1988, 54(4):1030-1032.
[16] Sánchezcarrón G, Garcíagarcía MI, Zapatapérez R, et al.Biochemical and mutational analysis of a novel nicotinamidase from Oceanobacillus iheyensis HTE831[J]. PLoS One, 2013, 8(2):e56727 .
[17] Pardee AB, Benz EJ, Stpeter DA, et al.Hyperproduction and purification of nicotinamide deamidase, a microconstitutive enzyme of Escherichia coli[J]. Journal of Biological Chemistry, 1971, 246(22):6792-6796.
[18] Yan C, Sloan DL.Purification and characterization of nicotinamide deamidase from yeast[J]. Journal of Biological Chemistry, 1987, 262(19):9082-9087.
[19] Somoskovi A, Wade MM, Sun Z, et al.Iron enhances the antituberculous activity of pyrazinamide[J]. Journal of Antimicrobial Chemotherapy, 2004, 53(2):192-196.
[20] Zhang H, Deng JY, Bi LJ, et al.Characterization of Mycobacterium tuberculosis nicotinamidase/pyrazinamidase[J]. FEBS Journal, 2008, 275(4):753-762.
[21] Ion BF, Kazim E, Gauld JW.A multi-scale computational study on the mechanism of streptococcus pneumoniae nicotinamidase(SpNic)[J]. Molecules, 2014, 19(10):15735-15752.
[22] Frothingham R, Meekero’Connell WA, Talbot EA, et al. Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA[J]. Antimicrobial Agents & Chemotherapy, 1996, 40(6):1426-1431.
[23] Sheen P, Ferrer P, Gilman RH, et al.Role of metal ions on the activity of Mycobacterium tuberculosis pyrazinamidase[J]. American Journal of Tropical Medicine & Hygiene, 2012, 87(1):153-161.
[24] Ahmady A, Poolad T, Rafee P, et al.Study of pyrazinamidase structural changes in pyrazinamide resistant and susceptible isolates of Mycobacterium tuberculosis[J]. Tuberk Toraks, 2013, 61(2):110-114.

Ensifer meliloti 1021烟酰胺酶的酶学特性及3-氰基吡啶调控机理的研究

Research on the NAMase of Ensifer meliloti 1021 and Regulation Mechanism of 3-Cyanopyridine

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 9

编辑推荐

Metrics

本文评价

[1]	张开平, 刘燕丽, 涂绵亮, 李继伟, 吴文标. 烟曲霉A-16产纤维素酶工艺优化及酶学特性[J]. 生物技术通报, 2022, 38(9): 215-225.
[2]	付巧, 林啟兰, 薛强, 熊海容, 王亚伟. N端截短CBM41对枯草芽孢杆菌来源普鲁兰酶酶学性质的影响[J]. 生物技术通报, 2022, 38(6): 245-251.
[3]	秦日甜, 谢占玲. 镰刀菌Q7-31T果胶酶PGL1的分离纯化、酶学性质鉴定及结构分析[J]. 生物技术通报, 2018, 34(4): 151-160.
[4]	于林港, 宿玲恰, 吴敬. Escherichia coli str. K-12 substr. MG1655海藻糖酶Tre F的重组表达及性质研究[J]. 生物技术通报, 2017, 33(4): 177-184.
[5]	贾博涵,周伟,赵罗迪,杨埔,苟敏. 一株产纤维素酶细菌的分离鉴定及酶学特性研究[J]. 生物技术通报, 2014, 0(11): 187-192.
[6]	蔡刘体;胡重怡;. 烟草烟酰胺酶基因的克隆及序列分析[J]. , 2011, 0(06): 76-79.
[7]	张丽靖;沈江锋;金庆超;杨郁;. 一株酸性淀粉酶产生菌的分离、鉴定及酶学特性初步研究[J]. , 2011, 0(05): 142-145.
[8]	谢振荣;慕跃林;闫丽娟;赵春雷;黄遵锡;. α-葡萄糖苷酶高产菌株HB-9-5的选育及产酶条件的优化[J]. , 2010, 0(06): 206-211.
[9]	梅岩;陈丽梅;. 植物甲酸脱氢酶的研究进展[J]. , 2010, 0(05): 23-26.