醌氧化还原酶的异源表达及其在偶氮染料脱色方面的研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-1454

摘要/Abstract

摘要：

为研究NAD（P）H:醌氧化还原酶在染料处理方面的潜力,选用大肠杆菌异源表达人来源的NAD（P）H:醌氧化还原酶（hNQO1）,对6种介体小分子（1,4-萘醌、蒽醌-2-磺酸钠盐、甲基对苯醌、1,4-二羟基蒽醌、蒽醌、甲萘醌）进行筛选,选择适合浓度的介体小分子辅助hNQO1全细胞对偶氮染料进行脱色。结果表明,利用1,4-二羟基蒽醌作为介体小分子,hNQO1全细胞对刚果红的脱色效果较好,菌体能够重复利用3次,脱色率（反应6 h）依次为94.0%,79.7%和20.3%。与刚果红相比,hNQO1全细胞对苋菜红、活性黑5的脱色率（6 h）分别为18.8%,16.4%。综上所述,该重组hNQO1在染料废水处理方面具有较好的潜力和前景。

关键词: 醌氧化还原酶, 染料脱色, 介体小分子

Abstract:

In order to study the potential of NAD（P）H:quinone oxidoreductase in dye decolorization,heterologous expressed human source NAD（P）H:quinone oxidoreductase（hNQO1）in Escherichia coli was used to decolorize the azo dye in the presence of mediator at proper concentration. Six small molecular mediators（1,4-naphquinone,sodium anthraquinone-2-sulfonate,2-methlcyclohexa-2,5-diene-1,4-dione,1,4-dihydroxyanthracene-9,10-dion,anthraquinone,and menadione）were investigated for hNQO1. The results showed that hNQO1 whole cell decolorized Congo red very well with 1,4-dihydroxyanthracene-9,10-dion as the small molecular mediator. The hNQO1 whole cells could be reused for three times and the decolorization rate（6 h）was 94.0%,79.7%,and 20.3% respectively. Compared with Congo red,the decolorization rate of hNQO1 whole cells on amaranth and reactive black 5（6 h）was only 18.8% and 16.4%,respectively. In conclusion,the recombinant hNQO1 presents great potential for dye wastewater treatment.

Key words: quinone oxidoreductase, dye decolorization, mediator

刘谦谦, 唐梓静, 李天真, 李宝库, 朱蕾蕾. 醌氧化还原酶的异源表达及其在偶氮染料脱色方面的研究[J]. 生物技术通报, 2021, 37(10): 128-136.

LIU Qian-qian, TANG Zi-jing, LI Tian-zhen, LI Bao-ku, ZHU Lei-lei. Heterologous Expression of Quinone Oxidoreductase and Its Role in the Decolorization of Azo Dyes[J]. Biotechnology Bulletin, 2021, 37(10): 128-136.

图/表 8

图1 异源表达 hNQO1 的 SDS-PAGE A:M :蛋白 marker;1:诱导后的 hNQO1;2:破碎后的上清。B:M : 表示标记;1:纯化后的 hNQO1

Fig.1 SDS-PAGE of hNQO1 heterologously expressed A:M:protein marker;1:induced hNQO1;2: supernatant after crushing. B: M:protein marker;1: purified hNQO1

图2 热稳定性对酶活力的影响

Fig. 2 Influence of thermal stability on enzyme activity

表1 染料名称及其最大吸收波长

Table 1 Dye names and their maximum absorption wavelengths

名称Name	分子质量Molecular mass/kD	吸收波长Absorption wavelength/nm
刚果红 Congo red	696.7	434
苋菜红 Amaranth	604.47	520
活性黑5 Reactive black 5	991.82	596

表2 小分子介体的名称及结构

Table 2 Names and structures of small molecular mediators

名称Name	化学结构Chemical structural formula	分子质量Molecular mass/kD
1,4-二羟基蒽醌 1,4-Dihydroxyant- hracene-9,10-dion		240.2109
1,4-萘醌 1,4-Naphthoqu- inone		158.15
甲萘醌 Menadione		172.18
蒽醌 Anthraquinone		208.2121
甲基对苯醌 2-Methlcyclohexa-2,5-diene-1,4-dione		122.1213
蒽醌-2-磺酸钠 Sodium anthraqu-inone-2-Sulfonate		310.2571

图3 不同介体小分子对hNQO1脱色刚果红影响 1,4-萘醌,1,4-Naphthoquinone;1,4-萘醌（对照）,1,4-Naphthoquinone (control);甲萘醌,Menadione;甲萘醌（对照）,Menadione (control);1,4-二羟基蒽醌,1,4-Dihydroxyanthracene-9,10-dion;1,4-二羟基蒽醌（对照）,1,4-Dihydroxyanthracene-9,10-dion (control);蒽醌,Anthraquinone;蒽醌（对照）,Anthraquinone (control);甲基对苯醌,2-Methlcyclohexa-2,5-diene-1,4-dione;甲基对苯醌（对照）,2-Methlcyclohexa-2,5-diene-1,4-dione (control);蒽醌-2-磺酸钠,Sodium anthraquinone-2-Sulfonate;蒽醌-2-磺酸钠（对照）,Sodium anthraquinone-2-Sulfonate (control)

Fig. 3 Influence of different medium small molecules on the decolorization of hNQO1 Congo red

图4 不同浓度的1,4-二羟基蒽醌对hNQO1脱色刚果红影响

Fig. 4 Effects of 1,4-Dihydroxyanthracene-910-dion at different concentrations on the decolorization of hNQO1 Congo red

图5 重复利用hNQO1全细胞对刚果红染料脱色的研究

Fig. 5 Decolorization of Congo red dye by reusing whole cells of hNQO1

图6 hNQO1全细胞对苋菜红、活性黑5等两种偶氮染料的脱色

Fig. 6 Decolorization of two azo dyes such as amaranth and reactive black 5 by whole cells of hNQO1

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