矢野鞘氨醇菌酯酶SyEst870的重组表达及其对氨基甲酸酯类农药的降解

Recombinant expression of Sphingobium yanoikuyae esterase SyEst870 capable of degrading carbamate pesticides

摘要:

氨基甲酸酯类农药是针对有机氯和有机磷农药的缺点而开发的一种新型广谱杀虫、杀螨、除草剂，其广泛使用及缓慢降解导致环境污染，对生态系统和人类健康造成损害，残留农药的处理是目前环境保护中亟待解决的问题。本研究通过原核表达矢野鞘氨醇菌(Sphingobium yanoikuyae)SGNH/GDSL水解酶家族蛋白SyEst870，研究重组蛋白对氨基甲酸酯类农药的降解能力。构建原核表达载体pET-32a-SyEst870，转化大肠杆菌(Escherichia coli) BL21进行异源表达并纯化；以对硝基苯酚乙酸酯为底物结合对硝基苯酚标准曲线测定酶活性以及温度、pH、金属离子的影响；通过液相色谱-质谱联用技术(liquid chromatography-mass spectrometry,LC-MS)检测SyEst870对甲萘威、速灭威、异丙威的降解能力，并使用气相色谱-质谱联用技术(gas chromatography-mass spectrometry,GC-MS)检测SyEst870对3种农药的降解产物。结果显示，通过大肠杆菌异源表达得到SyEst870可溶性蛋白，经过亲和层析得到酶活为677.5 U的SyEst870，SyEst870在30℃、pH 7.0条件下，24 h内对起始浓度为100 mg/L的甲萘威、速灭威和异丙威降解率分别为82.34%、84.43%、92.87%；甲萘威的降解产物主要为α-萘酚和异氰酸甲酯；速灭威的降解产物主要为间甲酚和异氰酸甲酯；异丙威的主要降解产物为2-异丙基苯酚和异氰酸甲酯。相较于‌氨基甲酸酯类农药在自然环境中数天到数周的半衰期，‌重组蛋白SyEst870可快速消除氨基甲酸酯类农药的残留。本研究为解决环境和果蔬中的农药残留问题奠定了基础。

Abstract:

Carbamate pesticides, a new type of broad-spectrum pesticides for controlling pests, mites, and weeds, are developed to address the shortcomings of organochlorine and organophosphorus pesticides. Their widespread use and slow degradation have led to environmental pollution, causing damage to ecosystems and human health. Managing pesticide residues is a pressing issue in the current environmental protection. This study aims to investigate the expression of SyEst870, a member of the SGNH/GDSL hydrolase family in Sphingobium yanoikuyae, in a prokaryotic system and evaluate the ability of the recombinant protein to degrade carbamate pesticides. The prokaryotic expression vector pET-32a-SyEst870 was constructed and transformed into the Escherichia coli BL21 for heterologous expression. The purified protein was studied in terms of enzyme activity and effects of temperature, pH, and metal ions on the enzyme activity, with p-nitrophenol acetate as the substrate and based on the standard curve of p-nitrophenol. LC-MS (liquid chromatography-mass spectrometry) was employed to examine the degradation effects of SyEst870 on carbaryl, metolcarb, and isoprocarb. GC-MS (gas chromatography-mass spectrometry) was employed to detect the degradation products of SyEst870 for the three pesticides. The soluble protein SyEst870 was successfully obtained through the heterologous expression in Escherichia coli, which yielded an enzyme with the activity of 677.5 U after affinity chromatography. SyEst870 exhibited degradation rates of 82.34%, 84.43%, and 92.87% for carbaryl, metolcarb, and isoprocarb, respectively, at an initial concentration of 100 mg/L within 24 h at 30 ℃ and pH 7.0. The primary degradation products of carbaryl were identified as α-naphthol and methyl isocyanate. Metolcarb was mainly degraded into m-cresol and methyl isocyanate, and isoprocarb was mainly degraded into 2-isopropylphenol and methyl isocyanate. Compared with the half-life of carbamate pesticides in the natural environment, which ranges from a few days to several weeks, the recombinant protein SyEst870 can rapidly eliminate the residues of carbamate pesticides. This study lays a foundation for addressing pesticide residues in the environment and in fruits and vegetables.