Cloning of a dibutyl phthalate hydrolase gene from Acinetobacter sp. strain M673 and functional analysis of its expression product in Escherichia coli

A dibutyl phthalate (DBP) transforming bacterium, strain M673, was isolated and identified as Acinetobacter sp. This strain could not grow on dialkyl phthalates, including dimethyl, diethyl, dipropyl, dibutyl, dipentyl, dihexyl, di(2-ethylhexyl), di-n-octyl, and dinonyl phthalate, but suspensions of cells could transform these compounds to phthalate via corresponding monoalkyl phthalates. During growth in Luria–Bertani medium, M673 produced the high amounts of non-DBP-induced intracellular hydrolase in the stationary phase. One DBP hydrolase gene containing an open reading frame of 1,095 bp was screened from a genomic library, and its expression product hydrolyzed various dialkyl phthalates to the corresponding monoalkyl phthalates.     

Biodegradation and kinetic analysis of phthalates by an Arthrobacter strain isolated from constructed wetland soil

A bacterial strain C21 isolated from constructed wetland soil was identified as Arthrobacter sp. based on 16S rRNA gene sequence analysis and physio-biochemical characteristics and was capable of utilizing di-n-butyl phthalate (DBP) as a carbon and energy source for growth. Strain C21 can also utilize other phthalates (PAEs) up to a molecular weight of 390.56 and phthalic acid (PA). The biodegradability of these compounds decreased with the increase in the length of phthalate alkyl chains and molecular weight. Kinetic analysis indicated that the strain C21 cell growth on DBP fitted well with Haldane-Andrews’ model (R ²?>?0.98) with μ _max, K _s, and K _i of 0.12/h, 4.2 mg/L, and 204.6 mg/L, respectively. When the initial DBP concentration was lower than 100 mg/L, DBP biodegradation reaction fitted with the first-order kinetics. The results suggested that Arthrobacter strain C21 played an active role in the bioremediation of the wetland contaminated with phthalates.     

Characterization of Ligninolytic Bacteria and Analysis of Alkali-Lignin Biodegradation Products

Ligninolytic bacteria degrading lignin were isolates and identified, and their biodegradation mechanism of alkaline-lignin was investigated. Four strains with lignin degradation capability were screened and identified from the soil, straw, and silage based on their decolorizing capacity of aniline blue and colony size on alkaline-lignin medium. The degradation ratio of Bacillus aryabhattai BY5, Acinetobacter johnsonii LN2, Acinetobacter lwoffii LN4, and Micrococcus yunnanensis CL32 have been assayed using alkaline-lignin as the unique carbon source. Further, the Lip (lignin peroxidase) and Mnp (manganese peroxidase) activities of strains were investigated. Lip activity of A. lwoffii LN4 was highest after 72 h of incubation and reached 7151.7 U · l^–1. Mnp activity of M. yunnanensis CL32 was highest after 48 h and reached 12533 U · l^–1. The analysis of alkaline-lignin degradation products by GC-MS revealed that the strains screened could utilize aromatic esters compounds such as dibutyl phthalate (DBP), and decomposite monocyclic aromatic compounds through the DBP aerobic metabolic pathway. The results indicate that B. aryabhattai BY5, A. johnsonii LN2, A. lwoffii LN4, and M. yunnanensis CL32 have high potential to degrade alkaline-lignin, and might utilize aromatic compounds by DBP aerobic metabolic pathway in the process of lignin degradation.Key words: isolation, bacteria, alkali-lignin, biodegradation products     

Kinetics and pathway of biodegradation of dibutyl phthalate by Pleurotus ostreatus

Dibutyl phthalate (DBP) is a plasticizer, whose presence in the environment as a pollutant has attained a great deal of attention due to its reported association with endocrine system disturbances on animals. Growth parameters, glucose uptake, percentage of removal efficiency (%E) of DBP, biodegradation constant of DBP (k) and half-life of DBP biodegradation (t_1/2) were evaluated for Pleurotus ostreatus grown on media containing glucose and different concentrations of DBP (0, 500 and 1000 mg l^?1). P. ostreatus degraded 99.6 % and 94 % of 500 and 1000 mg of DBP l^?1 after 312 h and 504 h, respectively. The k was 0.0155 h^?1 and 0.0043 h^?1 for 500 and 1000 mg of DBP l^?1, respectively. t_1/2 was 44.7 h and 161 h for 500 and 1000 mg of DBP l^?1, respectively. Intermediate compounds of biodegraded DBP were identified by GC-MS and a DBP biodegradation pathway was proposed using quantum chemical calculation. DBP might be metabolized to benzene and acetyl acetate, the first would be oxidated to muconic acid and the latter would enter into the Krebs cycle. P. ostreatus has the ability to degrade DBP and utilizes it as source of carbon and energy.     

Cytotoxic acridone and indoloquinazoline alkaloids from Zanthoxylum poggei

Two new alkaloids, poggeicridone (1) and 2-methoxy-7,8- dehydroruteacarpine (6), together with nine known compounds, were isolated from the dichloromethane (DCM) extract of the bark of Zanthoxylum poggei (Engl.) P. G. Waterman. The structures of all compounds were determined by comprehensive spectroscopic analyses (1D and 2D NMR and EI- and ESI⿿MS). Compounds 5-9 exhibited strong suppressive effects on the phagocytosis response upon activation with serum opsonized zymosan in the in vitro oxidative burst studies using whole blood. The IC₅₀ values were in the range of 12.0⿿25.9 μM. These compounds displayed a moderate level of cytotoxic activity against the human Caucasian prostate adenocarcinoma cell line PC-3, with IC₅₀ values of 15.8 and 22.1 μM (the IC₅₀ value of the positive control standard doxorubicin was IC₅₀ 0.9 μM). All isolated compounds were also tested against plant pathogenic bacteria, fungi and oomycetes using the paper disk agar diffusion assay, resulting in no significant activities (MICs > 1 mg/mL).     

Rates of Transformation of Methyl Parathion and Diethyl Phthalate by Aufwuchs Microorganisms

Using batch cultures, we determined transformation rates for low concentrations of two toxicants—an insecticide, methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate), and a plasticizer, diethyl phthalate—by aufwuchs, aquatic microbial growth attached to submerged surfaces or suspended in streamers or mats. Aufwuchs samples were collected from field sites, an indoor channel, and a continuous-flow fermentor. Aufwuchs fungi, protozoa, and algae did not transform methyl parathion or diethyl phthalate, but bacteria rapidly transformed both chemicals. Second-order transformation rate coefficients, K_b, based on total plate counts of bacteria in aufwuchs, were determined for potential use in a mathematical model capable of predicting the transport and fate of chemicals in aquatic systems. K_b for both methyl parathion and diethyl phthalate decreased as the concentration of total bacteria, [B], increased in aufwuchs. This effect resulted from the proportion of nontransformer to transformer bacteria increasing as [B] increased and from the rate of transformation per transformer cell decreasing as [B] increased. First-order transformation rate coefficients, K₁, were relatively stable per unit of surface area colonized by aufwuchs, because K_b decreased as [B] increased (K₁ = K_b × [B]).     

An immunoassay for dibutyl phthalate based on direct hapten linkage to the polystyrene surface of microtiter plates

Background

Dibutyl phthalate (DBP) is predominantly used as a plasticizer inplastics to make them flexible. Extensive use of phthalates in both industrial processes and other consumer products has resulted in the ubiquitous presence of phthalates in the environment. In order to better determine the level of pollution in the environment and evaluate the potential adverse effects of exposure to DBP, immunoassay for DBP was developed.

Methodology/Principal Findings

A monoclonal antibody specific to DBP was produced from a stable hybridoma cell line generated by lymphocyte hybridoma technique. An indirect competitive enzyme-linked immunosorbent assay (icELISA) employing direct coating of hapten on polystyrene microtiter plates was established for the detection of DBP. Polystyrene surface was first oxidized by permanganate in dilute sulfuric acid to generate carboxyl groups. Then dibutyl 4-aminophthalate, which is an analogue of DBP, was covalently linked to the carboxyl groups of polystyrene surface with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Compared with conjugate coated format (IC₅₀ = 106 ng/mL), the direct hapten coated format (IC₅₀ = 14.6 ng/mL) improved assay sensitivity after careful optimization of assay conditions. The average recovery of DBP from spiked water sample was 104.4% and the average coefficient of variation was 9.95%. Good agreement of the results obtained by the hapten coated icELISA and gas chromatography-mass spectrometry further confirmed the reliability and accuracy of the icELISA for the detection of DBP in certain plastic and cosmetic samples.

Conclusions/Significance

The stable and efficient hybridoma cell line obtained is an unlimited source of sensitive and specific antibody to DBP. The hapten coated format is proposed as generally applicable because the carboxyl groups on modified microtiter plate surface enables stable immobilization of aminated or hydroxylated hapten with EDC. The developed hapten coated icELISA can be used as a convenient quantitative tool for the sensitive and accurate monitoring DBP in water, plastic and cosmetic samples.     

Co-Metabolic Biodegradation of DBP by Paenibacillus sp. S-3 and H-2

Two di-n-butyl phthalate (DBP)-degrading strains, designated as S-3 and H-2, were isolated from DBP-polluted soil and both identified as Paenibacillus sp. When DBP was provided as the sole carbon source, about 45.5 and 71.7 % of DBP (100 mg/L) were degraded by strain S-3 and H-2, respectively, after incubation for 48 h. However, DBP (100 mg/L) was degraded completely by co-culture of strain S-3 and H-2 after incubation for 60 h. Four phthalic acid (PA) esters could be utilized by co-metabolism in the study and the degradation rates followed the order of dimethyl phthalate > diethyl phthalate > DBP > dioctyl phthalate. The metabolic pathway of DBP was elucidated based on the results of metabolites identification and enzyme assays. For strain S-3, DBP was degraded into butyl hydrogen phthalate which was degraded to PA by carboxyesterase further. But PA could be not hydrolyzed further because strain S-3 lacked 3,4-phthalate dioxygenase. Different with S-3, strain H-2 could hydrolyze PA into 3,4-dihydroxy-PA by 3,4-phthalate dioxygenase. Then 3,4-dihydroxy-PA was converted to protocatechuate and benzoic acid. Finally, the aromatic ring was cleavage and mineralized to CO₂ and H₂O. Above all, co-metabolism could increase the activity of 3,4-phthalate dioxygenase and accelerated the degradation of DBP. This study highlights an important potential use of co-metabolic biodegradation for the in situ bioremediation of DBP and its metabolites-contaminated environment.     

黄芩内生真菌N.SBA10代谢产物抗氧化活性及结构鉴定

黄芩内生真菌N.SBA10固体发酵培养物的乙酸乙酯提取物有较好的抗氧化活性。从乙酸乙酯提取物中分离得到3个化合物,根据波谱数据,化合物1–3的结构分别被鉴定为：邻苯二甲酸二正丁酯（DBP）,alternariol methyl ether（AME）和alternariol（AOH）。     

Alkynyl and β-ketophosphonates: Selective and potent butyrylcholinesterase inhibitors

A series of thirty-three alkynyl and β-ketophosphonates were evaluated for their in vitro acetyl- and butyryl-cholinesterase (AChE and BChE) inhibitory activities using Ellman’s spectrophotometric method. None of the examined compounds inhibited AChE activity at tested concentrations while twenty-nine of them showed significant and selective inhibition of BChE with IC₅₀ values between 38.60 µM and 0.04 µM. In addition, structure-activity relationships were discussed. The most effective inhibitors were the dibutyl o-methoxyphenyl alkynylphosphonate 3dc and dibutyl o-methoxyphenyl β-ketophosphonate 4dc. Activities of most potent compounds were also compared with a commercial organophosphorus compound. These results could inspire the design of new inhibitors with stronger activity against BChE.     

Potential of Ipomoea aquatica cultivars in phytoremediation of soils contaminated with di-n-butyl phthalate

Five different genotypic cultivars of Ipomoea aquatica commonly grown in Southeast Asia were cultivated to investigate their accumulation variation of di-n-butyl phthalate (DBP) and their potential for phytoremediation of three soils contaminated with DBP (4.5, 10.3 and 22.5 mg kg^?1). The results indicated different cultivar tolerance to DBP. DBP concentration in the shoots of the cultivars and residual DBP concentration in the soil were proportional to initial DBP concentrations in the soil and significantly different with different genotypic cultivars, indicating that the removal of DBP is cultivar-specific. DBP removal in the soil with indigenous DBP was higher than that in freshly DBP-spiked soils. The cultivars of local white-skin I. aquatica (cultivar V₅) and Taiwan filiform-leaf I. aquatica (cultivar V₁) presented the highest phytoremediation potential in the soil containing indigenous DBP and in freshly DBP-spiked soil, respectively. The translocation factor (TF, DBP concentration ratio of the shoots to the roots) and bioconcentration factor (BCF, DBP concentration ratio of the plant to the soil) also significantly varied with different cultivars, and they did not follow distribution profiles correlated to DBP removal indicating that phytoextraction was not the dominant DBP removal mechanism. Finally, the potential ability of different cultivars of enhancing biodegradation varied widely.     

Differential fluctuation in virulence and VOC profiles among different cultures of entomopathogenic fungi

Insect-passaged cultures of entomopathogenic fungi grown on potato dextrose agar media have been shown to have altered virulence and profiles of volatile compounds. The present study demonstrated the pathogenic status of FS₀ (in vitro) and FS₁ and FS₂ (insect-passaged cultures grown on PDA) cultures of Metarhizium anisopliae (strains 406 and 02049) and Beauveria bassiana by a non-choice assay, in which filter paper was inoculated with fungal spores at a concentration of 1 × 10⁷ spores/ml. The FS₁ and FS₂ cultures of M. anisopliae strain 02049 and B. bassiana produced conidia with high virulence, and the volatile profiles of these conidia comprised relatively lower percentages of branched-alkanes than conidia from the FS₀ cultures. In contrast, the conidia from an FS₀ culture of M. anisopliae strain 406 had somewhat elevated virulence levels, but their volatile profile had <2% branched-alkanes. The FS₁ and FS₂ cultures of M. anisopliae strain 406 did not gain virulence, and these cultures showed a decline in virulence along with major alteration of their volatile profiles. Their volatile profiles mainly comprised branched-alkanes. The volatile profiles of the FS₁ and FS₂ cultures lacked n-tetradecane, which was an important component of all the virulent cultures. Four compounds, 2-phenylpropenal, 2,5,5-trimethyl-1-hexene, n-tetradecane and 2,6-dimethylheptadecane, were detected only from the virulent cultures, suggesting that low LT₅₀ values were probably due to the production of these compounds. This is the first report to characterize volatiles from FS₀, FS₁ and FS₂ cultures of entomopathogenic fungi; its utility in different aspects opens an interesting area for further investigations.     

BIODEGRADATION OF PHTHALATE ESTERS BY CYANOBACTERIA1

Phthalate esters (PEs) are endocrine‐disrupting pollutants that are ubiquitous in the environment and can be degraded by microorganisms. In this study, we investigated the kinetics and pathway of biodegradation of di‐n‐butyl phthalate (DBP), diethyl phthalate (DEP), and dimethyl phthalate (DMP) by cyanobacteria Anabaena flos‐aquae G. S. West (strain 4054) and two strains of Microcystis aeruginosa (Kütz.) Kütz. (strain 2396 and strain SM). Gas chromatography/mass spectroscopy (GC/MS) and a deuterium‐labeled compound were used to analyze the degrading intermediates. The findings revealed that all three organisms were capable of metabolizing PE, and that among these organisms, A. flos‐aquae achieved the highest degradation. Additionally, the biodegradation of DBP, DEP, and DMP followed first‐order kinetics. Moreover, the results of the enzymatic study suggested that PE was degraded through transesterification on the side chains rather than deesterification. Finally, experiments using deuterium‐labeled DBP showed that there were two degradation pathways: C₁₆→ C₁₄→ C₁₂→ C₁₀→ C₈ and C₁₆→ C₁₅→ C₁₃→ C₁₁→ C₉. Based on our results, the biodegradation pathway of PE for cyanobacteria was suggested.     

Production profiles of phenolics from fungal tannic acid biodegradation in submerged and solid-state fermentation

Potent antioxidant phenolics are derived from tannin biodegradation. Understanding of biodegradation pathways through the identification of the intermediates molecules of great value like tannins is important to pursuit the production of bioactive monomers. Biodegradation of tannins remains poorly understood due to their chemical complexity and reactivity. Tannic acid biodegradation by Aspergillus niger GH1 in submerged fermentation (SF) and solid state fermentation (SSF) was evaluated by liquid chromatography coupled to mass spectrometry (LC–MS). Both cultures were kinetically monitored for the biodegradation profiles during 72 h. Differences in tannic acid composition were evidenced and the consumption of substrate and identification of biodegradation intermediates were achieved. The mechanism of tannic acid degradation by A. niger GH1 is by degradation of high molecular weight gallotannins and highly polymerized tannins to small molecules like gallic acid, digalloyl glucose and trigalloyl glucose. Important differences on time of substrate uptake and product release were revealed.     

Structure-dependent inhibition of human and rat 11β-hydroxysteroid dehydrogenase 2 activities by phthalates

Phthalates are diesters of phthalic acid and an alcohol moiety. Phthalates have been classified as endocrine disruptors and have a broad range of effects with unknown mechanisms. Some of the effects of phthalate are consistent with disruptions of normal glucocorticoid homeostasis, and in particular, with defective function of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2). In the present study, we tested 12 phthalate diesters and four monoesters for the inhibition of human and rat kidney 11β-HSD2. We examined the modes of inhibition and looked for a relationship between the potency for inhibition and the chemical structures. Of the phthalate diesters we tested, dipropyl phthalate (DPrP) and di-n-butyl phthalate (DBP) significantly inhibited both human and rat 11β-HSD2 activities. The IC₅₀s were 85.59 μM for DPrP and 13.69 μM for DBP when calculated for rat 11β-HSD2. As diesters, 8 of the phthalates did not affect 11β-HSD2 enzyme activity. Compared to the diesters that were inhibitory, the 8 non-inhibitory phthalates, had either fewer carbons, that is 1 or 2 carbons in the alcohol moiety, or more carbons, 5–10, as a branched or unbranched chain in the alcohol moeity. However, phthalates could be inhibitors with six carbons in the alcohol moiety if the carbons were cyclized, as in dicyclohexyl phthalate (DCHP), which inhibited rat 11β-HSD2 with an IC₅₀ of 32.64 μM. Thus, whether a phthalate is an inhibitor may reflect the size and shape of the compound. Although the diesters are the compounds used in manufacturing and present as environmental contaminants, it is the monoester metabolites that are detected in human serum and urine. We showed that mono (2-ethylhexyl) phthalate (MEHP) significantly inhibited human (IC₅₀ = 110.8 ± 10.9) and rat (121.8 ± 8.5 μM) 11β-HSD2 activity even though its parent compound, di(2-ethylhexyl) phthalate (DEHP) did not. MEHP was a competitive inhibitor of 11β-HSD2 enzymatic activity. We conclude that phthalates of a certain size act as competitive inhibitors.     

Evaluation of Mongolian compound library for potential antimalarial and anti-Toxoplasma agents

179 compounds in a Mongolian compound library were investigated for their inhibitory effect on the in vitro growth of Plasmodium falciparum and Toxoplasma gondii. Among these compounds, brachangobinan A at a half-maximal inhibition concentration (IC₅₀) of 2.62 μM and a selectivity index (SI) of 27.91; 2-(2′-hydroxy-5′-O-methylphenyl)-5-(2″,5″-dihydroxyphenyl)oxazole (IC₅₀ 3.58 μM and SI 24.66); chrysosplenetin (IC₅₀ 3.78 μM and SI 15.26); 4,11-di-O-galloylbergenin (IC₅₀ 3.87 μM and SI 13.38); and 2-(2′,5′-dihydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole (IC₅₀ 6.94 μM and SI 11.48) were identified as potential inhibitors of P. falciparum multiplication. Additionally, tricin (IC₅₀ 12.94 μM and SI > 23.40) was identified as a potential inhibitor of T. gondii multiplication. Our findings represent a good starting point for developing novel antimalarial and anti-Toxoplasma therapeutics from Mongolian compounds.     

Discovery of dicephalosterol, a new testosterone 5α-reductase inhibitor, and some new mycological aspects of its producer,Dicephalospora rufocornea (sclerotiniaceae, discomycetes)

Dicephalosterol, a new testosterone 5α-reductase inhibitor, was found from isolates ofDicephalospora rufocornea, a sclerotiniaceous discomycete widely distributed, but not previously cultured. Under SEM observation, the polar appendage of the ascospores inD. rufocornea was found to be more solid than was hitherto reported. Dicephalosterol was produced by submerged fermentation for 7 d. This new analogue of testosterone showed an IC₅₀ of 5.7 μg/ml for rat prostatic 5α-reductase, but no antimicrobial activity against bacteria or fungi.     

Design,synthesis and fungicidal evaluation of novel pyraclostrobin analogues

A series of novel pyraclostrobin derivatives were designed and prepared as antifungal agents. Their antifungal activities were tested in vitro with five important phytopathogenic fungi, namely, Batrylis cinerea, Phytophthora capsici, Fusarium sulphureum, Gloeosporium pestis and Sclerotinia sclerotiorum using the mycelium growth inhibition method. Among these compounds, 5s displayed IC₅₀ value of 0.57?μg/mL against Batrylis cinerea and 5k-II displayed IC₅₀ value of 0.43?μg/mL against Sclerotinia sclerotiorum, which were close to that of the positive control pyraclostrobin (0.18?μg/mL and 0.15?μg/mL). Other compounds 5f, 5k-II, 5j, 5m and 5s also exhibited strong antifungal activity. Further enzymatic assay demonstrated compound 5s inhibited porcine bc₁ complex with IC₅₀ value of 0.95?μM. The statistical results from an integrated computational pipeline demonstrated the predicted total binding free energy for compound 5s is the highest. Consequently, compound 5s with the biphenyl-4-methoxyl side chain could serve as a new motif as inhibitors of bc₁ complex and deserve to be further investigated.     

A pair of new sesquiterpene isomers containing spiro heterocyclic skeleton from plant-derived fungus Botryosphaeria dothidea

A pair of new sesquiterpene isomers containing a spiro heterocyclic skeleton, dothimes A (1) and B (2), together with six known compounds, quindoline (3), (S)-3-(3-indolyl)lactic acid methyl ester (4), dankasterone B (5), dibutyl phthalate (6), (1S,3R,4R,7S)-3,4-dihydroxy-α-bisabolol (7), and p-hydroxybenzaldehyde (8), were isolated from the plant-derived fungus Botryosphaeria dothidea. The structures of all isolated compounds were determined based on extensive spectroscopic analyses, including 1D/2D nuclear magnetic resonance (NMR), and high resolution electrospray ionization mass spectrometry (HRESIMS) data, as well as by comparison with literature reports. Compounds 1 and 2 exhibited inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production with IC₅₀ values of 63.66 and 58.29 μM, respectively.     

In vitro saprotrophic basidiomycetes tolerance to pendimethalin

Pendimethalin is a dinitroaniline herbicide classified among the persistent-bioaccumulative toxics. In this paper, the tolerance to this herbicide has been studied in isolates of basidiomycetes (10 species including 9 wood-rotting and 1 litter fungi), collected in different areas of the Campania region (South Italy). The isolates were grown on two different agar media, rich and poor for the presence/absence of dextrose and NH₄NO₃, and amended with 0, 100 and 500 ppm herbicide. The mycelial growth was recorded daily, and statistical analysis of fungal growth rates, determined via linear regression, allowed us to compare the hyphal extension of various macrofungi in presence of herbicide. These data represent the adaptation capacity of the fungal organisms to the peculiar environmental situation. In amended agar, all fungi exhibited a certain tolerance to pendimethalin and, normally, the fungal growth decreased with the increasing of pollutant concentration. Nevertheless, in some cases, the difference of ground reflected the different agar media. In fact, the growth of Agrocybe aegerita, in rich agar decreased with the increase of herbicide dose, while in poor agar it increased with the pollutant. Among the examined fungi, A. aegerita was the species which resulted the most tolerant to herbicide, showing growth to about 70% of the control to the highest concentrations of pollutant and for the two different agar media.Scientific relevance of the paperNo published report is available regarding pendimethalin herbicide biodegradation with basidiomycetes; therefore this work represented the first and preliminary investigation as regards. It's directed to determine the fungal tolerance to the pollutant.