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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (9): 17-26    DOI: 10.13523/j.cb.2204041
研究报告     
盐穗木金属硫蛋白HcMT的体外自由基清除活性及抗氧化能力*
刘阳,彭翠,吴彦辰,邓夕莞,毛新芳,刘忠渊**()
四川轻化工大学化学工程学院 自贡 643000
Free Radical Scavenging Activity and Antioxidant Capacity of Metallothionein HcMT from Halostachys caspica in vitro
LIU Yang,PENG Cui,WU Yan-chen,DENG Xi-wan,MAO Xin-fang,LIU Zhong-yuan**()
College of Chemical Engineering, Sichuan University of Science & Engineering, Zigong 643000, China
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摘要：

目的: 原核表达盐穗木(Halostachys caspica C. A. Mey.)金属硫蛋白HcMT并探究其抗氧化活性。方法: 构建原核表达载体pET-32a-HcMT,转化至大肠杆菌Escherichia coli BL21,加入Zn2+胁迫培养(终浓度为200 μmol/L),分离纯化得到Zn-HcMT,测定Zn-HcMT自由基清除活性和总抗氧化能力,制备复合物Zn-HcMT/TiO2并做FTIR表征。结果: 通过原核表达获得融合蛋白Zn-HcMT,对·OH、O2·-、DPPH自由基具有较强的清除活性,对·OH、O2·-的IC50分别为0.386 mg/mL、0.038 mg/mL。融合蛋白浓度为0.01 mg/mL时,对DPPH清除率达(37.43 ± 0.006 8)%,浓度为0.3mg/mL时TEAC(trolox-equivalent antioxidant capacity)值为(1.023 ± 0.01)mmol/L,融合蛋白还原力A700为0.142 ± 0.055,FTIR图谱同时表现了Zn-HcMT和TiO2吸收特性。结论: Zn-HcMT具有良好的清除ROS活性及较强的抗氧化能力,在化妆品领域有潜在应用前景。
关键词： 盐穗木金属硫蛋白;  原核表达;  自由基;  功能活性;  防晒霜    
Abstract:

Objective: To express metallothionein HcMT of Halostachys caspica in prokaryotic cells and explore its antioxidant activity, so as to lay a foundation for its application in the field of cosmetics. Methods: the prokaryotic expression vector pET-32a-HcMT was constructed, transformed into Escherichia coli BL21, cultured under Zn2+ stress (the final concentration was 200 μmol/L), separated and purified to obtain Zn-HcMT, and its free radical scavenging activity and total antioxidant capacity were measured. The complex Zn-HcMT/TiO2 was prepared and characterized by FTIR. Results: The fusion protein Zn-HcMT was obtained by prokaryotic expression. It had strong scavenging activity for ·OH,O2·- and DPPH free radicals. The IC50 of ·OH and O2·-were 0.386 mg/mL, 0.038 mg/mL, respectively. The DPPH clearance rate of 0.01 mg/mL fusion protein was (37.43 ± 0.006 8)%, the TEAC value of 0.3mg/mL fusion protein was (1.023 ± 0.01)mmol/L, and the reducing power of 0.3 mg/mL fusion protein A700 was 0.142 ± 0.055. FTIR spectra showed the absorption characteristics of Zn-HcMT and TiO2 at the same time. Conclusion: Zn-HcMT has a good ROS scavenging activity and strong antioxidant capacity. It has great application potential in the field of cosmetics.
Key words: Halostachys caspica metallothionein    Prokaryotic expression    Free radical    Functional activity    Sunscreen cream
收稿日期: 2022-04-19 出版日期: 2022-10-10
ZTFLH:  Q819  
基金资助: * 四川省“千人计划”(E90112);四川省应用基础研究(2019YJ0459)
通讯作者: 刘忠渊     E-mail: lzy1168@163.com
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引用本文:

刘阳,彭翠,吴彦辰,邓夕莞,毛新芳,刘忠渊. 盐穗木金属硫蛋白HcMT的体外自由基清除活性及抗氧化能力*[J]. 中国生物工程杂志, 2022, 42(9): 17-26.

LIU Yang,PENG Cui,WU Yan-chen,DENG Xi-wan,MAO Xin-fang,LIU Zhong-yuan. Free Radical Scavenging Activity and Antioxidant Capacity of Metallothionein HcMT from Halostachys caspica in vitro. China Biotechnology, 2022, 42(9): 17-26.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2204041    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I9/17

图1  重组蛋白Zn-HcMT的表达
图2  重组蛋白Zn-HcMT的纯化
图3  Zn-HcMT对羟自由基的清除率
图4  不同蛋白质对邻苯三酚自氧化速率抑制曲线
图5  Zn-HcMT对超氧自由基清除率
图6  DPPH清除率曲线
图7  不同浓度的样品还原力曲线
图8  不同浓度样品的总抗氧化能力曲线
图9  Zn-HcMT/TiO2复合材料的FTIR光谱
No Observed
frequency /cm		 Peak assignment Visible Founctional group
intensity
1 3 396.02 O-H Stretching Strong Phenols
2 3 233.96 N-H Stretching Strong Benzenering
3 2 985.10 C-H Stretching Medium Alkanes
4 2 596.10 S-H Stretching Medium Sulfhydryl group
5 1 630.98 C=O Bond Strong Amines
6 1 551.94 C=C Stretching Strong Phenols
7 1 396.29 C-N Stretching Strong Alipahtic amines
8 1 297.51 C-O Stretching Strong Secondary alcohol
9 1 042.85 C-N Stretching Strong Alipahtic amines
10 597.93 Ti-O-Ti Stretching Strong Mental oxide
表1  Zn-HcMT/TiO2复合材料的FTIR图谱分析
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