3-苯氧基苯甲酸降解菌的分离及降解特性研究

从农药厂污泥中分离到一株3-苯氧基苯甲酸（3-Phenoxybenzoic acid,3-PBA）降解菌PBM11。经生理生化试验和16S rDNA测定初步鉴定为假单胞菌属（Pseudomonas sp．）。PBM11能以3-PBA为唯一碳源生长,降解试验表明：在基础盐培养基中,初始pH7．0,3＆C,160r／rain,装瓶量为100mL／250mL的条件下,PBM11经过24h可完全降解200mg／L的3-PBA;添加低浓度的外源营养物质能够在一定程度上促进降解;Cu^2＋和Co^2＋等对其降解性能有抑制作用,而Fe^3＋有明显的促进作用;通过分析底物利用情况,初步推断3-PBA降解的中间产物能为原儿茶酸和苯酚。经过7d的处理,PBM11对土壤中100mg／L3-PBA的降解率为66．25％。从PBM11中检测到一个质粒。     

3-苯氧基苯甲酸降解菌的分离及降解特性研究*

从农药厂污泥中分离到一株3-苯氧基苯甲酸(3-Phenoxybenzoicacid,3-PBA)降解菌PBM11。经生理生化试验和16SrDNA测定初步鉴定为假单胞菌属(Pseudomonassp·)。PBM11能以3-PBA为唯一碳源生长,降解试验表明:在基础盐培养基中,初始pH7·0,30℃,160r/min,装瓶量为100mL/250mL的条件下,PBM11经过24h可完全降解200mg/L的3-PBA;添加低浓度的外源营养物质能够在一定程度上促进降解;Cu²⁺和     

微生物降解3-苯氧基苯甲酸的研究进展

摘要:3-苯氧基苯甲酸(3-phenoxybenzoic acid,3-PBA)作为大多数拟除虫菊酯类农药的降解产物之一,在自然环境中难以降解,具有雌激素毒性,严重威胁到食品安全及人体健康。微生物对拟除虫菊酯及其中间产物(3-PBA)的降解已成为近年来的研究热点。本文从降解3-PBA的微生物种类、降解酶及降解基因、降解途径等方面进行了综述,对3-PBA生物降解机理、3-PBA降解酶基因工程菌构建的研究方向进行了展望,以期为微生物降解3-PBA的研究提供参考。     

一株路德维希肠杆菌的筛选及其对3-苯氧基苯甲酸的降解特性分析

【目的】3-苯氧基苯甲酸(3-phenoxybenzoic acid,3-PBA)的消除是解决拟除虫菊酯类农药污染的关键,目的是从受拟除虫菊酯类农药污染植物根系土壤中分离出3-PBA高效降解菌株。【方法】采用富集驯化、筛选纯化方法,以3-PBA为唯一碳源、能源筛选3-PBA降解菌株;菌株鉴定采用形态、生理生化和16S r RNA序列分析法;并研究其生长降解动力学特性,最后采用Box-Behnken响应面分析确定最佳降解条件。【结果】从川北地区大豆根系土壤中筛选得到1株高效降解菌BPBA031,经鉴定为路德维希肠杆菌(Enterobacter ludwigii);该菌株耐3-PBA浓度达1600 mg/L,其生长降解过程分别符合Logistic生长动力学(μm=0.09149 h~(–1),X_m=1.1145)和一级降解动力学模型(k=0.02085,t_(1/2)=33.24 h);对3-PBA降解的最适条件为34–37°C、3-PBA浓度25–200 mg/L和p H 7.5–8.5;在35.19°C、30.0 mg/L 3-PBA和p H 7.58条件下,该菌株48 h对3-PBA的降解率达83.75%。【结论】路德维希肠杆菌BPBA031是1株高效3-PBA降解菌,可作为生物修复受3-PBA或拟除虫菊酯类农药污染环境的潜在菌株资源。     

三氯卡班降解菌株Sphingomonas sp. YL-JM2C中多个邻苯二酚双加氧酶的功能

【目的】研究Sphingomonas sp.YL-JM2C菌株的生长特性,确定以三氯卡班作为碳源的生长情况。挖掘菌株YL-JM2C潜在的邻苯二酚1,2-双加氧酶及邻苯二酚2,3-双加氧酶基因,在大肠杆菌(Escherichia coli)中异源表达邻苯二酚双加氧酶基因并研究其酶学性质。【方法】优化S.sp.YL-JM2C菌株以三氯卡班作为碳源时的培养条件,并利用全自动生长曲线测定仪测定菌株生长情况,绘制生长曲线。通过生物信息学方法挖掘潜在的邻苯二酚双加氧酶基因,并分别在Escherichia coli BL21(DE3)中进行异源表达,通过AKTA快速纯化系统纯化蛋白,分别以邻苯二酚、3-和4-氯邻苯二酚为底物检测重组蛋白的酶学特性。【结果】菌株在pH为7.0-7.5时生长最优。在以浓度为4-8 mg/L的三氯卡班做为底物时,菌株适宜生长。当R2A培养基仅含有0.01%酵母提取物和无机盐时,加入终浓度为4 mg/L的三氯卡班可促进菌株生长。挖掘到6个潜在的邻苯二酚双加氧酶基因stcA1、stcA2、stcA3、stcE1、stcE2和stcE3,表达并通过粗酶液分析证明其中5个基因stcA1、stcA2、stcA3、stcE1和stcE2编码的酶均具有邻苯二酚双加氧酶和氯邻苯二酚双加氧酶的活性;纯化酶的底物范围研究揭示了StcA1、StcA2和StcA3均属于Ⅱ型邻苯二酚1,2-双加氧酶,StcE1和StcE2为两个新型邻苯二酚2,3-双加氧酶;它们酶动力学分析研究证明了5个酶对邻苯二酚的亲和力和催化效率最高,4-氯邻苯二酚次之。【结论】在同一菌株中发现了5个具有功能的邻苯二酚双加氧酶基因,stcA1、stcA2和stcA3编码的酶均属于Ⅱ型邻苯二酚1,2-双加氧酶,stcE1和stcE2为两个新型邻苯二酚2,3-双加氧酶编码基因。5个酶均具有催化邻苯二酚和氯邻苯二酚开环反应的功能,这为更好地理解微生物基因组内代谢邻苯二酚及其衍生物氯代邻苯二酚基因的多样性奠定了基础。     

六六六(HCH)降解菌Sphingomonas sp. BHC-A的分离与降解特性的研究

从长期受六六六污染的土壤中分离得到一株能以HCH为唯一碳源的高效降解菌株BHC-A。通过对其主要生理生化特征分析,以及16S rDNA序列的测定和同源性比较分析,将BHC-A鉴定为鞘氨醇单胞菌属(Sphingomonassp.)。BHC-A菌株在12h以内能够完全矿化浓度分别为5mg/L的α-、β-、γ-、δ-HCH4种异构体,特别是对β-HCH的降解在国际上也属少例。而前人所报道的γ-HCH降解菌Sphingomonas paucimobilisUT26菌株对β-HCH和δ-HCH不产生降解作用,即使经过24h的培养,对5mg/L的α-HCH的降解率也只有12.6%。在黄瓜的盆钵试验中发现,15d后BHC-A在土壤中对α、β-、γ-、δ-HCH4种异构体的降解率为84.3%,能够有效地消除土壤中六六六的污染,缓解植株受药害症状。     

不动杆菌3-苯氧基苯甲酸降解基因的克隆与表达

3-苯氧基苯甲酸（3-PBA）作为拟除虫菊酯类农药的非特异性降解中间产物,具有抗雌激素特性,可扰乱生物体内分泌系统,比菊酯类农药迁移更广,半衰期更长,生物毒性更大,是拟除虫菊酯类农药在生物体中暴露的标志。通过构建4-D菌(Acinetobacter sp.)基因组文库,混合池驯化筛选得到4-D菌中降解3-PBA的关键酶基因,其开放阅读框为921 bp,编码306个氨基酸,Genbank登录号为KR024742。经同源比对和酶活验证,证实该酶为邻苯二酚双加氧酶。根据该ORF序列设计引物,引物两端分别加上NdeⅠ和Hind Ⅲ酶切位点,以4-D菌基因组DNA为模板,成功克隆到D34基因序列。构建表达载体pET-21b-D34并转化进宿主大肠杆菌BL21（DE3）,经IPTG（0.1 mmol/L）诱导后,表达重组菌对3-PBA降解率为18.7%。研究结果为3-PBA污染的微生物环境修复提供了理论参考。     

炔草酯降解菌Sphingopyxis sp. WP68的分离鉴定与降解特性

【背景】炔草酯可以高效防除麦田恶性杂草,但炔草酯的生产和使用也对环境造成了破坏,对动物和人类健康造成了威胁。【目的】分离筛选炔草酯高效降解菌株,研究其降解特性,为炔草酯污染生物修复提供优良菌种资源。【方法】采集农药厂活性污泥样品,通过富集培养和含有炔草酯的LB培养基进行炔草酯降解菌株的分离,通过形态和生理生化特性以及16S rRNA基因序列分析确定其分类学地位,通过单因素试验从温度、pH、接种量和底物浓度等方面考察菌株对炔草酯的降解特性,并利用UPLC-MS分析降解产物。【结果】筛选出一株炔草酯高效降解菌株WP68,经鉴定为鞘氨醇盒菌(Sphingopyxis sp.),该菌株在37°C和pH值为8.0时,10 h内可将200 mg/L的炔草酯降解98.26%。利用UPLC-MS鉴定菌株WP68降解炔草酯的产物为炔草酸。确定了该菌株降解炔草酯的最适温度、pH值、接种量、底物浓度分别是37°C、8.0、5%、200mg/L。菌株WP68还能降解氰氟草酯和精喹禾灵。【结论】Sphingopyxis sp. WP68对炔草酯有较强的降解能力和较高耐受性,在炔草酯污染土壤修复中具有潜在的应用前景。     

一株3-苯氧基苯甲酸降解菌的筛选及其协同Bacillus licheniformis G-04降解高效氯氰菊酯的研究

【目的】从长期受拟除虫菊酯类农药污染的白菜根系土壤分离1株3-苯氧基苯甲酸(3-phenoxybenzoic acid, 3-PBA)降解菌,并探究其与Bacillus licheniformis G-04协同作用对高效氯氰菊酯(beta-cypermethrin,Beta-CP)的降解及污染土壤的生物修复,为土壤农药残留危害处理提供优良菌种。【方法】采用富集驯化、筛选纯化方法,筛选3-PBA降解菌,并通过形态和生理生化特征以及16S rRNA序列分析进行鉴定。利用Origin 8.0分析3-PBA降解菌与B. licheniformis G-04的生长降解动力学过程。同时,采用高效液相色谱法评估两菌株协同降解Beta-CP的能力及其对受Beta-CP污染土壤的修复作用。【结果】筛选得到1株3-PBA高效降解菌HA516,48 h对3-PBA (100 mg/L)的降解率达到87.73%,经鉴定为皮特不动杆菌(Acinetobacter pittii);构建了该菌株和B. licheniformis G-04的生长降解动力学方程,结果表明模型与实验数据能较好拟合;以6.7∶3.3的接种比例先接种B. licheniformis G-04,24 h后再接入A. pittii HA516协同作用,在48 h,Beta-CP (50 mg/L)的降解率达78.37%,较单菌株(B. licheniformisG-04)的降解率(40.47%)提高了37.90%,半衰期从58.39h缩短为24.51h。土壤修复实验表明,第7天协同组对Beta-CP(30mg/kg)的降解率较单菌株提高了33.26%,达到79.27%。【结论】A.pittiiHA516是1株3-PBA高效降解菌,能与B. licheniformis G-04协同增效降解Beta-CP,可作为修复3-PBA或拟除虫菊酯类农药污染的优良微生物资源。     

食源米曲霉菌株的分离及其降解高效氯氰菊酯和3-苯氧基苯甲酸的特性研究

[目的]通过研究不同食源米曲霉菌株对高效氯氰菊酯(beta-cypermethrin,β-CP)及其必经代谢产物3-苯氧基苯甲酸(3-phenoxybenzoic acid,3-PBA)的降解特性,了解不同菌株的降解共性及差异性,为农副产品和发酵食品的农残减除提供理论基础和食品用安全微生物资源.[方法]以发酵食品为菌源...     

好氧条件下Sphingomonas sp．XJ1降解DBP途径的研究

在三角瓶中采用Sphingomonas sp．XJ1对邻苯二甲酸丁酯（DBP）进行好氧降解,以考察DBP的降解途径。分别对降解16h、32h和40h的DBP样品进行代谢产物分析,可判定保留时间为4．79min和5．11min所对应的代谢产物分别为原儿茶酸和邻苯二甲酸。由此可知,菌株Sphingomonassp．XJ1对DBP的降解遵循DBP好氧生物降解途径的一般途径。即在菌株XJI的作用下,DBP首先水解为MBP,继而水解为PA,经由PCA最终完全降解为CO2和H2O。     

好氧条件下Sphingomonassp.XJ1降解DBP途径的研究

在三角瓶中采用Sphingomonas sp.XJ1对邻苯二甲酸丁酯(DBP)进行好氧降解,以考察DBP的降解途径。分别对降解16h、32h和40h的DBP样品进行代谢产物分析,可判定保留时间为4.79min和5.11min所对应的代谢产物分别为原儿茶酸和邻苯二甲酸。由此可知,菌株Sphingomonas sp.XJ1对DBP的降解遵循DBP好氧生物降解途径的一般途径。即在菌株XJ1的作用下,DBP首先水解为MBP,继而水解为PA,经由PCA最终完全降解为CO2和H2O。     

Identification of novel metabolites in the degradation of phenanthrene by Sphingomonas sp. strain P2

Sphingomonas sp. strain P2, which is capable of utilizing phenanthrene as a sole carbon and energy source, was isolated from petroleum-contaminated soil in Thailand. Gas chromatography-mass spectrometry and (1)H and (13)C nuclear magnetic resonance analyses revealed two novel metabolites from the phenanthrene degradation pathway. One was identified as 5,6-benzocoumarin, which was derived by dioxygenation at the 1- and 2-positions of phenanthrene, and the other was determined to be 1,5-dihydroxy-2-naphthoic acid. Other metabolites from phenanthrene degradation were identified as 7, 8-benzocoumarin, 1-hydroxy-2-naphthoic acid and coumarin. From these results, it is suggested that strain P2 can degrade phenanthrene via dioxygenation at both 1,2- and 3,4-positions followed by meta-cleavage.     

Mineralization of phenol and its derivatives by Pseudomonas sp. strain CP4

A Pseudomonas sp. strain, CP4, was isolated that used phenol up to 1.5 g/l as sole source of carbon and energy. Optimal growth on 1.5 g phenol/l was at pH 6.5 to 7.0 and 30°C. Unadapted cells needed 72 h to decrease the chemical oxygen demand (COD) of about 2000 mg/l (from 1 g phenol/l) to about 200 mg/l. Adapted cells, pregrown on phenol, required only 65 h to decrease the COD level to below 100 mg/l. Adaptation of cells to phenol also improved the degradation of cresols. Cell-free extracts of strain CP4 grown on phenol or o-, m- or p-cresol had sp. act. of 0.82, 0.35, 0.54 and 0.32 units of catechol 2,3-dioxygenase and 0.06, 0.05, 0.05 and 0.03 units of catechol 1,2-dioxygenase, respectively. Cells grown on glucose or succinate had neither activity. Benzoate and all isomers of cresol, creosote, hydroxybenzoates, catechol and methyl catechol were utilized by strain CP4. No chloroaromatic was degraded, either as sole substrate or as co-substrate.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India     

Isolation and characterization of a meta-cleavage product in the degradation of quinaldic acid by Azotobacter sp.

Abstract From different samples of soil seventeen strains were isolated which grew aerobically in mineral salts medium with quinaldic acid as sole carbon source. Mutants were induced with N -methyl- N '-nitro- N -nitrosoguanidine. One mutant could be isolated which accumulated a yellow compound. The properties of this purified compound were those expected for 2-oxo-3-(4'-hydroxy-2'-oxo-3',4'-en-butyrate)-pyridine-6-carboxylic acid.     

Isolation and identification of a carbazole degradation gene cluster from Sphingomonas sp.JS1

The carbazole degrading bacterium JS1 was isolated from carbazole polluted soil and identified as Sphingomonas sp. bacterium based on its 16S rDNA gene. The car gene cluster located in the genome of JS1 was isolated by PCR and its presence verified by Southern hybridization. Sequence analysis of the car gene cluster showed that the arrangement of elements in JS1 was different from that of Pseudomons sp. CA10 and Nocardioides aromaticivorans IC177, but car gene cluster and neighboring regions were nearly identical to that of Sphingomonas sp. KA1 and Sphingomonas sp.GTIN11. Each element of the car gene cluster was expressed in E. coli upon IPTG induction. The amount of CaBb protein expressed was higher than CarBa and the ratio of these two proteins was 1:1.5. CarC expression level was detected using anti-CarC antibody. The result showed that carbazole degrading proteins were induced by the substrate carbazole. The quantity of CarC at 0.5 mg/ml carbazole was five times more than that at 0.1 mg/ml. Meiying Yang and Wenming Li have the equal contribution for this work.     

Mucoromycotina sp. HS-3对苯胺蓝染料的降解

采用静置开敞式培养法研究了碳源、氮源、盐度、金属离子对Mucoromycotina sp.HS-3菌降解苯胺蓝的影响。结果表明,菌株脱色最适合条件为葡萄糖1 g/L,硫酸铵0.6 g/L,Fe3+0.15 mmol/L,盐度小于50 g/L,在上述各培养条件下,对浓度为100 mg/L不灭菌的苯胺蓝溶液静止培养5 d,脱色率达95%以上。此外,通过降解前后的苯胺蓝溶液对豇豆和枯草芽孢杆菌进行毒性测试发现,降解后的苯胺蓝溶液毒性明显降低。因此,该菌对处理以苯胺蓝为主要成分的印染废水具有较好的应用潜力。     

Isolation and characterization of 3-N-trimethylamino-1-propanol-degrading Rhodococcus sp. strain A2

The aerobic degradation of 3- N -trimethylamino-1-propanol (homocholine) as a sole source of carbon and nitrogen has been found for a Rhodococcus sp. bacterium isolated from soil. The isolate was identified as Rhodococcus sp. strain A2 based on its phenotypic features, physiological and biochemical characteristics, and results of phylogenetic analysis. The washed cells of strain A2 completely degraded homocholine within 6 h, with concomitant formation of several metabolites. Analysis of the metabolites using capillary electrophoresis, fast atom bombardment–MS, and GC–MS showed that trimethylamine was the major metabolite, in addition to β-alanine betaine (β-AB) and trimethylaminopropionaldehyde. Therefore, the possible degradation pathway of homocholine in the isolated strain is through consequent oxidation of the alcohol group (-OH) to aldehyde (-CHO) and acid (-COOH). Thereafter, the cleavage of β-AB C–N bonds yielded trimethylamine and alkyl chain.