生物技术通报 ›› 2022, Vol. 38 ›› Issue (4): 261-268.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0852
毛国涛(), 王杰, 王凯, 王方园, 曹乐言, 张宏森, 宋安东()
收稿日期:
2021-07-06
出版日期:
2022-04-26
发布日期:
2022-05-06
通讯作者:
宋安东,男,博士,教授,研究方向:农林废弃物的资源化利用;E-mail: song1666@126.com作者简介:
毛国涛,男,博士,讲师,研究方向:木质纤维素降解酶;E-mail: maoguotao@henau.edu.cn
基金资助:
MAO Guo-tao(), WANG Jie, WANG Kai, WANG Fang-yuan, CAO Le-yan, ZHANG Hong-sen, SONG An-dong()
Received:
2021-07-06
Published:
2022-04-26
Online:
2022-05-06
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℃,Km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。
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
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 |
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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