Expression Studies of Ascorbate Peroxidase from <em>Lycium chinense</em> Mill. in <em>E.coli</em> and Yeast

doi:10.13523/j.cb.20140704

China Biotechnology

2014, Vol. 34

Issue (7): 24-29 DOI: 10.13523/j.cb.20140704

Expression Studies of Ascorbate Peroxidase from Lycium chinense Mill. in E.coli and Yeast

GAO Hai-ling¹, JI Jing¹, WANG Gang¹, WU Guang-xia², RONG Fei¹, GUAN Chun-feng¹, JIN Chao¹

1. School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China;
2. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

Download:

HTML

PDF(696KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract

Objective:The aim of the study is to investigate the expression and enzymatic properties in E.coli and oxidation resistance in yeast of ascorbate peroxidase (LmAPX) cloned from Lycium chinense Mill., which will provide the theory foundation of antioxidant stress for future studying.Method: LmAPX was transformed into heterologous expression system E.coli BL21 and wild type yeast strain W303. The recombinant protein was purified by Ni²⁺ affinity chromatography and the enzyme activities at different temperature and pH were detected. The enzyme kinetics constant Km and Vmax were calculated by double-reciprocal plot. Results: The results showed that the optimal pH and temperature for this enzyme were 6.5 and 40℃, respectively.At fixed concentration of AsA, the Km and Vmax values of the enzyme for H₂O₂were 0.17±0.02 mmol/L and 11.78±1.88 mmol/min·mg. And at fixed concentration of H₂O₂, the Km and Vmax values for AsA were 2.19±0.40 mmol/L and 58.82±3.51 mmol/min·mg. Tansgenic yeast stain transformed with LmAPX gene showed higher stress resistance than the control in the medium containing 8 mmol/L H₂O₂ or 100 mmol/L NaCl under the inducement of β-galactose. Conclusion: LmAPX enhanced the tolerance to oxidative stresses.

Key words： Lycium chinense Mill. Ascorbate peroxidase Enzymatic analysis Oxidative stress

Received: 12 May 2014 Published: 25 July 2014

ZTFLH:

Q78

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors

Cite this article:

GAO Hai-ling, JI Jing, WANG Gang, WU Guang-xia, RONG Fei, GUAN Chun-feng, JIN Chao. Expression Studies of Ascorbate Peroxidase from Lycium chinense Mill. in E.coli and Yeast. China Biotechnology, 2014, 34(7): 24-29.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20140704 OR https://manu60.magtech.com.cn/biotech/Y2014/V34/I7/24

[1] Zhao C Y, Wang Y C, Song Y D, et al. Biological drainage characteristics of alkalized desert soils in north-western China. Journal of Arid environments, 2004, 56(1): 1-9.

[2] Mittler R, Vanderauwera S, Gollery M, et al. Reactive oxygen gene network of plants. Trends Plant Science, 2004, 9(10): 490-498.

[3] Wu G X, Wang G, JiJ, et al. Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobacco. Gene, 2014, 543(1): 85-92.

[4] Ashraf M. Biotechnological approach of improving plant salt tolerance using antioxidants as markers. Biotechnology Advances, 2009, 27(1): 84-93.

[5] Marcia C P, Mariana B O, Pedro L. Hydrogen peroxide detoxification in the midgut of the blood-sucking insect, Rhodnius prolixus. Insect Biochemistry and Physiology, 2001, 48(2): 63-71.

[6] Shigeoka S, Ishikawa T, Tamoi M, et al. Regulation and function of ascorbate peroxidase isoenzymes. Journal of Ex Perimental Botany, 2002, 53(372): 1305-1319.

[7] 孙卫红,王伟青,孟庆伟.植物抗坏血酸过氧化物酶的作用机制、酶学及分子特性.植物生理学报,2005,41(2):143-147. Sun W H, Wang W Q, Meng Q W. Functional mechanism and enzymatic and molecular characteristic of ascorbate peroxidase in plants.Plant Psysiology Journal, 2005, 41(2): 143-147.

[8] 张维静,陆海,杜希华.抗坏血酸过氧化物酶在植物抵抗氧化胁迫中的作用.山东师范大学学报(自然科学版),2008,23(4):113-115. Zhang W J, Lu H, Du X H. The function of ascorbate peroxidases in plant resistance to oxidative stress. Journal of ShanDong Normal University(Natural Science), 2008, 23(4): 113-115.

[9] 杜秀敏,殷文璇,赵彦修,等. 植物中活性氧的产生及清除机制. 生物工程学报,2001,17(2): 121-125. Du X M, Yin W X, Zhao Y X, et al. The production and Scavenging of reactive oxygen species in plants. Chinese Journal of Biotechnology, 2001, 17(2): 121-125.

[10] Mittler R, Zilinskas B A. Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate per oxidase, FEBS Lett., 1991, 289(2): 257-259.

[11] Yoo T H, Park C J, Lee G L, et al. A hot pepper cDNA encoding ascorbate per oxidase is induced during the incompatible interaction with virus and bacteria. Molecular Cell, 2002, 14(1): 75-84.

[12] Lin L, Wang X P, Wang Y J. cDNA clone, fusion expression and purification of the novel gene related to ascorbate peroxidase from Chinese wild Vitis pseudoreticulata in E.coli. Molecular Biology Rep. 2006, 33(3): 197-206.

[13] Yoshimura K, Ishikawa T, Nakamura Y, et al. Comparative study on recombinant chloroplastic and cytosolic ascorbate peroxidase isozymes of spinach. Arch. Biochem. Biophys, 1998, 353(1): 55-63.

[14] Nishikawa F, Kata M, Wang R, et al. Two ascorbate peroxidases from broccoli: identification, expression and characterization of their recombinant preteins Postharv. Biol. Technol, 2003, 27(2): 147-156.

[15] Li H B, Qin Y M, Pang Y, et al. A cotton ascorbate peroxidase is involved in hydrogen peroxide homeostasis during fibre cell development. New Phytologist, 2007, 175(3): 462-471.

[16] Kazuyoshi O, Satoshi M, Hidenori Tanaka, et al. Purification and characterization of an acidophilic xylanase from Aureobasidium pullulan var. melanigenum and sequence analysis of the encoding gene. Journal of Bioscience and Bioengineering, 2001, 92(3): 262-270.

[17] Lu H, Han R L, Jiang X N. Heterologous expression and characterization of a proxidomal ascorbate peroxidase from Populus tomentosa. Molecular biology reports, 2009, 36(1): 21-27.

[18] 管清杰,李琳,高野哲夫,等. 朝鲜碱茅抗坏血酸过氧化物酶的基因克隆和表达. 基因组学与应用生物学,2009,28(4):631-629. Guan Q J, Li L,Tetsuo T, et al. Cloning of an ascorbate peroxidase gene from puccinellia tenuiflora and its expression analysis. Genomics and Applied Biology, 2009, 28(4): 631-639.

[1]	QIAO Sheng-tai,WANG Man-qi,XU Hui-ni. Functional Analysis of Prokaryotic Expression Protein of Tomato SlTpx in Vitro[J]. China Biotechnology, 2021, 41(8): 25-32.

[2]	XUE Zhi-yong,DAI Hong-sheng,ZHANG Xian-yuan,SUN Yan-ying,HUANG Zhi-wei. Effects of Vitreoscilla Hemoglobin Gene on Growth and Intracellular Oxidation State of Saccharomyces cerevisiae[J]. China Biotechnology, 2021, 41(11): 32-39.

[3]	HAO Xiao-ting,LIU Jun-jie,DENG Yu-lin,ZHANG Yong-qian. Radiation Biosensor Based on Promoter of SOS Reaction and Oxidative Stress Reaction[J]. China Biotechnology, 2020, 40(7): 30-40.

[4]	SHAN hong-yu, LIU Ren-ze, HAO Meng-qi, DONG Xiao-yu, GUO Chang-hong, GUO Dong-lin. Phytoferritin and the Response to Oxidative Stress[J]. China Biotechnology, 2017, 37(2): 121-126.

[5]	HU Yan-zhen, WEI Jun-ying, LUO Guang-ming. Research on Glutathione-related Signaling Pathway in Liver Diseases[J]. China Biotechnology, 2015, 35(10): 72-77.

[6]	JIN Chao, LIU Jie, JI Jing, WANG Gang, CAO Hai-yan, WU Jiang. Overexpression of LcCHYB to Enhance the Tolerance to Oxidative Stress of Eustoma Grandiflorum[J]. China Biotechnology, 2015, 35(1): 27-33.

[7]	MA Pan, LIU Hong-tao, XU Qing-song, BAI Xue-fang, DU Yu-guang. Effects of Chitosan Oligosaccharides Attenuating Menadione-induced Injury in Macrophages[J]. China Biotechnology, 2011, 31(06): 18-21.

[8]	HU Xiu-Li- Liu-Jian-Li- Cao-Xiang-Yu- Hou-Fang-Fang- Gao-Bing. Identification of a functional domain with in DHCR24 that is required for its Antioxidative function[J]. China Biotechnology, 2009, 29(05): 50-54.

[9]	Sang-Soo KWAK Haeng-Soon LEE Xiao-Li Yang. Improving potato plants oxidative stress and salt tolerance by gene transfer both of Cu/Zn superoxide dismutase and ascorbate peroxidase[J]. China Biotechnology, 2008, 28(3): 25-31.

Viewed

Full text

Abstract

Cited

Shared

Discussed