鲤鱼来源乙酰胆碱酯酶的高效表达及分子对接模拟
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河北省高等学校科学技术研究项目 (BJ2014036)


High-efficiency Expression and Molecular Docking of Acetylcholinesterase from the Carp
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    摘要:

    乙酰胆碱酯酶介导的农药残留检测方法在食品安全中发挥着越来越重要的作用。本研究以鲤鱼来源的乙酰胆碱酯酶基因(CpAChE)为试验材料,通过基因密码子优化来提高酶的表达量;通过表征鲤鱼乙酰胆碱酯酶的酶学性质探究其在农药残留检测中的应用潜力。采用计算机模拟技术寻找有机磷农药克百威与酶结合的氨基酸残基位点。结果表明:经密码子优化的重组酶乙酰胆碱酯酶的酶活力为7.4 U/mL,表达量提高约40倍,最适反应温度25 ℃,最适pH 8.0。动力学参数Km和Vmax 分别是1.639 mg/mL和8.071 μmol/min/mg。不同有机溶剂和金属离子对酶活力的影响存在差异,大多数有机试剂对乙酰胆碱酯酶活性有抑制作用,而Mg2+、K+、Ca2+和Li+离子对该酶活性有促进作用,其中Mg2+(5 mmol/L)的促进最为明显,使该酶活力提高约1.7倍。重组酶对8种有机磷和8种氨基甲酸酯类农药都表现出较强的敏感性,其中,克百威抑制作用最强,最低检测限为0.044 μg/mL。此外,分子对接发现,克百威与重组酶中的19个氨基酸之间形成氢键和疏水作用,其中氨基酸Tyr146、Ser147和Tyr355很可能是与克百威结合的靶位点。本研究成功实现了乙酰胆碱酯酶的高效表达,探究了乙酰胆碱酯酶与克百威的结合位点,为该酶在食品中有机磷和氨基甲酸类农药残留检测的应用及理性改造提供了一定的理论指导。

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    Acetylcholinesterase(AChE) has been widely used for the detection of organophosphate and carbamate pesticides. In this work, the CpAChE gene has been constructed to match P. pastoris-preferred codon usage for simple scale-up fermentation. We have analyzed the characterization of CpAChE and evaluate the application effect in pesticide residue detection. Meanwhile, the computer simulation technique was used to investigate carbofuran-binding amino acid residues of CpAChE. The results showed that the codon-optimized recombinase CpAChE activity was 7.4 U/mL (increasing 40-fold). The optimum temperature and pH value for activity are 25 ℃ and pH 8.0, respectively. The kinetic parameters of CpAChE are found to be Km of 1.639 mg/mL, and Vmax of 8.071 μmol/min/mg, respectively. The inhibitory effects of most organic reagents on the enzyme activity were examined. By contrast, the activity of the enzyme is strongly activated by Mg2+, K+, Ca2+ and Li+. When Mg2+ (5 mmol/L) was added to the enzyme, it is obvious that a 1.7-fold increase in the activity of the enzyme can be seen. The recombinant CpAChE showed high sensitivity to organophosphorus and carbamate pesticides, and the detection limit of carbofuran was 0.044 μg/mL. The results of molecular docking indicate that the carbofuran and the 19 amino acids of recombinase CpAChE were tightly bound by the hydrogen bond network and hydrophobic interaction, and the Tyr146, Ser147 and Tyr355 are likely to play a more important role. In conclusion, the optimized CpAChE gene is a high-level expression. Meanwhile, the mechanism between the recombinant CpAChE and carbofuran were investigated. This work provides theoretical support for the application and molecular evolution of the CpAChE.

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卢海强;陈伟;黄蕾;谷新晰;田洪涛.鲤鱼来源乙酰胆碱酯酶的高效表达及分子对接模拟[J].中国食品学报,2021,21(6):92-100

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  • 在线发布日期: 2021-07-16
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