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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2022, Vol. 42 Issue (9): 17-26    DOI: 10.13523/j.cb.2204041
    
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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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     
Received: 19 April 2022      Published: 10 October 2022
ZTFLH:  Q819  
Corresponding Authors: Zhong-yuan LIU     E-mail: lzy1168@163.com
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Yang LIU
Cui PENG
Yan-chen WU
Xi-wan DENG
Xin-fang MAO
Zhong-yuan LIU
Cite this article:

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.

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

Fig.1 Expression of recombinant protein Zn-HcMT M: Protein molecular weight marker; 1: Lysis of Escherichia coli without induced by IPTG; 2: Lysis of Escherichia coli after induced by IPTG
Fig.2 Purification of recombinant protein Zn-HcMT M: Protein molecular weight marker; 1: Lysis of Escherichia coli without induced by IPTG; 2: Lysis of Escherichia coli after induced by IPTG; 3: Flow through liquid component; 4: Eluate with 5 mmol/L imidazole; 5: Eluate with 5 mmol/L imidazole; 6: Eluate with 10 mmol/L imidazole; 7: Eluate with 100 mmol/L imidazole; 8: Eluate with 200 mmol/L imidazole; 9: Eluate with 500 mmol/L imidazole
Fig.3 Clearance rate of hydroxyl radical by Zn-HcMT
Fig.4 Inhibition curve of pyrogallol autoxidation rate of different proteins Group C: Pyrogallol autoxidation rate
Fig.5 Superoxide radical clearance rate of Zn-HcMT
Fig.6 DPPH clearance curves
Fig.7 Reduction force curves of samples with different concentrations
Fig.8 Total antioxidant capacity curve of samples with different concentrations
Fig.9 FTIR spectra of Zn-HcMT/TiO2 composites
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
Table 1 FTIR spectral analysis of Zn-HcMT/TiO2 composites
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