一株芘降解菌B2的降解条件优化及降解基因

芳香烃降解菌是石油污染土壤修复的主要生物资源。采用芘平板升华法对克拉玛依原油污染土壤样品进行驯化培养,分离得到一株芘降解菌B2,经16S rDNA基因序列比对及系统发育进化分析表明,该菌株为假单胞菌属(Pseudo-monas)。采用正交设计方法优化菌株B2对高分子量多环芳烃芘的降解条件,并构建多元非线性模型预测菌株B2对芘的最佳降解条件,结果表明:在接种量OD660 nm为0.60、降解温度为40℃、降解时间为6.0 d时,预测菌株B2对芘的降解最大达到38.214 mg/L,实际测得最大降解量为37.906 mg/L,预测准确率为99.19%。运用PCR技术克隆B2的邻苯二酚-2,3-双加氧酶基因(B2C23O)(I.2.A亚家族),核酸序列分析表明,该基因全长880 bp,具有一个完整的开放阅读框,编码246个氨基酸,与已报道的Pseudomonas putida W619同源性最高为97%;对B2C23O基因编码氨基酸序列进行分析,发现其具有邻位断裂双加氧酶模式结构,推测菌株B2通过邻位裂解途径降解芘代谢中间产物邻苯二酚。     

Persistence of herbicide fenoxaprop ethyl and its acid metabolite in soil and wheat crop under Indian tropical conditions

The persistence of fenoxaprop ethyl {Ethyl (RS)-2-[4-(6-chloro-1,3-benzoxazol-2-yloxy) phenoxy] propionate} herbicide and its active metabolite fenoxaprop acid was investigated in soil and wheat crop. Fenoxaprop acid was prepared by alkaline hydrolysis of fenoxaprop ethyl. A HPLC method was developed in which fenoxaprop ethyl herbicide and its acid metabolite showed sharp single peak at 6.44 and 2.61 min respectively. The sensitivity of the method for ester and acid was 2 and 1 ng respectively with limit of detection of 0.1 and 0.05 μg mL^?1. The recovery of fenoxaprop ethyl and fenoxaprop acid from soil, wheat straw and grain ranged between 73.8–80.2%. In a field experiment fenoxaprop ethyl (Puma super® 10 EC) when applied to wheat crop at the rate of 120 g and 240 g a.i. ha^-1 as post emergence spray, fenoxaprop ethyl converted to fenoxaprop acid. Residues of fenoxaprop ethyl and acid dissipated in soil with a half-life of 0.5 and 7.3 days, respectively. At harvest no detectable residues of fenoxaprop ethyl or acid were observed in soil, wheat grain and straw samples.     

乳酸乳球菌SM526产生乳链菌肽的营养调控

自酸泡菜中分离并鉴定了一株产生乳链菌肽的菌株－乳酸乳球菌（Ｌａｃｔｏｃｏｃｃｕｓ ｌａｃｔｉｓ ｓｕｂｓｐ．ｌａｃｔｉｓ）ＳＭ５２６。研究了不同种类与浓度的碳源、氮源和磷源对该菌产生乳链菌肽的影响,结果表明乳链菌肽的生物合成受碳源、氮、磷源的调控,蔗糖和磷酸二氢钾分别为其最佳碳源和磷源,大豆蛋白胨和酵母粉为其理想氮源。得用分批培养也链菌肽合成的代谢动力学则表明,乳链菌肽在ＳＭ５２６菌株的指数生长期     

代谢产物积累对二氯甲烷生物降解的影响

研究了代谢调节产物(NaCl)浓度积累对生物法降解二氯甲烷效率的影响。试验表明,一定浓度的NaCl对二氯甲烷降解菌(GD11)的生长有不同程度的抑制作用。摇瓶试验中NaCl对二氯甲烷降解菌的抑制浓度为35.1g/L;当空塔气速91.7m/h、二氯甲烷进口浓度2.02g/m3、温度28.5℃、循环液流量0.06m3/h、pH值6.5～7.5时,生物滴滤塔中NaCl对二氯甲烷降解菌的抑制浓度为23.4g/L,两者相比有一定差异。NaCl浓度对生物膜的厚度有一定影响,这从侧面反映了NaCl浓度对微生物活性的影响。     

Enantioselectivity in toxicity and degradation of dichlorprop-methyl in algal cultures

Enantioselectivity in the toxicity and degradation of the herbicide dichlorprop-methyl (2,4-DCPPM) in algal cultures was studied. Enantioselectivity was clearly observed in the toxicity of racemic 2,4-DCPPM and its two enantiomers. R-2,4-DCPPM showed low toxicity to Chlorella pyrenoidosa and Chlorella vulgaris, but higher toxicity to Scenedesmus obliquus. The observed toxicity was ranked: R-2,4-DCPPM > S-2,4-DCPPM ? Rac-2,4-DCPPM; the toxicity of R-2,4-DCPPM was about 8-fold higher than that of Rac-2,4-DCPPM. Additionally, 2,4-DCPPM was quickly degraded, in the initial 12 h, and different algae cultures had different enantioselectivity for the 2,4-DCPPM enantiomers. There was no significant enantioselectivity for 2,4-DCPPM in Chlorella vulgaris in the initial 7 h. However, racemic 2,4-DCPPM was degraded by Scenedesmus obliquus quickly, in the initial 4 h, much quicker, in fact, than the S- or R-enantiomers (racemate > R- > S-), indicating that the herbicide 2,4-DCPPM was absorbed enantioselectively by Scenedesmus obliquus. The rapid formation of 2,4-DCPP suggested that 2,4-DCPPM adsorbed by algal cells was catalytically hydrolyzed to the free acid, a toxic metabolite. The production rates of 2,4-DCPP were as follows: Scenedesmus obliquus > Chlorella pyrenoidosa > Chlorella vulgaris, consistent with the degradability of 2,4-DCPPM. Scenedesmus obliquus had quick, but different, degradative and uptake abilities for R-, S-, and Rac-2,4-DCPPM. The R- and S- enantiomers were not hydrolyzed in the first 12 h, while both enantiomers were hydrolyzed slowly after that. These results indicate that some physical and chemical properties of compounds are of importance in determining their enantioselective toxicity and degradation. The ester and its metabolite likely played an important role in enantioselective toxicity to the three algae.     

Development of competitive immunoassays to hydroxyl containing fungicide metabolites

This paper describes the isolation of monoclonal antibodies and the development of competitive immunoassays to pesticide metabolites of the fungicides imazalil, carbendazim and thiabendazole. The metabolite specific hydroxyl residues were used as the reactive group with which to link the metabolite to the carrier proteins Keyhole Limpet Haemocyanin (KLH) and Bovine Serum Albumin (BSA). In each case immune responses in mice were raised and monoclonal antibodies were produced. Antibodies were developed into competitive ELISAs to the appropriate metabolite. The antibody raised to a metabolite of imazalil was optimised into a competitive ELISA format which had an assay IC50 of 7.5 μg/L and a limit of detection (LOD) of 1.1 μg/L. A single antibody isolated against the metabolite of carbendazim had assay IC50s of 3.2 and 2.7 μg/L for the metabolites of carbendazim and thiabendazole respectively with an LOD of 0.38 μg/L for both. These sensitive immunoassays may have application in the monitoring of human exposure to these fungicide residues either by occupational or non-occupational routes.     

光照对芦丁稳定性及抑菌活性的影响

黄酮类化合物具有多羟基结构易被氧化,为了解其羟基被氧化后,是否还具有抑菌、抗癌等多种生物学活性,对芦丁光照处理前后的含量、抗氧化活性(清除DPPH.和还原能力)、分子结构以及抑菌活性进行了比较研究.结果表明,随着光照时间的延长,芦丁含量和抗氧化活性均呈下降趋势,降解反应属于一级反应,半衰期为187.3 d.结构鉴定表明,光照处理芦丁母核结构没有降解,只是A环5、7-OH和B环3′、4′-OH氢解离,芦丁在溶液中以负离子或自由基形式存在.芦丁氧化前后的浓度与抑菌效果均成良好的量效关系,其相关系数分别为0.933 2和0.933 0.但氧化芦丁的EC50值(15.58 g/L)与对照(6.34 g/L)相比差异显著(P<0.01).可见,氧化芦丁的抑菌活性明显降低.这进一步表明,芦丁分子结构的细微改变与体外的抗氧化活性和抑菌活性三者之间的构效关系基本一致.     

The effect of multiple soil applications of disulfoton on enhanced microbial degradation in soil and subsequent uptake of insecticidal chemicals by potato plants

Abstract

Potatoes were grown during 1992 in 2 m² plots of loam which had received 1, 2 or 3 annual treatments of Di‐Syston 15G, equivalent to 3.36 kg AI/ha, in furrow at planting. The presence of enhanced degradative activity to the sulfoxide and sulfone metabolites of disulfoton in the soil treated in the previous two years was confirmed by laboratory tests prior to the 1992 treatments. Soil, seed potato and foliage from the three treatments were analyzed for disulfoton and its sulfoxide and sulfone metabolites for 12 wk following planting/treatment. Disulfoton was the major insecticidal component of the soil, a minor component of the seed piece and was not detected (<0.02 ppm) in potato foliage. Disulfoton concentrations in each of the three substrates sampled were similar for the three treatments. Disulfoton sulfoxide and sulfone were the major insecticidal components of the seed piece and foliage. Their maximum concentrations in 1st year soil, seed pieces and foliage were ca. 2x, 2x and 6x, respectively, those measured in the 2nd and 3rd year treatments. The results demonstrate that enhanced microbial degradation of relatively minor insecticidal compounds in the soil can profoundly affect insecticide levels in the plant when these compounds are the major insecticidal components accumulated. The broader implications for crop protection using soil‐applied systemic insecticides are discussed.     

磺胺二甲基嘧啶(SM2)高效降解菌J2的分离筛选及降解特性研究

从城市污水处理厂活性污泥中分离得到一株能以磺胺二甲基嘧啶(SM2)为唯一碳源的菌株,经生理生化鉴定和16S rRNA基因序列同源性分析,将此菌鉴定为蜡样芽孢杆菌(Bacillus cereus),命名为J2.不同条件下的降解特性研究结果表明,J2菌株具有极高的SM2耐受性(100 mg·L~(-1))、较广的温度(20～30℃)和pH(6～8)适用范围;在温度30℃、pH=8.0、初始OD_(600)=0.1、SM2起始浓度为50 mg·L~(-1)的条件下,J2菌株在36 h内对SM2的降解率可达100%,降解效率远超目前已报道的其他SM2降解菌株,展现出了良好的应用潜力.J2菌株降解SM2过程中产生了5种主要中间代谢产物,分析推断其降解SM2的途径分为两条:①磺胺二甲基嘧啶分子首先在酶促反应作用下脱除SO_2,生成嘧啶环和苯胺环自由基,这两种自由基再经过环间耦合生成N-(4,6-二甲基嘧啶-2基)-1,4-二苯胺,该分子中的C—N键在活性氧物种的作用下断开生成苯胺和2-氨基-4,6-二甲基嘧啶;②在漆酶的作用下N~4键断裂,产生N-(3,5-二甲基嘧啶)-苯磺酰胺,之后N-(3,5-二甲基嘧啶)-苯磺酰胺的N—S键断裂,进一步形成2-氨基-4,6-二甲基嘧啶和苯亚砜.     

采用代谢流量分析方法评估二噁英对细胞的代谢干扰

以肝癌细胞HepG2为供试细胞,选用5个不同浓度的二噁英（0、0.001、0.01、0.1和1.0 nmol.L-1）,探讨2,3,7,8-TCDD对HepG2细胞的毒性效应.采用90%甲醇离心法实现蛋白与代谢产物的分离,建立了高效液相色谱-串联三重四极杆质谱（HPLC-MS/MS）分析胞外液中的23种代谢产物（20种氨基酸、乳酸、甘油和尿素）的方法.建立的HPLC-MS/MS分析方法对各化合物的加标回收率可达91%—105%之间,化合物浓度的标准偏差均小于10%.在此基础上,结合代谢流量分析系统研究了2,3,7,8-TCDD对HepG2细胞内代谢流的影响.结果表明,随着浓度的增高,2,3,7,8-TCDD对HepG2细胞代谢的干扰作用呈增强趋势.2,3,7,8-TCDD降低了细胞对葡萄糖的吸收,并进一步抑制了HepG2细胞糖酵解的速率.此外,2,3,7,8-TCDD导致了HepG2细胞乳酸代谢流的明显增加,由此使细胞产生了过量的乳酸.