乙虫腈是拜耳公司开发的第二代作用于GABA受体的苯基吡唑类杀虫杀螨剂，可有效防治蚜虫、褐飞虱、蓟马和蝽象等害虫，广泛应用于甘蔗、水稻等作物。然而，乙虫腈的过度使用对环境存在较高风险，蜜蜂、鱼类和家蚕会受到影响。因此，有必要寻找能够消减环境中残留乙虫腈的有效策略。微生物降解农药作为自然界中本身存在的农药分解方式，不仅经济高效、靶向性强，而且对环境友好，无二次污染。目前，能够降解乙虫腈的微生物尚未见报道。本研究从土壤中筛选能够高效降解乙虫腈的微生物，研究其种类、生长特性、降解特性、代谢途径和代谢机理，为阐明乙虫腈微生物降解代谢机制提供理论依据，为乙虫腈污染的微生物修复提供技术支持。主要研究结果如下：

1、从江苏土壤中筛选获得了一株能够高效降解乙虫腈的菌株NC1，并通过表型鉴定和16S rRNA鉴定方法，最终确认该菌株归属于恶臭假单胞菌（Pseudomonas putida）。在不同条件下的生长特性研究表明，菌株NC1在温度为37 ℃、pH 7时生长情况最佳，且生长过程需要充足的氧气供应。该菌株具有良好的渗透压耐受性，在含2%以下氯化钠的培养基中仍能正常增殖，表明该菌株具有较强的环境适应能力。

2、通过响应曲面优化分析，对微生物降解的温度、pH和接种量进行优化，发现在温度为25 °C，pH为9，接种量为0.5%时，菌株NC1在24 h对乙虫腈的降解率达到79.7%。同时，研究了菌株NC1对其它苯基吡唑类杀虫剂降解的特异性，发现该菌株能够降解乙虫腈和丁虫腈，但对氟虫腈没有降解能力。

3、通过高分辨质谱分析，鉴定了菌株NC1代谢乙虫腈的中间产物，其中代谢物M1为乙虫腈的腈基被水解成酰胺，代谢物M2为乙虫腈末端亚砜被氧化成砜，初步推测腈水合酶和氧化还原酶参与了乙虫腈的代谢过程。从菌株NC1的基因组文库中筛选出一个新的氧化还原酶基因，该基因大小为1601个碱基对（bp），编码的酶由391个氨基酸组成，G+C含量为63%，将该酶命名为GmcF。GmcF基因的序列比对结果表明，在氨基酸水平上与已知的GMC氧化还原酶家族具有98.06%的最高相似性。GMC家族是一类依赖FAD的氧化还原酶，而GmcF含有FAD的α-β折叠保守结合域，因此确定GmcF属于GMC氧化还原酶家族。

4、构建了gmcF基因的重组表达载体pET28a-gmcF，并在大肠杆菌E.coli BL21(DE3)中实现了该基因的表达。通过Ni-NAT亲和层析柱对重组表达的GmcF酶进行纯化后获得纯酶。酶学特性研究表明，GmcF酶的最适催化温度为30 ℃，在pH 8.0时表现出最高酶活性。热稳定性和pH稳定性试验中，在较宽的温度和pH范围内保持催化活性。GmcF酶以乙虫腈为底物时的酶活为2.66 mmol·mg^-1·min^-1，显示出对乙虫腈的强效降解能力。

5、初步研究了降解菌NC1在污染土壤和生菜中的实际应用效果。降解菌NC1经过秸秆颗粒固定后，7天能降解土壤中12.1%的30 mg/kg的乙虫腈。在生菜中，添加降解菌NC1的处理组的半衰期比对照组缩短了37.7%，表明添加菌株NC1可显著加快乙虫腈在生菜中的降解速率。

Ethiprole is the second generation of phenylpyrazole insecticides and acaricides acting on GABA receptors developed by Bayer. It can effectively control pests such as aphids, brown planthoppers, thrips and bugs, and is widely used in sugarcane, rice and other crops. However, excessive use of ethiprole poses a high risk to the environment, affecting bees, fish and silkworms. Therefore, it is necessary to find effective strategies to eliminate residual ethiprole in the environment. Microbially-mediated pesticide decomposition represents a sustainable bioremediation strategy characterized by cost-effectiveness, operational efficiency, and environmental compatibility without secondary contamination. At present, the microorganisms that can degrade ethiprole have not been reported. In this paper, microorganisms capable of efficiently degrading ethiprole were screened from soil, and the microbial species, growth characteristics, degradation characteristics, metabolic pathways and metabolic mechanisms were studied. It provides a theoretical basis for elucidating the metabolic mechanism of microbial degradation of ethiprole, and provides technical support for microbial remediation of ethiprole pollution. The main results are as follows:

1. A strain NC1 with high-efficiency degradation for ethiprole was screened from Jiangsu soil, and it was finally confirmed to belong to Pseudomonas putida by phenotypic identification and 16S rRNA identification. In the study of the growth of strain NC1 under different conditions, it was found that the strain exhibited optimal growth at 37 °C and pH 7, and the growth process required sufficient oxygen supply. The strain has osmotic pressure tolerance and can maintain normal proliferation in medium containing less than 2% NaCl, showing strong environmental adaptability.

2. Response surface optimization analysis was used to optimize the temperature, pH, and inoculation amount for the degradation process. Results showed that at 25 °C, pH 9, and an inoculation amount of 0.5%, strain NC1 degraded ethiprole with a rate of 79.7% within 24 h. Additionally, the degradation specificity of strain NC1 to other phenylpyrazole insecticides was studied. It was found that strain NC1 could degrade ethiprole and flufiprole, but had no degradation ability to fipronil.

3. The intermediate product of strain NC1 metabolizing ethiprole was identified by high resolution mass spectrometry. The nitrile group of the metabolite M1 was hydrolyzed into amide, and the metabolite M2 was oxidized to sulfone by the terminal sulfoxide of ethiprole. It was preliminarily inferred that nitrile hydratase and oxidoreductase were involved in the metabolic process of ethiprole. A new oxidoreductase gene was screened from the genomic library of NC1. The gene was 1601 bp in size, and the enzyme was composed of 391 amino acids. The G+C content was 63%, and the enzyme was named GmcF. The sequence alignment of GmcF gene showed that it had the highest similarity of 98.06% with the known GMC oxidoreductase family at the amino acid level. The GMC family is a class of FAD-dependent oxidoreductases, and GmcF contains the α-β fold conserved binding domain of FAD, so it is determined that GmcF belongs to the GMC oxidoreductase family.

4. The recombinant expression vector pET28a-gmcF was constructed and expressed in E.coli BL21(DE3). The recombinant GmcF enzyme was purified by Ni-NAT column to obtain pure enzyme. The study of enzymatic properties showed that the optimum catalytic temperature of GmcF enzyme was 30 °C, and it showed the highest enzyme activity at pH 8.0. In the thermal stability and pH stability tests, the catalytic activity was maintained over a wide temperature and pH range. The enzyme activity of GmcF was 2.66 mmol mg^-1 min^-1 when using ethiprole as the substrate, showing a strong ability to degrade ethiprole.

5. The practical application effect of degrading bacteria NC1 in contaminated soil and lettuce was preliminarily explored. The degrading bacteria NC1 could degrade 12.1% of 30 mg/kg ethiprole in the soil after 7 days of straw particle fixation. In lettuce, the half-life of the treatment group added with degrading bacteria NC1 was 37.7% shorter than the control group, indicating that the addition of strain NC1 can effectively accelerate the degradation rate of ethiprole in lettuce.

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