水生栖热菌漆酶TaLac的性质分析及对孔雀石绿染料的脱除

doi:10.13560/j.cnki.biotech.bull.1985.2021-0852

摘要/Abstract

摘要：

为挖掘适用于脱除有毒染料孔雀石绿（malachite green,MG）的DUF152家族漆酶,利用生物信息学方法在嗜热菌水生栖热菌中筛选候选酶TaLac,构建重组表达载体pET28a-TaLac,在大肠杆菌中进行过量表达和纯化,测定TaLac的酶学性质,探究TaLac对MG的脱色和脱毒能力。结果表明,TaLac具有DUF152家族保守铜原子结合位点,利用Ni亲和层析纯化得到高纯度的TaLac具有氧化2,6-二甲基苯酚（2,6-dimethylphenol,DMP）等漆酶底物的催化活性。以DMP为底物,该酶的最适pH为5.0,最适温度为60℃,K_m为0.74 mmol/L。此外,TaLac在50℃热处理4 h后,仍保留80%的原始活力;该酶可耐受高浓度的Mg²⁺、Ca²⁺、Mn²⁺、Zn²⁺和Ba²⁺等金属离子。TaLac可于3 h内使50 mg/L MG完全脱色,并彻底消除MG对玉米萌发的毒害作用。因此,TaLac是具有活性的DUF152家族漆酶,具有良好的热稳定性,可高效催化MG的脱色与脱毒。

关键词: 漆酶, DUF152家族, 水生栖热菌, 酶学性质, 孔雀石绿的脱色及脱毒

Abstract:

To mine the laccases of DUF152 family showing great potential in removing the hazardous dye MG,a candidate from the thermophile Thermus aquaticus（TaLac）was screened by bioinformatics methods,and the recombinant vector pET28a-TaLac was constructed and overexpressed in Escherichia coli. TaLac was purified to study its enzymatic characterization and the ability to decolorize and detoxify MG. The results showed that TaLac contained the conserved copper binding sites of DUF152 family. The purified TaLac with high purity using nickel chelating affinity chromatography catalyzed the oxidation of 2,6-dimethylphenol（DMP）,the substrate of typical laccases. The optimal pH,temperature,and K_m of TaLac were 5.0,60℃,and 0.74 mmol/L,respectively using DMP as the substrate. Besides,TaLac retained 80% of its original activity after incubation for 4 h at 50℃. TaLac tolerated high concentrations of Mg²⁺,Ca²⁺,Mn²⁺,Zn²⁺ and Ba²⁺. Furthermore,TaLac completely decolorized 50 mg/L of MG in 3 h,and completely eliminated the toxicity of MG to Zea mays. Taken together,TaLac is a functional and thermostable laccase of DUF152 family,showing great ability to decolorize and detoxify MG.

Key words: laccase, DUF152 family, Thermus aquaticus, enzymatic characterization, decolorization and detoxification of malachite green

毛国涛, 王杰, 王凯, 王方园, 曹乐言, 张宏森, 宋安东. 水生栖热菌漆酶TaLac的性质分析及对孔雀石绿染料的脱除[J]. 生物技术通报, 2022, 38(4): 261-268.

MAO Guo-tao, WANG Jie, WANG Kai, WANG Fang-yuan, CAO Le-yan, ZHANG Hong-sen, SONG An-dong. Characterization of Laccase TaLac from Thermus aquaticus and Its Application in Removing Malachite Green Dye[J]. Biotechnology Bulletin, 2022, 38(4): 261-268.

图/表 8

图1 TaLac的生物信息学分析 A：TaLac与GsYlmD（WP_053413740.1）、RL5（CAK32504.1）、Tfu1114（AAZ55152.1）和LaclK（WP_029500662）的序列比对;相同的氨基酸残基高亮为红色,可能的铜原子结合位点使用黑色三角标记;B：TaLac的三级结构模型

Fig.1 Bioinformatic analyses of TaLac A：Sequence alignment of TaLac,GsYlmD（WP_053413740.1）,RL5（CAK32504.1）,Tfu1114（AAZ55152.1）and LaclK（WP_029500662）. The identical residues are highlighted with red,and the possible copper ion binding sites are labelled with black triangles. B：Tertiary structure model of TaLac

图2 TaLac的克隆及纯化 A：pET28a-TaLac表达载体结构;B：纯化后TaLac的SDS-PAGE图;1：纯化后TaLac;M：蛋白分子量标准品

Fig.2 Cloning and purification of TaLac A：The expression vector structure of pET28a-TaLac. B：SDS-PAGE of the purified TaLac. 1：Purified TaLac. M：Protein marker

图3 pH和温度对TaLac活力的影响 A：最适pH;B：最适温度;C：热稳定性

Fig.3 Effects of pH and temperature on the activity of TaLac A：Optimal pH. B：Optimal temperature. C：Thermostability

图4 TaLac的底物特异性（A）和对DMP的动力学参数（B）

Fig.4 Substrate specificity of TaLac（A）and kinetic parameters against DMP（B）

图5 TaLac活力对Cu2+的依赖

Fig. 5 Dependence of TaLac activity on Cu2+

图6 金属离子对TaLac活力的影响

Fig. 6 Effects of metal ions on the activity of TaLac

图7 TaLac催化MG的脱色

Fig. 7 Decolorization of MG catalyzed by TaLac

表1 MG及其脱色产物的植物毒性实验

Table 1 Phytotoxicity test of MG and MG decolorized products

参数Parameter	对照组Control group	MG组MG group	MG脱色产物组MG decolorization group
发芽率 Germination/%	100 ^a	33 ^b	100 ^a
胚芽长度 Plumule length/cm	1.50 ± 0.82 ^a	0.40 ± 0.17 ^b	1.55 ± 0.90 ^a
胚根长 Radicle length/cm	5.75 ± 1.60 ^a	1.26 ± 0.64 ^b	5.25 ± 1.98 ^a

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