多芳环化合物降解菌的筛选、特性及降解途径研究
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摘要
多芳环类化合物是化学合成工业中的一大类重要原料,种类繁多,广泛应用于制药、化工和印染等行业,易造成水体、土壤等环境污染。丙酯草醚,[4-(2-(4,6-二甲氧基-2-嘧啶氧基)苄胺基)苯甲酸正丙酯],是我国具自主知识产权的新型高效除草剂,丙酯草醚原药、油力(10%乳油制剂)和油力Ⅱ(10%悬浮剂)已获农药临时登记证和农药生产批准证书。然而,关于丙酯草醚的微生物降解特性和生物降解途径及其降解蛋白质组学的相关研究迄今未见报道,关于同时降解萘和丙酯草醚的基因工程菌也未见报道。本研究综合运用传统微生物学实验方法、核素示踪技术、现代生物技术和现代仪器分析技术,分别从化工园区污水处理厂活性污泥和常州市南郊施用过丙酯草醚的农田土壤中筛选、纯化获得丙酯草醚高效降解菌株Amycolatopsis sp.M3-1和萘高效降解菌株Pseudomonadaceae sp. Nai8,系统研究丙酯草醚和萘的微生物降解特性,以[B环-4,6-14C]标记丙酯草醚和[C环-U-14C]标记丙酯草醚为示踪剂,研究丙酯草醚在水体和土壤中的生物降解途径,利用双向电泳技术初步鉴定了菌株M3-1降解丙酯草醚的降解功能酶,并利用原生质体融合技术,构建可同时降解丙酯草醚和萘的高效工程菌MN6。微生物降解丙酯草醚的特性及降解途径、代谢机制等进行系统的研究,有助于深入了解丙酯草醚在环境的行为,为合理、安全使用丙酯草醚和环境生物修复提供技术支持和理论指导,保证环境和农产品的安全性。
本研究的主要研究结果如下：
1.从自然界中富集、筛选出5株以萘为唯一碳源和15株以丙酯草醚作为唯一碳源生长的降解菌。对降解效果较好的菌株Nai8、M3-1和CY进行进一步研究,经生理生化和16S rDNA鉴定,菌株Nai8为Pseudomonadaceae sp.Nai8, M3-1为较罕见的地中海拟无枝酸菌Amycolatopsis sp.M3-1, CY为Bacillus sp. CY。菌株M3-1降解丙酯草醚的最适条件为35℃、pH6.0、接种量8%,底物浓度为100mg/L,培养25天,丙酯草醚降解率达63.30%。当丙酯草醚浓度高于300mg/L时,对菌株M3-1的降解效果有较强的抑制作用,并随着底物丙酯草醚浓度的增加而增强,而浓度较低时(<150ppm)降解效果较好,较高浓度的丙酯草醚可以抑制菌株M3-1的生长繁殖速度,大大降低细胞内降解酶的生物合成速度,甚至抑制酶的合成。菌株Nai8降解萘的最适条件为30℃,pH7.0,接种量8%,培养100h,降解率达96.24%。
2.以B-14C-ZJ0273和C-14C-ZJ0273为示踪剂,研究水体中丙酯草醚在Amycolatopsis sp.M3-1的催化下的代谢规律,在培养体系中有6种中间代谢产物被检测出有放射性活度,用LC-MS鉴定其结构,并推导出ZJ0273在水溶液中的代谢途径。首先其丙基酯键被水解形成M1—(4-(2-(4,6-二甲基-2-嘧啶氧基)苄胺基)苯甲酸；随后,M1上的侧链CH-NH键发生酰化反应生成M2-(4-(2-(4,6-二甲基-2-嘧啶氧基)苯甲酰氨基)苯甲酸；同时,M1通过水解CH-NH键生成M3—--4,6-二甲基-2-嘧啶氧基苯甲酸,M2的CO-NH键也在Amycolatopsis sp. M3-1的催化下,发生水解反应形成M3,M3也是丙酯草醚在杂草中最主要的代谢产物；M3结构的醚键发生断裂反应生成M4—2-羟-4,6-二甲基-嘧啶和苯甲酸；M3嘧啶环上的甲氧基通过水解形成M5,即6-甲基-4-羟-2-嘧啶氧基苯甲酸,然后再通过进一步的水解生成M6—尿嘧啶(2,4-二羟基嘧啶)和苯甲酸。在自然环境中(如土壤和淤泥中)M4、M6和苯甲酸均可以被其他微生物作为碳源和能源,促进微生物的生长与繁殖,并最终被降解成C02和H20。
3.菌株M3-1对5种灭菌土壤红砂田(S1)、黄松田(S2)、黄泥田(S3)、淡涂泥田(S4)和黄石土(s5)中的丙酯草醚均具有去除作用。培养90天后,S1、S4和S3中丙酯草醚的残留量分别为48.60%、23.32%和5.31%。空白土样中丙酯草醚残留量均在缓慢的下降过程中,表明丙酯草醚在土壤中的光解、水解及矿化等自然消解作用对丙酯草醚有一定的去除作用,但是去除效果不明显。M3-1在5种未灭菌土壤中对丙酯草醚也具有很好的去除作用,培养90d,丙酯草醚在五种土壤中的残留量分别为33.50%,31.14%,31.52%,22.03%和4.22%。
4.运用蛋白质双向电泳技术,丙酯草醚诱导菌株M3-1前后的蛋白变化进行了初步研究,结果表明,菌株M3-1经过丙酯草醚诱导之后,胞内蛋白图谱发生较大的变化,双向电泳检测出6个新蛋白点,5个表达量增加2倍以上的蛋白点(超过1倍上的蛋白质点多达16个以上),通过MALDI-TOF-MS鉴定出8个蛋白质,分别是Phosphoglycerate kinase、 Citrate sysnthase、Lipase、Biotin carboxylase、aldehydrogenase、Small subunit aromatic oxygenase等与细胞生长与代谢相关的酶系。
5.采用紫外诱变法,获得M3-1和Nai8的脯氨酸(Pro-)缺陷型菌株和天冬酰胺(Asn-)缺陷型菌株。通过原生质体融合,构建8株可以同时降解丙酯草醚和萘的工程菌,其中工程菌株MN2和MN6对萘和丙酯草醚的降解率较高,比原始菌株M3-1对丙酯草醚降解率(20d)分别提高了2.83%、6.87%,比菌株Nai5的降解率(48h)分别提高了10.82%和18.01%。
Polyaromatic compound has been produced and widely used in pharmaceutical, chemical and dyeing industries. Industrial wastewater containing polyaromatic compound such as naphthalene could lead to gross pollution of the environment. ZJ0273, propyl4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate, is a novel herbicide developed for oilseed crop in China.10%ZJ0273emulsifiable concentration and10%suspension concentration (the trade name is Youli) have been obtained registration of Ministry of Agriculture, China, and permitted to be used extensively for eliminating oil rape weed. However, Microbial degradation of ZJ0273, degradation pathway in aquatic system and microbial degradation proteome has not been reported until now. I n order to understand microbial behavior, biodegradation pathway and mechanism of ZJ0273in environment, biodegradation and bioremediation of ZJ0273contaminated water and soil need to be further studied. In this study, ZJ0273-degrading bacterium Amycolatopsis sp. M3-1and Naphthalene-degrading bacterium Pseudomonadaceae sp. Nai8were isolated from soil. Using traditional microbiological method, isotope tracer, modern biotechnology and modern instrument analysis, biodegrading characteristics, pathway and mechanism of ZJ0273in soils and water were studied. The degradation enzymes were identified by2-DE and MALDI-TOF-MS, gene engineering bacterium was constructed and it can degrade ZJ0273and naphthalene simultaneously. The results of this study will be will provide technical support and thoretical guidance for reasonable and safe use of ZJ0273, and it is very important for improving the quality of agricultural products, ecological environment and human health.
The main results are as follows
1. Five strains of naphthalene degrading bacteria and fifteen strains of ZJ0273degrading bacteria were screened, isolated and separated from soils by applying modern molecular biological technique with traditional microbiological method. The strain M3-1, Nai8and CY were identified as Amycolatopsis sp., Pseudomonadaceae sp. and Bacillus sp. by physiological, biochemical tests and16s rDNA sequence. The optimum ZJ0273degrading conditions of Amycolatopsis sp. M3-1were studied. The results showed that with condition of35°C, pH6.0,8%of inoculation and100mg/L of ZJ0273, the degrading ratio in25d was63.30%. The degradation ratio of ZJ0273by Amycolatopsis sp. M3-1was decreased with the increasing ZJ0273concentration,300mg/L of ZJ0273can strongly inhibit bio-activity of strain M3-1, which means that higher concentration of ZJ0273has higher toxicity to M3-1. And the optimum naphthalene degrading conditions of Pseudomonadaceae sp. Nai8was30℃, pH7.0,8%of inoculation and1000mg/L of naphthalene, the degrading ratio in100h was96.24%.
2.Six metabolites (M1-M6) during the degradation of ZJ0273by Amycolatopsis sp. M3-1were identified by combination with multi-poa'tion14C-labeled compounds (B-ZJ0273and C-ZJ0273), chromatography, liquid scintillation spectrometer and LC-MS, a novel pathway of ZJ0273degradation by Amycolatopsis sp. M3-1was proposed based on the identified metabolites and their biodegradation courses.ZJ0273was initially hydrolyzed into M1(4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzaoicacid), then further oxidized into M3(2-(4,6-dimethoxypyimidin-2-yloxy) benzoic acid); M1also could undergo a carbonylation into M2(4-(2-(4,6-dimethoxypyimidin-2-yloxy) benzamido) benzoicacid), and then its C-N and C-0bonds were deaved to yield M3(2-(4,6-dimethoxypyimidin-2-yloxy) benzoicadd) and M4(4,6-dimethoxypyrimidin-2-ol), respectively. Moreover, another two new metabolites, M5(2-(4-hydroxy,6-methoxypyimidin-2-yloxy) benzoicadd) and M6(2,4-dihydroxy-pyrimidine) were found. M5was formed through de-methyl of M3, and then hydrolyzed into M6.In the natural environment, M4and M6could be used as carbon and energy source and bread down to CO2and H2O.
3. ZJ0273could be degraded in the five sterile soil samples after inoculation of Amycolatopsis sp. M3-1. The residue of ZJ0273in S1, S4and S3was48.60%.23.32%and5.31%, respectively. Degradation rate of ZJ0273in S3and S4were higher than that of in add soil like S1, S2and S5. In the blank soil samples, the concentration of ZJ0273also decreased slowly, which showed that photodegradation, hydrolysis and mineralization in soil have certain effect to degrade ZJ0273. The residue of ZJ0273in unsterile S1, S2, S3, S4and S5after inoculation of Amycolatopsis sp.M3-1was33.50%,31.11%,31.52%,22.03%and4.22%.
4. To illuminate the mechanism of Z J0273-degrading pathway in bacteria, two-demensional protein SDS-PAGE electrophresis was used to identify variations in protein expression in ZJ0273degrading bacterium. The results showed that strain M3-1exposed to ZJ0273could lead to the6new proteins and the up-regulation of5proteins, and8proteins were indentified by MALDI-TOF-MS.
5. Two auxotrophic mutant strains (Pro-and Asn-) were gain through ultraviolet mutagenesis method and chose to mark protoplast fusion.8fusants were obtained with genetic stability on the degradation of ZJ0273and naphthalene by protoplast fusion, the fusant named M N6could degrade ZJ0273and naphthalene simultaneously and has higher degradation rate of degrading ZJ0273and naphthalene. The degradation ratio could improve6.87%(20d) and18.01%(48h).
本研究的主要研究结果如下：
1.从自然界中富集、筛选出5株以萘为唯一碳源和15株以丙酯草醚作为唯一碳源生长的降解菌。对降解效果较好的菌株Nai8、M3-1和CY进行进一步研究,经生理生化和16S rDNA鉴定,菌株Nai8为Pseudomonadaceae sp.Nai8, M3-1为较罕见的地中海拟无枝酸菌Amycolatopsis sp.M3-1, CY为Bacillus sp. CY。菌株M3-1降解丙酯草醚的最适条件为35℃、pH6.0、接种量8%,底物浓度为100mg/L,培养25天,丙酯草醚降解率达63.30%。当丙酯草醚浓度高于300mg/L时,对菌株M3-1的降解效果有较强的抑制作用,并随着底物丙酯草醚浓度的增加而增强,而浓度较低时(<150ppm)降解效果较好,较高浓度的丙酯草醚可以抑制菌株M3-1的生长繁殖速度,大大降低细胞内降解酶的生物合成速度,甚至抑制酶的合成。菌株Nai8降解萘的最适条件为30℃,pH7.0,接种量8%,培养100h,降解率达96.24%。
2.以B-14C-ZJ0273和C-14C-ZJ0273为示踪剂,研究水体中丙酯草醚在Amycolatopsis sp.M3-1的催化下的代谢规律,在培养体系中有6种中间代谢产物被检测出有放射性活度,用LC-MS鉴定其结构,并推导出ZJ0273在水溶液中的代谢途径。首先其丙基酯键被水解形成M1—(4-(2-(4,6-二甲基-2-嘧啶氧基)苄胺基)苯甲酸；随后,M1上的侧链CH-NH键发生酰化反应生成M2-(4-(2-(4,6-二甲基-2-嘧啶氧基)苯甲酰氨基)苯甲酸；同时,M1通过水解CH-NH键生成M3—--4,6-二甲基-2-嘧啶氧基苯甲酸,M2的CO-NH键也在Amycolatopsis sp. M3-1的催化下,发生水解反应形成M3,M3也是丙酯草醚在杂草中最主要的代谢产物；M3结构的醚键发生断裂反应生成M4—2-羟-4,6-二甲基-嘧啶和苯甲酸；M3嘧啶环上的甲氧基通过水解形成M5,即6-甲基-4-羟-2-嘧啶氧基苯甲酸,然后再通过进一步的水解生成M6—尿嘧啶(2,4-二羟基嘧啶)和苯甲酸。在自然环境中(如土壤和淤泥中)M4、M6和苯甲酸均可以被其他微生物作为碳源和能源,促进微生物的生长与繁殖,并最终被降解成C02和H20。
3.菌株M3-1对5种灭菌土壤红砂田(S1)、黄松田(S2)、黄泥田(S3)、淡涂泥田(S4)和黄石土(s5)中的丙酯草醚均具有去除作用。培养90天后,S1、S4和S3中丙酯草醚的残留量分别为48.60%、23.32%和5.31%。空白土样中丙酯草醚残留量均在缓慢的下降过程中,表明丙酯草醚在土壤中的光解、水解及矿化等自然消解作用对丙酯草醚有一定的去除作用,但是去除效果不明显。M3-1在5种未灭菌土壤中对丙酯草醚也具有很好的去除作用,培养90d,丙酯草醚在五种土壤中的残留量分别为33.50%,31.14%,31.52%,22.03%和4.22%。
4.运用蛋白质双向电泳技术,丙酯草醚诱导菌株M3-1前后的蛋白变化进行了初步研究,结果表明,菌株M3-1经过丙酯草醚诱导之后,胞内蛋白图谱发生较大的变化,双向电泳检测出6个新蛋白点,5个表达量增加2倍以上的蛋白点(超过1倍上的蛋白质点多达16个以上),通过MALDI-TOF-MS鉴定出8个蛋白质,分别是Phosphoglycerate kinase、 Citrate sysnthase、Lipase、Biotin carboxylase、aldehydrogenase、Small subunit aromatic oxygenase等与细胞生长与代谢相关的酶系。
5.采用紫外诱变法,获得M3-1和Nai8的脯氨酸(Pro-)缺陷型菌株和天冬酰胺(Asn-)缺陷型菌株。通过原生质体融合,构建8株可以同时降解丙酯草醚和萘的工程菌,其中工程菌株MN2和MN6对萘和丙酯草醚的降解率较高,比原始菌株M3-1对丙酯草醚降解率(20d)分别提高了2.83%、6.87%,比菌株Nai5的降解率(48h)分别提高了10.82%和18.01%。
Polyaromatic compound has been produced and widely used in pharmaceutical, chemical and dyeing industries. Industrial wastewater containing polyaromatic compound such as naphthalene could lead to gross pollution of the environment. ZJ0273, propyl4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate, is a novel herbicide developed for oilseed crop in China.10%ZJ0273emulsifiable concentration and10%suspension concentration (the trade name is Youli) have been obtained registration of Ministry of Agriculture, China, and permitted to be used extensively for eliminating oil rape weed. However, Microbial degradation of ZJ0273, degradation pathway in aquatic system and microbial degradation proteome has not been reported until now. I n order to understand microbial behavior, biodegradation pathway and mechanism of ZJ0273in environment, biodegradation and bioremediation of ZJ0273contaminated water and soil need to be further studied. In this study, ZJ0273-degrading bacterium Amycolatopsis sp. M3-1and Naphthalene-degrading bacterium Pseudomonadaceae sp. Nai8were isolated from soil. Using traditional microbiological method, isotope tracer, modern biotechnology and modern instrument analysis, biodegrading characteristics, pathway and mechanism of ZJ0273in soils and water were studied. The degradation enzymes were identified by2-DE and MALDI-TOF-MS, gene engineering bacterium was constructed and it can degrade ZJ0273and naphthalene simultaneously. The results of this study will be will provide technical support and thoretical guidance for reasonable and safe use of ZJ0273, and it is very important for improving the quality of agricultural products, ecological environment and human health.
The main results are as follows
1. Five strains of naphthalene degrading bacteria and fifteen strains of ZJ0273degrading bacteria were screened, isolated and separated from soils by applying modern molecular biological technique with traditional microbiological method. The strain M3-1, Nai8and CY were identified as Amycolatopsis sp., Pseudomonadaceae sp. and Bacillus sp. by physiological, biochemical tests and16s rDNA sequence. The optimum ZJ0273degrading conditions of Amycolatopsis sp. M3-1were studied. The results showed that with condition of35°C, pH6.0,8%of inoculation and100mg/L of ZJ0273, the degrading ratio in25d was63.30%. The degradation ratio of ZJ0273by Amycolatopsis sp. M3-1was decreased with the increasing ZJ0273concentration,300mg/L of ZJ0273can strongly inhibit bio-activity of strain M3-1, which means that higher concentration of ZJ0273has higher toxicity to M3-1. And the optimum naphthalene degrading conditions of Pseudomonadaceae sp. Nai8was30℃, pH7.0,8%of inoculation and1000mg/L of naphthalene, the degrading ratio in100h was96.24%.
2.Six metabolites (M1-M6) during the degradation of ZJ0273by Amycolatopsis sp. M3-1were identified by combination with multi-poa'tion14C-labeled compounds (B-ZJ0273and C-ZJ0273), chromatography, liquid scintillation spectrometer and LC-MS, a novel pathway of ZJ0273degradation by Amycolatopsis sp. M3-1was proposed based on the identified metabolites and their biodegradation courses.ZJ0273was initially hydrolyzed into M1(4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzaoicacid), then further oxidized into M3(2-(4,6-dimethoxypyimidin-2-yloxy) benzoic acid); M1also could undergo a carbonylation into M2(4-(2-(4,6-dimethoxypyimidin-2-yloxy) benzamido) benzoicacid), and then its C-N and C-0bonds were deaved to yield M3(2-(4,6-dimethoxypyimidin-2-yloxy) benzoicadd) and M4(4,6-dimethoxypyrimidin-2-ol), respectively. Moreover, another two new metabolites, M5(2-(4-hydroxy,6-methoxypyimidin-2-yloxy) benzoicadd) and M6(2,4-dihydroxy-pyrimidine) were found. M5was formed through de-methyl of M3, and then hydrolyzed into M6.In the natural environment, M4and M6could be used as carbon and energy source and bread down to CO2and H2O.
3. ZJ0273could be degraded in the five sterile soil samples after inoculation of Amycolatopsis sp. M3-1. The residue of ZJ0273in S1, S4and S3was48.60%.23.32%and5.31%, respectively. Degradation rate of ZJ0273in S3and S4were higher than that of in add soil like S1, S2and S5. In the blank soil samples, the concentration of ZJ0273also decreased slowly, which showed that photodegradation, hydrolysis and mineralization in soil have certain effect to degrade ZJ0273. The residue of ZJ0273in unsterile S1, S2, S3, S4and S5after inoculation of Amycolatopsis sp.M3-1was33.50%,31.11%,31.52%,22.03%and4.22%.
4. To illuminate the mechanism of Z J0273-degrading pathway in bacteria, two-demensional protein SDS-PAGE electrophresis was used to identify variations in protein expression in ZJ0273degrading bacterium. The results showed that strain M3-1exposed to ZJ0273could lead to the6new proteins and the up-regulation of5proteins, and8proteins were indentified by MALDI-TOF-MS.
5. Two auxotrophic mutant strains (Pro-and Asn-) were gain through ultraviolet mutagenesis method and chose to mark protoplast fusion.8fusants were obtained with genetic stability on the degradation of ZJ0273and naphthalene by protoplast fusion, the fusant named M N6could degrade ZJ0273and naphthalene simultaneously and has higher degradation rate of degrading ZJ0273and naphthalene. The degradation ratio could improve6.87%(20d) and18.01%(48h).
引文
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