氯氰菊酯（Cypermethrin）是拟除虫菊酯类杀虫剂的代表品种之一，其具有高效、广谱、低哺乳动物毒性的特点，被广泛用于农业、卫生和家庭害虫防治。但随着使用年限的不断延长和使用量的不断增加，使其在土壤中和农副产品表面大量残留。由氯氰菊酯残留造成的环境污染和食品安全问题，引起人们的广泛关注。研究表明微生物在氯氰菊酯降解过程中发挥重要作用。目前国内外对于氯氰菊酯的微生物降解已取得了系列进展，但是这些菌株与降解酶大多对氯氰菊酯降解活性较低。因此，筛选高效降解的微生物菌株和挖掘具有高催化活性和功能的基因和酶还需进一步研究。本论文分离得到两株氯氰菊酯降解菌株，从代谢途径，降解基因及酶学水平上解析了氯氰菊酯的微生物代谢机制；分析了降解菌株修复氯氰菊酯污染过程中土壤细菌群落结构的变化过程；同时，构建功能菌株实现降解基因的分泌表达。具体研究内容和结果如下：

1. 氯氰菊酯降解菌株的分离、降解特性及代谢途径研究

采用富集培养的方法，从长年喷洒氯氰菊酯的茶园土壤里分离得到了两株氯氰菊酯降解菌株。通过菌株的生理生化特征及细菌16S rRNA比对分析构建的系统发育树，两个菌株均被鉴定为Rhodococcus sp.，分别命名为Rhodococcus sp. H-3和Rhodococcus sp. H-4。菌株H-3在7 d内对30 mg/L的氯氰菊酯降解率达83.48%。其降解氯氰菊酯的最适温度和pH分别为35°C和7.0，添加Ca²⁺能促进菌株H-3对氯氰菊酯的降解，而Al³⁺、Cd²⁺、Co²⁺、Cu²⁺、Ni²⁺和Zn²⁺对氯氰菊酯降解有明显的抑制作用。菌株H-4在7 d内对初始浓度为30 mg/L氯氰菊酯的降解率为77.10%。最适反应条件为40°C和pH 7.0。金属离子对氯氰菊酯的降解几乎无促进效果，Al³⁺、Co²⁺、Cu²⁺和Zn²⁺有明显的抑制效果。通过HPLC和HPLC-MS/MS鉴定了菌株H-3和H-4降解氯氰菊酯的代谢产物并推测其代谢途径，发现其均可以将氯氰菊酯水解为3-苯氧基苯甲酸（3-PBA）和二氯菊酸。

2. 菌株H-3和H-4修复氯氰菊酯污染土壤的细菌群落结构分析

分别利用菌株H-3和H-4降解土壤中氯氰菊酯残留，结果发现10 d内，菌株H-3和H-4可将土壤中5 mg/kg的氯氰菊酯分别降解至1.71和2.06 mg/kg。通过16S rRNA高通量测序探究了菌株H-3和H-4修复氯氰菊酯污染过程中土壤细菌群落结构的动态变化过程。Alpha和Beta多样性分析表明氯氰菊酯处理的土壤样品中细菌群落结构多样性和丰富度降低，添加菌株H-3和H-4有助于其多样性和丰富度的恢复。门水平相对丰度结果推测，氯氰菊酯可能对Actinobacteriota门和Bacteroidota门的菌株生长存在抑制作用，而Proteoobacteria门的菌株可能能以氯氰菊酯为碳氮源生长。属水平相对丰度结果表明，氯氰菊酯对Dokdonella_A属、Ferruginibacter属的菌株生长可能存在抑制作用，Brevundimonas属的菌株可能能以氯氰菊酯为碳氮源刺激自身生长，推测Symbiobacterium属能够利用氯氰菊酯的代谢产物生长。

3. 酯酶基因的克隆表达、酶学特性研究及分泌表达

基于菌株H-3和H-4的基因组信息，通过比较基因组分析从菌株H-3和H-4中各鉴定到一个氯氰菊酯酯酶基因分别命名为cymE1和cymE2。经HPLC和HPLC-MS/MS验证纯酶对氯氰菊酯的降解效果，发现酯酶CymE1和CymE2降解氯氰菊酯的产物均为3-PBA和二氯菊酸。序列分析表明，酯酶基因cymE1全长822 bp，编码273个氨基酸，与已报道氨基酸序列最高同源性为33.5%；酯酶基因cymE2全长861 bp，编码286个氨基酸，与已报道氨基酸序列最高同源性为55.1%。两者均属于酯酶Family V，且均不存在跨膜区域和信号肽，属于胞内酶。两个酯酶都具有的Gly-X-Ser-X-Gly保守五肽结构以及催化活性位点Ser-Asp-His。

酶学研究表明，CymE1和CymE2的最适温度分别为30℃和40℃，均在4℃的时候稳定性最好。酶促反应最适pH均为8.0。添加Al³⁺、Co²⁺、Cu²⁺、Ni²⁺、Zn²⁺和Fe³⁺对CymE1和CymE2的活性都有抑制作用，Mn²⁺和Ca²⁺对CymE1和CymE2均有促进作用。CymE1水解氯氰菊酯的V_max、K_m和K_cat值分别为0.18 µM S^-1、32.31 µM和1.78×10^-2 S^-1。CymE2水解氯氰菊酯的V_max、K_m和K_cat值分别为0.22 µM S^-1、15.08 µM和2.24×10^-2 S^-1。CymE2对于氯氰菊酯的亲和力和催化效率优于CymE1。

采用双交换策略成功构建了功能菌株Bacillus sp. SCK6-E，实现了酯酶CymE1在菌株Bacillus sp. SCK6中的分泌表达。对分泌表达条件进行优化，发现其最佳培养基为SR培养基，最佳发酵温度为37℃。在最优条件下，菌株Bacillus sp. SCK6-E可在84 h内完全降解30 mg/L的氯氰菊酯。菌株Bacillus sp. SCK6-E发酵液对茶树叶片中残留的氯氰菊酯有良好的去除效果，在发酵液中浸泡2 h，茶树叶片上氯氰菊酯残留的相对去除率可达57.83%。

Cypermethrin is one of the representative species of synthetic pyrethroid insecticides, which is widely used in agriculture, health and household pest control because of its high efficiency, broad spectrum and low mammalian toxicity. However, with the prolonged service life and increasing use, it has caused a large number of residues in soil and on the surface of agricultural by-products, and the environmental pollution and food safety problems caused by cypermethrin residues have aroused widespread concern. Environmental pollution and food safety problems caused by cypermethrin residues have attracted widespread attention. Studies have shown that microorganisms play an important role in the degradation process of cypermethrin. A series of progress has been made in the microbial degradation of cypermethrin, but most of these strains and enzymes have low degradation activity for cypermethrin. Therefore, the identification of microbial strains with high degradation efficiency and the mining of genes and enzymes with high catalytic activity and function need to be further investigated. In this study, two cypermethrin-degrading bacterial strains were isolated, and the microbial metabolic mechanism of cypermethrin was analyzed in terms of metabolic pathways, degradation genes and enzymology; the changes in soil bacterial community structure during the remediation of cypermethrin contamination by degrading bacterial strains were analyzed; and at the same time, the functional strains were constructed to realize the secretion and expression of degradation genes. The details and results of the study are as follows:

1. Study on the isolation, degradation characteristics and metabolic pathway of cypermethrin-degrading bacterial strains

Two cypermethrin-degrading bacterial strains were isolated from the soil of tea plantations sprayed with cypermethrin for many years by enrichment culture method. Through the physiological and biochemical characteristics of the strains and the phylogenetic tree constructed by bacterial 16S rRNA comparison analysis, both strains were identified as Rhodococcus sp. and named Rhodococcus sp. H-3 and Rhodococcus sp. H-4, respectively. The degradation rate of cypermethrin by strain H-3 reached 83.48% at 30 mg/L within 7 d. The optimal temperature and pH for the degradation of cypermethrin were 35°C and 7.0, respectively. The addition of Ca²⁺ promoted the degradation of cypermethrin by strain H-3, while the degradation was inhibited by Al³⁺, Cd²⁺, Co²⁺, Cu²⁺, Ni²⁺ and Zn²⁺.The degradation of cypermethrin by strain H-4 was 77.10% at an initial concentration of 30 mg/L within 7 d. The optimum reaction conditions were 40°C and pH 7.0. The degradation of cypermethrin was hardly promoted by metal ions, while Al³⁺, Co²⁺, Cu²⁺ and Zn²⁺ had obvious inhibitory effects. The degradation products of strains H-3 and H-4 were identified by HPLC and HPLC-MS/MS, and the metabolic pathways of H-3 and H-4 were investigated. It was found that both strains could hydrolyze cypermethrin to 3-phenoxybenzoic acid (3-PBA) and 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylic acid (DCVA).

2. Analysis of the bacterial community structure in the remediation of cypermethrin-contaminated soil by strains H-3 and H-4

The strains H-3 and H-4 were utilized to degrade cypermethrin remains in soil, respectively, and it was found that the strains H-3 and H-4 could degrade the cypermethrin at a concentration of 5 mg/kg in the soil to 1.71 and 2.06 mg/kg respectively, in 10 d. The dynamics of soil bacterial community structure during the remediation of cypermethrin contamination by 16S rRNA high-throughput sequencing was investigated by strains H-3 and H-4. Alpha and Betadiversity analyses showed that the diversity and richness of bacterial community structure were reduced in cypermethrin-treated soil samples and that the addition of strains H-3 and H-4 helped to restore the diversity and richness of bacterial community structure. The results of relative abundance at the phylum level suggested that cypermethrin might have an inhibitory effect on the growth of the strains of the phylum Actinobacteriota and Bacteroidota, while the strains of the phylum Proteobacteria might be able to use cypermethrin as a carbon and nitrogen source for growth. The results of relative abundance at the genus level indicated that cypermethrin might have an inhibitory effect on the growth of strains of the genera Dokdonella_A and Ferruginibacter, and strains of the genus Brevundimonas might be able to stimulate their growth by using cypermethrin as a source of carbon and nitrogen, and it was hypothesized that the genus Symbiobacterium was able to utilize the metabolites of cypermethrin for growth.

3. Cloning, expression, characterization and secretion of esterase gene

Based on the genomic information of strains H-3 and H-4, a cypermethrin esterase gene was identified from each of strains H-3 and H-4 by comparative genomic analysis named cymE1 and cymE2, respectively. The degradation of cypermethrin by pure enzymes was verified by HPLC and HPLC-MS/MS, and it was found that the products of cypermethrin degradation by the esterases CymE1 and CymE2 were 3-PBA and DCVA. Sequence analysis showed that, the esterase gene cymE1 has a full length of 822 bp, encoding 273 amino acids, and has the highest identity of 33.5% with the reported amino acid sequence; the esterase gene cymE2 has a full length of 861 bp, encoding 286 amino acids, and has the highest identity of 55.1% with the reported amino acid sequence. Both esterases belong to esterase Family V and are intracellular enzymes without transmembrane region and signal peptide. Both esterases have the conventional pentapeptide Gly-X-Ser-X-Gly and the catalytic triad Ser-Asp-His.

Enzymatic studies showed that the optimal temperatures of CymE1 and CymE2 were 30℃ and 40℃, respectively, the enzyme stability is best at 4°C. The optimal pH of the enzymatic reaction is both 8.0. Al³⁺, Co²⁺, Cu²⁺, Ni²⁺, Zn²⁺and Fe³⁺ inhibits the activities of CymE1 and CymE2, while Mn²⁺ and Ca²⁺ enhances the activities of CymE1 and CymE2. The V_max, K_m and K_cat values of CymE1 hydrolyzed cypermethrin were 0.18 µM S^-1, 32.31 µM and 1.78 × 10^-2 S^-1, respectively. The V_max, K_m and K_cat values of CymE2 hydrolyzed cypermethrin were 0.22 µM S^-1, 15.08 µM and 2.24 × 10^-2 S^-1, respectively. CymE2 showed better affinity and catalytic efficiency for cypermethrin than CymE1.

A double crossover strategy was adopted to successfully construct a functional strain Bacillus sp. SCK6-E, achieve the secretion expression of esterase CymE1 in strain Bacillus sp. SCK6. The secretion expression conditions were optimized, and it was found that the optimal medium was SR medium, and the optimal fermentation temperature was 37°C. Under the optimal conditions, strain Bacillus sp. SCK6-E could completely degrade 30 mg/L of cypermethrin within 84 h. The fermentation temperature was 37℃. The fermentation broth of strain Bacillus sp. SCK6-E had a good effect on the removal of cypermethrin residue in tea leaves, and the relative removal rate of cypermethrin residue on tea leaves could reach 57.83% after 2 h of immersion in the fermentation broth.

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