<em>LmAPX</em>基因在大肠杆菌和酵母菌中的表达研究

doi:10.13523/j.cb.20140704

中国生物工程杂志

2014, Vol. 34

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

研究报告

LmAPX基因在大肠杆菌和酵母菌中的表达研究

高海伶¹, 季静¹, 王罡¹, 吴广霞², 荣非¹, 关春峰¹, 金超¹

1. 天津大学环境科学与工程学院天津 300072;
2. 天津大学化工学院天津 300072

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

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摘要：

目的：研究枸杞抗坏血酸过氧化物酶基因（ascorbate peroxidase，LmAPX）在原核中的表达和酶学特性以及在酵母菌中的抗氧化能力，为进一步研究逆境诱导的抗氧化胁迫的作用机理奠定理论基础。方法：将LmAPX转入大肠杆菌BL21中进行异源表达，采用 Ni²⁺亲和层析，纯化重组蛋白，并对不同温度和pH值下的酶活进行研究，Lineweaver-Burk双倒数作图法测定该酶的Km和Vmax值。将LmAPX转入酵母菌株W303中进行H₂O₂和NaCl氧化胁迫处理。结果：该酶的最适温度和最适pH值分别为40℃和6.5。当抗坏血酸（Ascorbic acid，AsA）浓度过量时，对H₂O₂的Km和Vmax分别是0.17±0.02 mmol/L和11.78±1.88 mmol/min·mg；当H₂O₂浓度过量时，对AsA的Km和Vmax分别是2.19±0.40 mmol/L和58.82±3.51 mmol/min·mg。含有LmAPX基因的酵母菌株，在半乳糖的诱导下在8 mmol/L H₂O₂和100 mmol/L NaCl的培养基上的生长都明显优于对照组。结论：LmAPX蛋白具有很好的抗氧化性和耐盐性。

关键词： 中华枸杞; 抗坏血酸过氧化物酶（APX）; 酶学分析; 氧化胁迫

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

收稿日期: 2014-05-12 出版日期: 2014-07-25

ZTFLH:

Q78

基金资助:

国家自然科学基金（31271793，31271419），国家转基因生物新品种培育重大专项（2014ZX08003-002B），国家水体污染控制与治理科技重大专项（2014ZX07203-009）

通讯作者: 季静, 王罡 E-mail: jijingtjdx@163.com;wanggangtjdx@126.com

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引用本文:

高海伶, 季静, 王罡, 吴广霞, 荣非, 关春峰, 金超. LmAPX基因在大肠杆菌和酵母菌中的表达研究[J]. 中国生物工程杂志, 2014, 34(7): 24-29.

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.

链接本文:

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

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