蜜蜂NADPH-细胞色素P450还原酶基因在大肠杆菌中的表达及酶学特性分析

doi:DOI:10.13523/j.cb.20161205

中国生物工程杂志

2016, Vol. 36

Issue (12): 28-35 DOI: DOI:10.13523/j.cb.20161205

研究报告

蜜蜂NADPH-细胞色素P450还原酶基因在大肠杆菌中的表达及酶学特性分析

武雪龙, 杨晓慧, 汪俊卿, 王瑞明

山东省齐鲁工业大学生物工程学院济南 250353

Expression and Characteristics of Apis mellifera NADPH-cytochrome P450 Reductase Gene in Escherichia coli

WU Xue-long, YANG Xiao-hui, WANG Jun-qing, WANG Rui-ming

Shandong Provincial Key Laboratory of Microbial Engineering, Department of Biology Engineering, Qilu University of Technology, Jinan 250353, China

全文: PDF(1310 KB) HTML

摘要：

10-羟基-2-癸烯酸（10-HDA）是蜂王浆中的主要脂肪酸成分，具有抗菌、抗癌、延缓衰老等多种生理活性，但目前关于10-HDA生物合成的分子机制还不清楚。通过克隆蜜蜂NADPH-细胞色素P450还原酶（EC 1.6.2.4，NADPH-cytochrome P450 reductase，CPR），在大肠杆菌中异源表达，并对其酶学特性进行分析。结果表明重组菌经IPTG诱导后表达蛋白的分子量与预期一致，为86.29 kDa，Ni-NTA亲和纯化后测得其比活性为77.33（EU of CPR）/μg。酶学性质分析结果表明蜜蜂CPR酶最适温度与pH分别为40℃和8.0，并对一些金属离子及有机溶剂具有不同程度的耐受性。其对底物细胞色素C的动力学参数K_m和k_cat分别为76 μM和268/min。以上研究为探究CPR在10-HDA生物合成途径中的功能奠定理论基础。

关键词： 酶学特性; 大肠杆菌; 异源表达; NADPH-细胞色素P450还原酶

Abstract:

10-Hydroxy-2-decenoic acid (10-HDA),a major fatty acid component from royal jelly,is known to have various biologic activities in antibacteria, antitumour and lifespan-extending.However, it is unclear about the underlying molecular mechanism of its biosynthesis. A NADPH-cytochrome P450 reductase (EC 1.6.2.4, NADPH-cytochrome P450 reductase, CPR) gene from Apis mellifera was cloned and expressed in Escherichia coli. Subsequently, the enzymatic properties of CPR were analyzed. Results showed that the target protein was successfully expressed in E. coli after induced by IPTG, and its molecular weight was 86.29 kDa. The CPR protein was then purified by Ni-NTA protein affinity chromatography and its specific activity was 77.33 (EU of CPR)/μg.The purified CPR protein displayed the maximum activity at pH 8.0 and 40℃. Its activity was not significantly affected by several metal ions except Ni²⁺, Zn²⁺ and Co²⁺ which caused a strong inhibition. The K_m and k_cat, towards cytochrome C at 30℃, were 76μM and 268/min, respectively. It lay a foundation for researching the function of CPR in 10-HDA biosynthesis pathway.

Key words: Heterologous expression Enzymatic properties Escherichia coli NADPH-cytochrome P450 reductase

收稿日期: 2016-05-25 出版日期: 2016-12-25

ZTFLH:

Q819

基金资助:

国家自然科学基金（31201281，31171727）、山东省自主创新及成果转化专项（201422CX02602）资助项目

通讯作者: 王瑞明 E-mail: ruiming3K@163.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章

引用本文:

武雪龙, 杨晓慧, 汪俊卿, 王瑞明. 蜜蜂NADPH-细胞色素P450还原酶基因在大肠杆菌中的表达及酶学特性分析[J]. 中国生物工程杂志, 2016, 36(12): 28-35.

WU Xue-long, YANG Xiao-hui, WANG Jun-qing, WANG Rui-ming. Expression and Characteristics of Apis mellifera NADPH-cytochrome P450 Reductase Gene in Escherichia coli. China Biotechnology, 2016, 36(12): 28-35.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/DOI:10.13523/j.cb.20161205 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I12/28

[1] 易胜.烟粉虱NADPH-细胞色素P450还原酶与NADPH-细胞色素b5还原酶基因的克隆机表达量分析.湖南:湖南农业大学,2014. Yi S. Molecular cloning and relative expression analysis of NADPH-cytochrome P450 reductase and NADPH-cytochrome b5 reductase gene in whitefly (Bemisia tabaci). Hunan:Hunan Agricultural University,2014.
[2] Rana S, Lattoo S K, Dhar N, et al. NADPH-cytochrome P450 reductase:molecular cloning and function characterization of two paralogs from Withania somnifera (L.) Dunal. PLoS One, 2013,8(2):e57068.
[3] Jensen K, Møller B L,Birger L.Plant NADPH-cytochrome P450 oxidoreductases.Phytochemistry,2010,71(2-3):132-141.
[4] Riddick D S, Ding X, Wolf C R,et al. NADPH-cytochrome P450 oxidoreductase:roles in physiology, pharmacology, and toxicology. Drug Metabolism and Disposition, 2013,41(1):12-23.
[5] Mizutani M. Impacts of diversification of cytochrome P450 on plant metabolism. Biol Pharm Bull, 2012,35(6):824-832.
[6] Cheng J, Wan D F, Gu J R,et al.Establishment of a yeast system that stably expresses human cytochrome P450 reductase:application for the study of drug metabolism of cytochrome P450s in vitro. Protein Expression and Purification,2006,47(2):467-476.
[7] Nonaka Y, Horike N, Takemori H, et al. Molecular cloning of NADPH-cytochrome P450 oxidoreductase from silkworm egg. European Journal of Biochemistry, 2000, 267(23):6914-6920.
[8] Hart S N,Wang S,Nakamoto K, et al. Genetic polymorphisms in cytochrome P450 oxidoreductase influence microsomal P450-catalyzed drug metabolism. Pharmacogenet Genomics, 2008, 18(1):11-24.
[9] Shackleton C, Marcos J, Arlt W, et al. Prenatal diagnosis of P450 oxidoreductase deficinecy (ORD):a discorder causing low pegnancy estriol, maternal and fetal virilization, and the Antley-Bixler syndrome phenotype. Am J Med Genet A, 2004,129A(2):105-112.
[10] Koo A J,Howe G A.Catabolism and deactivation of the lipid-derived hormone jasmonoyl-isoleucine. Frontiers in Plant Science, 2012,3:19.
[11] Heitz T,Widemann E,Lugan R,et al.Cytochromes P450 CYP94C1 and CYP94B3 catalyze two successive oxidation steps of plant hormone jasmonoyl-isoleucine for catabolic turnover. Journal of Biological Chemistry,2012, 287(9):6296-6306.
[12] Saurabh B,Kwangkyoung L,Tae-Jin O.Hydroxylation of long chain fatty acids by CYP147F1, a new cytochrome P450 subfamily protein from Streptomyces peucetius.Archives of Biochemistry and Biophysics,2013,539(1):63-69.
[13] Isoda Y.Antitumor activity of lipids antitumor activity of free fatty acid in mice with transplant stumors. Yukagaku,1993,42(11):923-928.
[14] 党亚丽,张中健,闫小伟.蜂王浆产品中10-羟基-2-癸烯酸的稳定性比较.食品研究与开发,2012,7(33):5-8. Dang Y L, Zhang Z J, Yan X W. Comparison on the stability of 10-hydroxy-2-decenoic acid in royal jelly product.Food Research and Development,2012,7(33):5-8.
[15] Plettner E, Slessor K N, Winston M L. Biosynthesis of mandibular acids in honeybees (Apis mellifera):De novo synthesis, route of fatty acid hydroxylation and caste selective β-oxidation. Insect Biochem Mol Biol, 1997, 1:31-42.
[16] Paine M J, Scrutton N S, Munro A W, et al. Electron transfer partners of cytochrome P450. In:Ortiz de Montellano. Cytochrome P450. Springer, 2005.115-148.
[17] Kim D, Cryle M J, De Voss J J. et al. Functional expression and characterization of cytochrome P45052A21 from Candida albicans. Arch Biochem Biophys, 2007, 464(2):213-220.
[18] Saurabh B,Kwangkyoung L,Tae-Jin O.Hydroxylation of long chain fatty acids by CYP147F1, a new cytochrome P450 subfamily protein from Streptomyces peucetius. Archives of Biochemistry and Biophysics,2013,539(1):63-69.
[19] Brogdon W G, McAllister J C, Vulule J. Heme peroxidase activity measured in single mosquitoes identifies individuals expressing an elevated oxidase for insecticide resistance. J Am Mosq Control Assoc, 1997, 13(3):233-237.
[20] Tiwari S,Pelz-Stelinski K, Mann R S, et al. Glutathione transferase and cytochrome P450(general oxidase) activity levels in Candidatus Liberibacter asiatcus-infected and uninfected Asian citrus psyllid (Hemiptera:Psyllidae). Ann Entomol Soc Am, 2011, 104(2):297-305.
[21] Koener J E, Cario F A, Feyereisen R. The cDNA and deduced protein sequence of house fly NADPH-cytochrome P450 reductase. Insect Biochem Mol Biol, 1993, 23(4):439-447.
[22] Zhu F, Sams S, Moural T, et al. RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius. PLoS One,2012,7(2), e31037.
[23] Dong L B,Xiaojie Zhoua B C,Mei L,et al.Characterization of NADPH-cytochrome P450 reductase gene from the cotton bollworm, Helicoverpa armigera.Gene,2014,545(2):262-270.
[24] Ga-Young L,Hyun M K,Sang H M,et al.Heterologous expression and functional characterization of the NADPH-cytochrome P450 reductase from Capsicum annuum.Plant Physiology and Biochemistry,2014,82:116-122.
[25] Zhou X,Li M,Sheng C,et al.NADPH-cytochrome P450 oxidoreductase from the chicken (Gallus gallus):Sequence characterization, functional expression and kinetic study.Comparative Biochemistry and Physiology Part C:Toxicology and Pharmacology,2011,153(1):53-39.

[1]	乔圣泰,王曼琦,徐慧妮. 番茄SlTpx原核表达蛋白的体外功能分析^*[J]. 中国生物工程杂志, 2021, 41(8): 25-32.
[2]	何若昱,林福玉,高向东,刘金毅. 信号肽在大肠杆菌分泌系统中的研究与应用进展[J]. 中国生物工程杂志, 2021, 41(5): 87-93.
[3]	吴弘轩, 杨金花, 沈培杰, 李清晨, 黄建忠, 祁峰. 利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究 ^*[J]. 中国生物工程杂志, 2021, 41(1): 12-19.
[4]	饶海密,梁冬梅,李伟国,乔建军,财音青格乐. 真菌芳香聚酮化合物的合成生物学研究进展^*[J]. 中国生物工程杂志, 2020, 40(9): 52-61.
[5]	闫伟欢,黄统,洪解放,马媛媛. 丁醇在大肠杆菌中的生物合成研究进展^*[J]. 中国生物工程杂志, 2020, 40(9): 69-76.
[6]	位薇,常保根,王英,路福平,刘夫锋. Tau蛋白核心片段306~378的异源表达、纯化及聚集特性验证^*[J]. 中国生物工程杂志, 2020, 40(5): 22-29.
[7]	童梅,程永庆,刘金毅,徐晨. 促进大肠杆菌周质空间小分子抗体表达的菌种构建方法^*[J]. 中国生物工程杂志, 2020, 40(5): 48-56.
[8]	杨丽,石晓宇,李文蕾,李剑,徐寒梅. 构建噬菌体展示抗体库过程中电穿孔法的条件优化[J]. 中国生物工程杂志, 2020, 40(4): 42-48.
[9]	乐易林,傅毓,倪黎,孙建中. 热稳定性丙酮酸:铁氧还蛋白氧化还原酶异源表达及其在乙酰辅酶A合成中的应用 ^*[J]. 中国生物工程杂志, 2020, 40(3): 72-78.
[10]	杭海英,刘春春,任丹丹. 流式细胞术的发展、应用及前景 ^*[J]. 中国生物工程杂志, 2019, 39(9): 68-83.
[11]	李吉萍,包昌杰,陈光,张斯童. 木聚糖酶异源表达的研究进展 ^*[J]. 中国生物工程杂志, 2019, 39(7): 91-99.
[12]	赵程程,孙长坡,常晓娇,伍松陵,林振泉. 大肠杆菌细胞裂解系统的构建及其在真菌毒素降解酶表达中的应用 ^*[J]. 中国生物工程杂志, 2019, 39(4): 69-77.
[13]	姚银,闵琪,熊海容,张莉. 木聚糖酶和甘露聚糖酶在毕赤酵母中的共表达及产酶分析 ^*[J]. 中国生物工程杂志, 2019, 39(3): 37-45.
[14]	史超硕,李登科,曹雪,袁航,张钰文,于江悦,路福平,李玉. 两个不同启动子及其组合对碱性蛋白酶AprE异源表达的影响 ^*[J]. 中国生物工程杂志, 2019, 39(10): 17-23.
[15]	陈子晗,周海胜,尹新坚,吴坚平,杨立荣. **Amphibacillus xylanus谷氨酸脱氢酶基因工程菌培养条件优化 ^***[J]. 中国生物工程杂志, 2019, 39(10): 58-66.

Viewed

Full text

Abstract

Cited

Shared

Discussed