Functional diversity of bacterial genes associated with aromatic hydrocarbon degradation in anthropogenic dark earth of Amazonia

The objective of this work was to evaluate the catabolic gene diversity for the bacterial degradation of aromatic hydrocarbons in anthropogenic dark earth of Amazonia (ADE) and their biochar (BC). Functional diversity analyses in ADE soils can provide information on how adaptive microorganisms may influence the fertility of soils and what is their involvement in biogeochemical cycles. For this, clone libraries containing the gene encoding for the alpha subunit of aromatic ring-hydroxylating dioxygenases (α-ARHD bacterial gene) were constructed, totaling 800 clones. These libraries were prepared from samples of an ADE soil under two different land uses, located at the Caldeirão Experimental Station - secondary forest (SF) and agriculture (AG) -, and the biochar (SF_BC and AG_BC, respectively). Heterogeneity estimates indicated greater diversity in BC libraries; and Venn diagrams showed more unique operational protein clusters (OPC) in the SF_BC library than the ADE soil, which indicates that specific metabolic processes may occur in biochar. Phylogenetic analysis showed unidentified dioxygenases in ADE soils. Libraries containing functional gene encoding for the alpha subunit of the aromatic ring-hydroxylating dioxygenases (ARHD) gene from biochar show higher diversity indices than those of ADE under secondary forest and agriculture.

Type II aromatic polyketide biosynthetic tailoring enzymes: diversity and adaptation in Streptomyces secondary metabolism.

Members of the bacterial genus Streptomyces are well known for their ability to produce an exceptionally wide selection of diverse secondary metabolites. These include natural bioactive chemical compounds which have potential applications in medicine, agriculture and other fields of commerce. The outstanding biosynthetic capacity derives from the characteristic genetic flexibility of Streptomyces secondary metabolism pathways: i) Clustering of the biosynthetic genes in chromosome regions redundant for vital primary functions, and ii) the presence of numerous genetic elements within these regions which facilitate DNA rearrangement and transfer between non-progeny species. Decades of intensive genetic research on the organization and function of the biosynthetic routes has led to a variety of molecular biology applications, which can be used to expand the diversity of compounds synthesized. These include techniques which, for example, allow modification and artificial construction of novel pathways, and enable gene-level detection of silent secondary metabolite clusters. Over the years the research has expanded to cover molecular-level analysis of the enzymes responsible for the individual catalytic reactions. In vitro studies of the enzymes provide a detailed insight into their catalytic functions, mechanisms, substrate specificities, interactions and stereochemical determinants. These are factors that are essential for the thorough understanding and rational design of novel biosynthetic routes. The current study is a part of a more extensive research project (Antibiotic Biosynthetic Enzymes; www.sci.utu.fi/projects/biokemia/abe), which focuses on the post-PKS tailoring enzymes involved in various type II aromatic polyketide biosynthetic pathways in Streptomyces bacteria. The initiative here was to investigate specific catalytic steps in anthracycline and angucycline biosynthesis through in vitro biochemical enzyme characterization and structural enzymology. The objectives were to elucidate detailed mechanisms and enzyme-level interactions which cannot be resolved by in vivo genetic studies alone. The first part of the experimental work concerns the homologous polyketide cyclases SnoaL and AknH. These catalyze the closure of the last carbon ring of the tetracyclic carbon frame common to all anthracycline-type compounds. The second part of the study primarily deals with tailoring enzymes PgaE (and its homolog CabE) and PgaM, which are responsible for a cascade of sequential modification reactions in angucycline biosynthesis. The results complemented earlier in vivo findings and confirmed the enzyme functions in vitro. Importantly, we were able to identify the amino acid -level determinants that influence AknH and SnoaL stereoselectivity and to determine the complex biosynthetic steps of the angucycline oxygenation cascade of PgaE and PgaM. In addition, the findings revealed interesting cases of enzyme-level adaptation, as some of the catalytic mechanisms did not coincide with those described for characterised homologs or enzymes of known function. Specifically, SnoaL and AknH were shown to employ a novel acid-base mechanism for aldol condenzation, whereas the hydroxylation reaction catalysed by PgaM involved unexpected oxygen chemistry. Owing to a gene-level fusion of two ancestral reading frames, PgaM was also shown to adopt an unusual quaternary sturucture, a non-covalent fusion complex of two alternative forms of the protein. Furthermore, the work highlighted some common themes encountered in polyketide biosynthetic pathways such as enzyme substrate specificity and intermediate reactivity. These are discussed in the final chapters of the work.

Synthesis and NMR characterization of aliphatic-aromatic copolyesters by reaction of poly(ethylene terephthalate) post-consumer and poly(ethylene adipate)

An aliphatic-aromatic copolyester of poly(ethylene terephthalate), PET, and poly(ethylene adipate), PEA, PET-co-PEA, was synthesized by the high temperature melt reaction of post-consumer PET and PEA. As observed by NMR spectroscopy, the reaction yielded random copolyesters in a few minutes through ester-interchange reactions, even without added catalyst. The copolyesters obtained in the presence of a catalyst presented higher intrinsic viscosity than that obtained without the addition of catalyst, due to simultaneous polycondensation and ester-interchange reactions. The structure of the aliphatic-aromatic copolyesters obtained in different PET/PEA ratio is random as observed by NMR analysis.

A convenient method for the determination of moisture in aromatic plants

A method is proposed for the determination of the moisture content of aromatic plants. This method is based on the co-distillation of the starting material in a modified Clevenger apparatus with four organic solvents (toluene, cyclohexane, dichloromethane and carbon tetrachloride). The results were compared with those obtained by oven drying at 105 ºC and steam distillation of the essential oil. The efficiencies of the methods were shown to be equivalent. The solvent distillation method was more practical, especially with respect to operating time (2 h).

Database of the Amazon aromatic plants and their essential oils

The aromatic flora of the Amazon has been inventoried for 30 years. In this sense, were made over 500 field trips to collect over 2500 plants and to obtain more than 2000 essential oils and aroma concentrates, all of them submitted to GC and GC-MS. This work led to the creation of a database for the aromatic plants of the Amazon, which catalogs general information about 1250 specimens. The database has allowed the publication of the chemical composition of the oils and aromas of more than 350 species, associated with a larger number of chemical types. The essential oils of many species offer optimum conditions for economic exploitation and use in national and international market of fragrances, cosmetics, agricultural and household pesticides.

Aromatic compounds from three Brazilian Lauraceae species

Phytochemical investigations on three Brazilian Lauraceae species from the Cerrado region of São Paulo State, Ocotea corymbosa (Meins) Mez., O. elegans Mez. and Persea pyrifolia Nees & Mart. ex Nees resulted in the isolation of flavonoids, an ester of the 4-O-E-caffeoylquinic acid, an aromatic sesquiterpene besides furofuran lignans. This is the first chemical study on the leaves of Ocotea elegans and O. corymbosa as well as the first report of non-volatile compounds from Persea pyrifolia.

Removal of high-molecular weight polycyclic aromatic hydrocarbons

Alternatives for the removal of high-molecular weight polycyclic aromatic hydrocarbons (HWM-PAH) from soil were tested by adding fertilizer or glycerol, as well as the combination of both. Experiments were carried out for 60 days in reactors containing a HWM-PAH-contaminated soil (8030 μg kg-1), accompanied by pH monitoring, humidity control and quantification of total heterotrophic bacteria and total fungus. Fertilizer addition removed 41.6% of HWM-PAH. Fertilizer and glycerol in combination removed 46.2%. When glycerol was added individually, degradation reached 50.4%. Glycerol also promoted the increase of degradation rate during the first 30 days suggesting the HMW-PAH removal occurred through cometabolic pathways.

Synthesis and antifungal activity of halogenated aromatic bis-γ-lactones analogous to avenaciolide

Here we describe the total syntheses and characterization by elemental analyses, infrared and NMR spectroscopy of three new compounds analogous to avenaciolide, a bis-γ-lactone isolated from Aspergillus avenaceus that possesses antifungal activity, where the octyl group of the natural product was replaced by aromatic groups containing chlorine and fluorine atoms. The effects of the avenaciolide, the novel compounds and their synthetic precursors on mycelia development and conidia germination of Colletotrichum gloeosporioides and Fusarium solani were evaluated in vitro. The title compounds were almost as active as avenaciolide. The absolute structures of the chlorinated analogs were determined by X-ray diffraction analysis.

Electrochemical evidence of sulfinic acid derivative as an intermediate in the reduction of aromatic sulfonyl chloride in an aprotic medium

The electrochemical reduction of p-nitrobenzenesulfonyl chloride (NBSCl) in dimethylsulfoxide (DMSO) solution is used here as a model to investigate the role of sulfinic acid derivative in this compound's global reduction process. Cyclic voltammetric experiments reveal the production of sulfinic acid derivative, which is important in chemical reactions involving the original compound and other intermediates. This paper also discusses the probable mechanisms of the reduction.

Self-assembly of supermolecular species directed by hydrogen bonding and aromatic π-π stacking interactions

A new Cu(II) trimers, [Cu3(dcp)2(H2O)8]. 4DMF, with the ligand 3,5-pyrazoledicarboxylic acid monohydrate (H3dcp) has been prepared by solvent method. Its solid-state structure has been characterized by elemental analysis, thermal analysis (TGA and DSC), and single crystal X-ray diffraction. X-ray crystallographic studies reveal that this complex has extended 1-D,2-D and 3-D supramolecular architectures directed by weak interactions (hydrogen bond and aromatic π-π stacking interaction) leading to a sandwich solid-state structure.

Sensory evaluation of black instant coffee beverage with some volatile compounds present in aromatic oil from roasted coffee

The oil obtained from Brazilian roasted coffee by supercritical CO2 extraction shows considerable aromatic properties, mainly composed by five aromatic compounds, 2-methylpyrazine; 2-furfurylalcohol, 2,5-dimethylpyrazine; γ-butyrolactone and 2-furfurylacetate. Sensory analyses were used to verify the influence of a mixture of these important classes of aromatic coffee compounds (pyrazines, furans and lactones) and of the roasted coffee aromatic oil on the coffee aroma and flavour of black instant freeze and spray-dried coffee beverages. In the acceptance evaluation of the aroma, the samples prepared with freeze-dried instant coffee without the mixture of volatile compounds (sample 4) were not significantly different from the freeze-dried instant coffee in which the aromatic coffee oil was added (sample 5) and from the sample prepared with freeze-dried coffee in which the mixture of the five volatile was added (sample 3), coincidentally from the same drying process. Therefore, sample (3) did not differ from samples prepared with spray dried instant coffee without (sample 1) and to which (sample 2) the mixture of volatile was added. Therefore, with respect to this attribute, the addition of this mixture did not interfere in this drink acceptance. Taking into consideration the flavor, samples prepared with freeze-dried instant coffee in which the aromatic coffee oil was added (5) and the samples with (3) and without (4) the mixture of the five volatile was added did not differ significantly, however sample (4) did not differ from samples (1) and (2). Regarding this attribute, the addition of the aromatic oil of roasted coffee or a mixture of volatile in samples of freeze-dried instant coffee had a better acceptance than those dried by spray dryer (1) and (2). Thus, the enrichment of drinks with the aromatic oil of roasted coffee, or even with the mixture of the five components did not influence the consumer acceptance with respect to the aroma, but exerts influence with respect to flavour.

Biotransformation of polycylic aromatic compounds by fungi and an investigation into the oxidation of alkylbenzenes by Mortierella isabellina NRRL 1757

Incubations of several polycyclic heteroaromatic compounds and two polycyclic aromatic hydrocarbons with a series of common fungi have been performed. The fungi Cunninghamella elegans ATCC 26269, Rhizopus arrhizus ATCC 11145, and Mortierella isabellina NRRL 1757 were studied in this regard. Of the aza heteroaromatics, only dibenzopyrrole gave a ring hydroxylated product following the incubation with C. elegans. From the thio heteroaromatics studied, dibenzothiophene was metabolized by all the three fungi and thioxanthone by C. elegans and M. isabellina giving sulfones and sulphoxides. Thiochromanone was metabolized stereoselectively to the corresponding sulphoxide by C. elegans. Methyl substituted thioxanthones on incubation with C. elegans produced oxidative products, arising from S -oxidation and hydroxylation at the methyl group. Of the cyclic ketones studied, only fluorenone was reduced to hydroxyfluorene and this metabolism is compared with that reported with cytochrome P-450 monooxygenases of hepatic microsomes. A series of para-substituted ethylbenzenes has been transformed stereoselectively to the 1-phenylethanols by incubation with M. isabellina. Comparisons of the enantiomeric purities obtained from products with their respective para substituent of the same steric size but different electronic properties indicate that the stereoselectivity of hydroxylation at benzylic carbon may be susceptible to electron donating or withdrawing factors in some cases, but that observation is not va lid in all the comparisons. The stereochemistry of the reaction is discussed in terms of three possible steps, ethylbenzene ---) 1-phenylethanol ---) acetophenone ---) 1-phenylethanol. This metabolic pathway could account for the inconsistencies observed in the comparisons of optical purities and electronic character of para substituents. Furthermore, formation of 2-phenylethanol (in some cases), l-(p-acetylphenyl)ethanol from p-diethylbenzene, and N-acetylation of p-ethylaniline was observed. n-Propylbenzene was also converted to optically active 1-phenylpropanol. Acetophenone, p-ethylacetophenone, and o(,~,~-trifluoroacetophenone were transformed to 1-phenylethanol, l-(p-ethylphenyl)ethanol, and 1-phenyl-2,2,2-trifluoroethanol, respectively, with high chemical and excellent optical yields. The 13 C NMR spectra of several substrates and metabolic products have been reported and assigned for the first time.

Investigations into the extraction and the determination of polycyclic aromatic hydrocarbons (PAHs) from water by solid-phase extraction and capillary gas chromatography/ mass spectrometry

Factors affecting the detennination of PAHs by capillary GC/MS were studied. The effect of the initial column temperature and the injection solvent on the peak areas and heights of sixteen PAHs, considered as priority pollutants, USillg crosslinked methyl silicone (DB!) and 5% diphenyl, 94% dimethyl, 1% vinyl polysiloxane (DBS) columns was examined. The possibility of using high boiling point alcohols especially butanol, pentanol, cyclopentanol, and hexanol as injection solvents was investigated. Studies were carried out to optimize the initial column temperature for each of the alcohols. It was found that the optimum initial column temperature is dependent on the solvent employed. The peak areas and heights of the PAHs are enhanced when the initial column temperature is 10-20 c above the boiling point of the solvent using DB5 column, and the same or 10 C above the boiling point of the solvent using DB1 column. Comparing the peak signals of the PAHs using the alcohols, p-xylene, n-octane, and nonane as injection solvents, hexanol gave the greatest peak areas and heights of the PAHs particularly the late-eluted peaks. The detection limits were at low pg levels, ranging from 6.0 pg for fluorene t9 83.6 pg for benzo(a)pyrene. The effect of the initial column temperature on the peak shape and the separation efficiency of the PARs was also studied using DB1 and DB5 columns. Fronting or splitting of the peaks was obseIVed at very low initial column temperature. When high initial column temperature was used, tailing of the peaks appeared. Great difference between DB! and.DB5 columns in the range of the initial column temperature in which symmetrical.peaks of PAHs can be obtained is observed. Wider ranges were shown using DB5 column. Resolution of the closely-eluted PAHs was also affected by the initial column temperature depending on the stationary phase employed. In the case of DB5, only the earlyeluted PAHs were affected; whereas, with DB1, all PAHs were affected. An analytical procedure utilizing solid phase extraction with bonded phase silica (C8) cartridges combined with GC/MS was developed to analyze PAHs in water as an alternative method to those based on the extraction with organic solvent. This simple procedure involved passing a 50 ml of spiked water sample through C8 bonded phase silica cartridges at 10 ml/min, dried by passing a gentle flow of nitrogen at 20 ml/min for 30 sec, and eluting the trapped PAHs with 500 Jll of p-xylene at 0.3 ml/min. The recoveries of PAHs were greater than 80%, with less than 10% relative standard deviations of nine determinations. No major contaminants were present that could interfere with the recognition of PAHs. It was also found that these bonded phase silica cartridges can be re-used for the extraction of PAHs from water.

Reactions of polyhalogenated aromatic compounds and related ethers with metal/ammonia solutions

This work contains the results of a series of reduction studies on polyhalogenated aromatic compounds and related ethers using alkali metals in liquid ammonia. In general, polychlorobenzenes were reduced to t he parent aromatic hydrocarbon or to 1 ,4-cyc1ohexadiene, and dipheny1ethers were cleaved to the aroma tic hydrocarbon and a phenol. Chlorinated dipheny1ethers were r eductive1y dechlorinated in the process. For example, 4-chlorodipheny1- ether gave benzene and phenol. Pentach1orobenzene and certain tetrachlorobenzenes disproportionated to a fair degree during the reduction process if no added proton source was present. The disproportionation was attributed to a build-up of amide ion. Addition of ethanol completely suppressed the formation of any disproportionation products. In the reductions of certain dipheny1ethers , the reduction of one or both of the dipheny1ether rings occurred, along with the normal cleavage. This was more prevalent when lithium was the metal used . As a Sidelight, certain chloropheno1s were readily dechlorinated. In light of these results, the reductive detoxification of the chlorinated dibenzo-1,4-dioxins seems possible with alkali metals in l iquid ammonia.

Biotransformation of aromatic hydrocarbons and organic sulphides by fungi

Toluene is converted to benzyl alcohol by the fungi Mortierella isabellina and Helminthosporium species; in the latter case, the product is further metabolized. Toluene-a -d 1 , toluene-a,a-d2, and toluene-a,a,a-d 3 have been used with Mortierellaisabellina in a series of experiments to determine both primary and secondary deuterium kinetic isotope effects for the enzymic benzylic hydroxylation reaction. The values obtained, intermolecular primary kH/kD = intramolecular p rim a r y kH r kD = 1. 0 2 + O. 0 5, and sec 0 n dar y k H I kD = 1. 37 .:!. 0.05, suggest a mechanism for the reaction involving benzylic proton removal from a radical intermediate in a non-symmetrical transition state. 2H NMR (30.7 MHz) studies using ethylbenzene-l,1-d 2 , 3 -fluoroethylbenzene-l,1-d 2 , 4 -fluoroethylbenzene-l,1-d 2 , and toluene-dB as substrates with Mortierella isabellina suggest, based on the observable differences in rates of conversion between the substrates, that the hydroxylation of hydrocarbons at the benzylic position proceeds via a one electron abstraction from the aromatic ring, giving a radical cation. A series of 1,3-oxathiolanes (eight) were incubated with Mortierella isabellina , Helminthosporium , Rhizopus arrhizus , and Aspergillus niger . Sulphoxides were obtained from Mortierella isabellina and Rhizopus arrhizus using the substrates 2-phenyl-, 2-methyl-2-phenyl-, and 2-phenyl-2-tert. butyl-l,3-oxathiolane. The relative stereochemistry of 2-methyl-2-phenyl-l,3-oxathiolan-l-oxide was assigned based on lH decoupling, n.O.e, 1 and H NMR experiments. The lH NMR (200 MHz) of the methylene protons of 2-methyl-2-phenyl-l,3-oxathiolan-l-oxide was used as a diagnostic standard in assigning the relative stereochemistry of 2-phenyl-l,3-oxathiolan-l-oxide and 2-phenyl-2-tert. butyl-l,3-oxathiolan-l-oxide. The sulphoxides obtained were consistent with an oxidation occurring from the opposite side of the molecule to the phenyl substituent.