Differential Degradation of Nonylphenol Isomers by Sphingomonas xenophaga Bayram

Sphingomonas xenophaga Bayram, isolated from the activated sludge of a municipal wastewater treatment plant, was able to utilize 4-(1-ethyl-1,4-dimethylpentyl)phenol, one of the main isomers of technical nonylphenol mixtures, as a sole carbon and energy source. The isolate degraded 1 mg of 4-(1-ethyl-1,4-dimethylpentyl)phenol/ml in minimal medium within 1 week. Growth experiments with five nonylphenol isomers showed that the three isomers with quaternary benzylic carbon atoms [(1,1,2,4-tetramethylpentyl)phenol, 4-(1-ethyl-1,4-dimethylpentyl)phenol, and 4-(1,1-dimethylheptyl)phenol] served as growth substrates, whereas the isomers containing one or two hydrogen atoms in the benzylic position [4-(1-methyloctyl)phenol and 4-n-nonylphenol] did not. However, when the isomers were incubated as a mixture, all were degraded to a certain degree. Differential degradation was clearly evident, as isomers with more highly branched alkyl side chains were degraded much faster than the others. Furthermore, the C₉ alcohols 2,3,5-trimethylhexan-2-ol, 3,6-dimethylheptan-3-ol, and 2-methyloctan-2-ol, derived from the three nonylphenol isomers with quaternary benzylic carbon atoms, were detected in the culture fluid by gas chromatography-mass spectrometry, but no analogous metabolites could be found originating from 4-(1-methyloctyl)phenol and 4-n-nonylphenol. We propose that 4-(1-methyloctyl)phenol and 4-n-nonylphenol were cometabolically transformed in the growth experiments with the mixture but that, unlike the other isomers, they did not participate in the reactions leading to the detachment of the alkyl moiety. This hypothesis was corroborated by the observed accumulation in the culture fluid of an as yet unidentified metabolite derived from 4-(1-methyloctyl)phenol.     

A novel metabolic pathway for degradation of 4-nonylphenol environmental contaminants by Sphingomonas xenophaga Bayram: ipso-hydroxylation and intramolecular rearrangement

Several nonylphenol isomers with alpha-quaternary carbon atoms serve as growth substrates for Sphingomonas xenophaga Bayram, whereas isomers containing hydrogen atoms at the alpha-carbon do not. Three metabolites of 4-(1-methyloctyl)-phenol were isolated in mg quantities from cultures of strain Bayram supplemented with the growth substrate isomer 4-(1-ethyl-1,4-dimethyl-pentyl)-phenol. They were unequivocally identified as 4-hydroxy-4-(1-methyl-octyl)-cyclohexa-2,5-dienone, 4-hydroxy-4-(1-methyl-octyl)-cyclohex-2-enone, and 2-(1-methyl-octyl)-benzene-1,4-diol by high pressure liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, two metabolites originating from 4-n-nonylphenol were identified as 4-hydroxy-4-nonyl-cyclohexa-2,5-dienone and 4-hydroxy-4-nonyl-cyclohex-2-enone by high pressure liquid chromatography-mass spectrometry. We conclude that nonylphenols were initially hydroxylated at the ipso-position forming 4-alkyl-4-hydroxy-cyclohexa-2,5-dienones. Dienones originating from growth substrate nonylphenol isomers underwent a rearrangement that involved a 1,2-C,O shift of the alkyl moiety as a cation to the oxygen atom of the geminal hydroxy group yielding 4-alkoxyphenols, from which the alkyl moieties can be easily detached as alcohols by known mechanisms. Dienones originating from nongrowth substrates did not undergo such a rearrangement because the missing alkyl substituents at the alpha-carbon atom prevented stabilization of the putative alpha-carbocation. Instead they accumulated and subsequently underwent side reactions, such as 1,2-C,C shifts and dihydrogenations. The ipso-hydroxylation and the proposed 1,2-C,O shift constitute key steps in a novel pathway that enables bacteria to detach alpha-branched alkyl moieties of alkylphenols for utilization of the aromatic part as a carbon and energy source.     

Elucidation of the ipso-substitution mechanism for side-chain cleavage of alpha-quaternary 4-nonylphenols and 4-t-butoxyphenol in Sphingobium xenophagum Bayram

Recently we showed that degradation of several nonylphenol isomers with alpha-quaternary carbon atoms is initiated by ipso-hydroxylation in Sphingobium xenophagum Bayram (F. L. P. Gabriel, A. Heidlberger, D. Rentsch, W. Giger, K. Guenther, and H.-P. E. Kohler, J. Biol. Chem. 280:15526-15533, 2005). Here, we demonstrate with 18O-labeling experiments that the ipso-hydroxy group was derived from molecular oxygen and that, in the major pathway for cleavage of the alkyl moiety, the resulting nonanol metabolite contained an oxygen atom originating from water and not from the ipso-hydroxy group, as was previously assumed. Our results clearly show that the alkyl cation derived from the alpha-quaternary nonylphenol 4-(1-ethyl-1,4-dimethyl-pentyl)-phenol through ipso-hydroxylation and subsequent dissociation of the 4-alkyl-4-hydroxy-cyclohexadienone intermediate preferentially combines with a molecule of water to yield the corresponding alcohol and hydroquinone. However, the metabolism of certain alpha,alpha-dimethyl-substituted nonylphenols appears to also involve a reaction of the cation with the ipso-hydroxy group to form the corresponding 4-alkoxyphenols. Growth, oxygen uptake, and 18O-labeling experiments clearly indicate that strain Bayram metabolized 4-t-butoxyphenol by ipso-hydroxylation to a hemiketal followed by spontaneous dissociation to the corresponding alcohol and p-quinone. Hydroquinone effected high oxygen uptake in assays with induced resting cells as well as in assays with cell extracts. This further corroborates the role of hydroquinone as the ring cleavage intermediate during degradation of 4-nonylphenols and 4-alkoxyphenols.     

Impact of bio-augmentation with Sphingomonas sp. strain TTNP3 in membrane bioreactors degrading nonylphenol

This study evaluates the potential of bio-augmentation to improve the degradation of recalcitrant nonylphenol during the wastewater treatment in membrane bioreactors (MBR). One MBR containing activated sludge was bio-augmented using multistep inoculation with freeze dried Sphingomonas sp. strain TTNP3, whereas a second control reactor contained activated sludge solely. The ¹⁴C-labeled-nonylphenol isomer (4-[1-ethyl-1,3-dimethylpentyl]phenol) was applied as a single pulse. Bio-augmentation resulted in an immediate increase of dissolved radioactivity in the effluent in comparison to the control reactor (13% and 2% of initially applied radioactivity after 1 day, respectively). After 5 days of operation, the retentate of the bio-augmented reactor contained only 7% of the initial radioactivity in contrast to 50% in the control reactor. The radioactivity associated to the mixed liquor suspended solids, i.e., the suspension of biomass and other solids on the retentate side of the membrane, was mainly found as non-extractable residues that were increasingly formed during prolonged reactor operation, especially for the control MBR. HPLC-LSC and GC-MSⁿ analyses revealed that the bio-augmented reactor produced more polar hydroquinone as main degradation intermediate, whereas the control reactor effluent contained a complex mixture of apolar compounds with shortened oxidized alkyl chains. Thus, the apparent differences in the behavior of nonylphenol between the reactors were due to the catabolism of nonylphenol conferred by bio-augmentation with Sphingomonas sp. strain TTNP3. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Elucidation of the ipso-Substitution Mechanism for Side-Chain Cleavage of α-Quaternary 4-Nonylphenols and 4-t-Butoxyphenol in Sphingobium xenophagum Bayram

Recently we showed that degradation of several nonylphenol isomers with α-quaternary carbon atoms is initiated by ipso-hydroxylation in Sphingobium xenophagum Bayram (F. L. P. Gabriel, A. Heidlberger, D. Rentsch, W. Giger, K. Guenther, and H.-P. E. Kohler, J. Biol. Chem. 280:15526-15533, 2005). Here, we demonstrate with ¹⁸O-labeling experiments that the ipso-hydroxy group was derived from molecular oxygen and that, in the major pathway for cleavage of the alkyl moiety, the resulting nonanol metabolite contained an oxygen atom originating from water and not from the ipso-hydroxy group, as was previously assumed. Our results clearly show that the alkyl cation derived from the α-quaternary nonylphenol 4-(1-ethyl-1,4-dimethyl-pentyl)-phenol through ipso-hydroxylation and subsequent dissociation of the 4-alkyl-4-hydroxy-cyclohexadienone intermediate preferentially combines with a molecule of water to yield the corresponding alcohol and hydroquinone. However, the metabolism of certain α,α-dimethyl-substituted nonylphenols appears to also involve a reaction of the cation with the ipso-hydroxy group to form the corresponding 4-alkoxyphenols. Growth, oxygen uptake, and ¹⁸O-labeling experiments clearly indicate that strain Bayram metabolized 4-t-butoxyphenol by ipso-hydroxylation to a hemiketal followed by spontaneous dissociation to the corresponding alcohol and p-quinone. Hydroquinone effected high oxygen uptake in assays with induced resting cells as well as in assays with cell extracts. This further corroborates the role of hydroquinone as the ring cleavage intermediate during degradation of 4-nonylphenols and 4-alkoxyphenols.     

Degradation pathway of bisphenol A: does ipso substitution apply to phenols containing a quaternary alpha-carbon structure in the para position?

The degradation of bisphenol A and nonylphenol involves the unusual rearrangement of stable carbon-carbon bonds. Some nonylphenol isomers and bisphenol A possess a quaternary alpha-carbon atom as a common structural feature. The degradation of nonylphenol in Sphingomonas sp. strain TTNP3 occurs via a type II ipso substitution with the presence of a quaternary alpha-carbon as a prerequisite. We report here a new degradation pathway of bisphenol A. Consequent to the hydroxylation at position C-4, according to a type II ipso substitution mechanism, the C-C bond between the phenolic moiety and the isopropyl group of bisphenol A is broken. Besides the formation of hydroquinone and 4-(2-hydroxypropan-2-yl)phenol as the main metabolites, further compounds resulting from molecular rearrangements consistent with a carbocationic intermediate were identified. Assays with resting cells or cell extracts of Sphingomonas sp. strain TTNP3 under an (18)O(2) atmosphere were performed. One atom of (18)O(2) was present in hydroquinone, resulting from the monooxygenation of bisphenol A and nonylphenol. The monooxygenase activity was dependent on both NADPH and flavin adenine dinucleotide. Various cytochrome P450 inhibitors had identical inhibition effects on the conversion of both xenobiotics. Using a mutant of Sphingomonas sp. strain TTNP3, which is defective for growth on nonylphenol, we demonstrated that the reaction is catalyzed by the same enzymatic system. In conclusion, the degradation of bisphenol A and nonylphenol is initiated by the same monooxygenase, which may also lead to ipso substitution in other xenobiotics containing phenol with a quaternary alpha-carbon.     

Quantification of the Influence of Extracellular Laccase and Intracellular Reactions on the Isomer-Specific Biotransformation of the Xenoestrogen Technical Nonylphenol by the Aquatic Hyphomycete Clavariopsis aquatica

The aquatic hyphomycete Clavariopsis aquatica was used to quantify the effects of extracellular laccase and intracellular reactions on the isomer-specific biotransformation of technical nonylphenol (t-NP). In laccase-producing cultures, maximal removal rates of t-NP and the isomer 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP112) were about 1.6- and 2.4-fold higher, respectively, than in laccase-lacking cultures. The selective suppression of either laccase or intracellular reactions resulted in essentially comparable maximal removal rates for both compounds. Evidence for an unspecific oxidation of t-NP isomers was consistently obtained from laccase-expressing fungal cultures when intracellular biotransformation was suppressed and from reaction mixtures containing isolated laccase. This observation contrasts with the selective degradation of t-NP isomers by bacteria and should prevent the enrichment of highly estrogenic isomers in remaining t-NP. In contrast with laccase reactions, intracellular fungal biotransformation caused a significant shift in the isomeric composition of remaining t-NP. As a result, certain t-NP constituents related to more estrogenic isomers were less efficiently degraded than others. In contrast to bacterial degradation via ipso-hydroxylation, the substitution pattern of the quaternary α-carbon of t-NP isomers does not seem to be very important for intracellular transformation in C. aquatica. As-yet-unknown intracellular enzymes are obviously induced by nonylphenols. Mass spectral data of the metabolites resulting from the intracellular oxidation of t-NP, NP112, and 4-(1-ethyl-1,3-dimethylpentyl)phenol indicate nonyl chain hydroxylation, further oxidation into keto or aldehyde compounds, and the subsequent formation of carboxylic acid derivatives. Further metabolites suggest nonyl chain desaturation and methylation of carboxylic acids. The phenolic moieties of the nonylphenols remained unchanged.Nonylphenol ethoxylates (NPEOs) represent a major group of industrial nonionic surfactants. Technical nonylphenol (t-NP), used for the production of NPEOs, is synthesized by Friedel-Crafts alkylation of phenol with a mixture of differently branched nonenes. It therefore comprises a great variety of mainly para-substituted isomers, with variously branched nonyl chains. About 50 to 80 t-NP isomers were estimated to occur in environmentally relevant matrices (19). The incomplete bioconversion of NPEOs in wastewater treatment plants results in the formation of the less biodegradable t-NP and is considered a major source of this contaminant in the aquatic environment (57). The recalcitrance of t-NP to biodegradation is partly due to the presence in more than 85% of the t-NP isomers of a quaternary α-carbon in the branched nonyl chain. Such structural characteristics are considered to limit biological nonyl chain oxidation (11, 53, 55). Nonylphenols are known to disrupt normal endocrine functions in vertebrates (57). Certain isomers contained in t-NP have been reported to possess a considerably higher estrogenic activity than the t-NP mixture (15). Due to increasing concerns with respect to their largely unknown environmental fate and potentially adverse environmental and human health effects, nonylphenols have been listed as priority hazardous substances in the EU water framework directive.In light of the concerns above, microbial reactions with the potential to reduce nonylphenol concentrations in the environment but also offering new possibilities for applications such as effluent treatment have received increasing attention (11). Among environmental microorganisms, both aquatic and terrestrial fungi, as well as bacteria, have been shown to degrade t-NP (11). Fungal attack on nonylphenols differs from bacterial nonylphenol degradation. In the case of intracellular nonylphenol biotransformation reactions catalyzed by fungi, only metabolites modified in the alkyl chain have been described (23, 52). Metabolites indicative of oxidation of the phenolic ring have not been described to date. Bacterial degradation pathways have only been documented in the genera Sphingomonas and Sphingobium. Bacterial mineralization of the aromatic moiety of t-NP isomers to CO₂ and H₂O is initiated via ring hydroxylation at the ipso (C-4) position of the phenolic ring, and nonanols are produced from the nonyl chains (10, 11, 15, 16). Bacteria have been shown to utilize branched-chain nonylphenols as growth substrates (11, 12, 17, 43). In contrast, only one report describes the growth of a fungus, the yeast Candida aquaetextoris, on nonylphenol (the isomer 4-n-NP containing a linear nonyl chain) (52). With respect to fungal attack on t-NP, cometabolism seems to be the dominating process (11). Recent literature data indicate that certain t-NP isomers with an estrogenic potency higher than those of the original t-NP mixture can be enriched in remaining t-NP. This results from the selective removal of individual isomers upon bacterial ipso-substitution degradation mechanisms (15). However, the effects of fungal biotransformation reactions on the isomeric profile of t-NP have not yet been quantified.Laccases are extracellular multicopper oxidases. These have most frequently been described in white-rot basidiomycetes, which unspecifically oxidize via one-electron abstraction certain lignin constituents, as well many xenobiotic compounds. Thereby, organic radicals are generated as the primary oxidation products (3). Among the several groups of fungi found in aquatic environments, aquatic hyphomycetes (AQH) are a phylogenetically diverse group of mitosporic fungi specifically adapted to their exclusively aquatic lifestyle. AQH have been shown to metabolize several organic environmental pollutants, including t-NP (23), polycyclic musk fragrances (31), pesticide metabolites (2), and synthetic dyes (22). Therefore, with respect to the fungal attack on organic pollutants found in aquatic ecosystems, AQH are of special importance. Laccase production by strictly aquatic fungi such as AQH has already been demonstrated and discussed in the context of lignocellulose decay in aquatic ecosystems (1). A role of this enzyme in the AQH-catalyzed breakdown of aquatic environmental pollutants has been recently suggested. Here, laccase isolated from the AQH Clavariopsis aquatica was shown to act on nonylphenol (23) and polycyclic musk fragrances (31). Laccase has also been implicated in nonylphenol degradation by white-rot fungi (44, 45). Isolated extracellular laccases from several aquatic and terrestrial fungi were shown to catalyze the formation of oligomeric coupling products from nonylphenols via organic radical intermediates (6, 11). However, the effects of laccase reactions on the isomeric patterns of t-NP have not been assessed to date.The aim of the present study was to quantify the influence of extracellular laccase catalysis and intracellular biotransformation on nonylphenol removal rates and on the isomeric composition of t-NP. For this, C. aquatica was used as a model organism. The derived data were compared to effects of bacteria on nonylphenol isomers reported by other authors (15), and environmental and biotechnological implications of fungal t-NP biotransformation were deduced. At the same time we addressed metabolite formation from t-NP and the two major t-NP isomers 4-(1-ethyl-1,3-dimethylpentyl)phenol (NP111) and 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP112) (Fig. ​(Fig.1).1). This was done to substantiate the apparent differences between fungi and bacteria in the intracellular oxidation of t-NP (11, 15).Open in a separate windowFIG. 1.Chemical structures of the nonylphenol isomers NP111 and NP112.     

Degradation Pathway of Bisphenol A: Does ipso Substitution Apply to Phenols Containing a Quaternary α-Carbon Structure in the para Position?

The degradation of bisphenol A and nonylphenol involves the unusual rearrangement of stable carbon-carbon bonds. Some nonylphenol isomers and bisphenol A possess a quaternary α-carbon atom as a common structural feature. The degradation of nonylphenol in Sphingomonas sp. strain TTNP3 occurs via a type II ipso substitution with the presence of a quaternary α-carbon as a prerequisite. We report here a new degradation pathway of bisphenol A. Consequent to the hydroxylation at position C-4, according to a type II ipso substitution mechanism, the C-C bond between the phenolic moiety and the isopropyl group of bisphenol A is broken. Besides the formation of hydroquinone and 4-(2-hydroxypropan-2-yl)phenol as the main metabolites, further compounds resulting from molecular rearrangements consistent with a carbocationic intermediate were identified. Assays with resting cells or cell extracts of Sphingomonas sp. strain TTNP3 under an ¹⁸O₂ atmosphere were performed. One atom of ¹⁸O₂ was present in hydroquinone, resulting from the monooxygenation of bisphenol A and nonylphenol. The monooxygenase activity was dependent on both NADPH and flavin adenine dinucleotide. Various cytochrome P450 inhibitors had identical inhibition effects on the conversion of both xenobiotics. Using a mutant of Sphingomonas sp. strain TTNP3, which is defective for growth on nonylphenol, we demonstrated that the reaction is catalyzed by the same enzymatic system. In conclusion, the degradation of bisphenol A and nonylphenol is initiated by the same monooxygenase, which may also lead to ipso substitution in other xenobiotics containing phenol with a quaternary α-carbon.     

Phenol--another cockchafer attractant shared by Melolontha hippocastani Fabr. and M. melolontha L

The response of the two most abundant cockchafer species in central Europe, Melolontha hippocastani and M. melolontha, towards phenol, mixtures of phenol with the leaf alcohol (Z)-3-hexen-1-ol and the known cockchafer pheromones, 1,4-benzoquinone (M. hippocastani) and toluquinone (M. melolontha), was investigated in the field. During the swarming period at dusk, phenol attracted males of both species, and enhanced the known attraction of cockchafer males towards (Z)-3-hexen-1-ol. A mixture of phenol plus (Z)-3-hexen-1-ol was less attractive for M. hippocastani males than a mixture of (Z)-3-hexen-1-ol plus 1,4-benzoquinone, whereas phenol plus (Z)-3-hexen-1-ol attracted as many M. melolontha males as a mixture of (Z)-3-hexen-1-ol plus toluquinone. In both species three component mixtures containing phenol, (Z)-3-hexen-1-ol, and the respective benzoquinone did not capture more males than two component mixtures consisting of only (Z)-3-hexen-1-ol and the benzoquinone. A possible role of phenol as another cockchafer sex pheromone component is discussed.     

Biodegradation of commercial linear alkyl benzenes byNocardia amarae

Laboratory degradation studies of two indigeneously produced linear alkyl benzenes byNocardia amarae MB-11 isolated from soil showed an overall degradation of linear alkyl benzenes isomers to the extent of 57–70%. Degradation of 2-phenyl isomers of linear alkyl benzenes was complete and faster than that of other phenyl position (C3–C7) isomers which were degraded to the extent of 40–72% only. Length of alkyl side chains (C₁₀–C₁₄) had little or no impact on the degradation pattern. Major metabolities detected were 2-, 3-and 4-phenyl butyric acids, phenyl acetic acid and cis, cis-muconic acid. Minor metabolites weretrans-cinnamic acid, 4-phenyl 3-butenoic acid and 3-phenyl pentanoic acid along with two unidentified hydroxy acids. On the basis of the formation pattern of these metabolities, three catabolic pathways of linear alkyl benzenes isomers inNocardia amarae MB-11 were postulated. All the phenyl position (C2–C7) isomers of C₁₀, C₁₂, and C₁₄ linear alkyl benzenes along with 3-phenyl and 5-phenyl isomers of C₁₁ and C₁₃ linear alkyl benzenes were degraded viacis,cis-muconic acid pathway. Other phenyl position isomers of C₁₁ and C₁₃ linear alkyl benzenes with phenyl substitution at even number carbon atoms were principally degraded via phenyl acetic acid pathway whiletrans-cinnamic acid formation provided a minor pathway     

Essential carboxyl residues in yeast enolase.

Yeast enolase (EC 4.2.1.11) is rapidly inactivated at pH 6.1 by three different water-soluble carbodiimides — 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, and 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide. Inactivation is most likely due to the modification of essential carboxyl residues at the enzyme active site.     

Sulfation of "estrogenic" alkylphenols and 17beta-estradiol by human platelet phenol sulfotransferases

We have investigated the ability of alkylphenols to act as substrates and/or inhibitors of phenol sulfotransferase enzymes in human platelet cytosolic fractions. Our results indicate: (i) straight chain alkylphenols do not interact with the monoamine-sulfating phenol sulfotransferase (SULT1A3); (ii) short chain 4-n-alkylphenols (C < 8) are substrates for the phenol-sulfating enzymes (SULT1A1/2), which exhibit two activity maxima against substrates with alkyl chain lengths of C1-2 and C4-5; (iii) long chain 4-n-substituted alkylphenols (C >/= 8) are poor substrates and act as inhibitors of SULT1A1/2; (iv) human platelets contain two activities, of low and high affinity, capable of sulfating 17beta-estradiol, and 4-n-nonylphenol is a partial mixed inhibitor of the low affinity form of this activity. We conclude that by acting either as substrates or inhibitors of SULT1A1/2, alkylphenols may influence the sulfation, and hence the excretion, of estrogens and other phenol sulfotransferase substrates in humans.     

Degradation of the Radioactive and Non-labelled Branched 4(3',5'-dimethyl 3'-heptyl)-phenol Nonylphenol Isomer by Sphingomonas TTNP3

The degradation of the 4(3',5'-dimethyl-3'-heptyl)-phenol (p353NP) nonylphenol isomer in cultures of Sphingomonas TTNP3 supplemented with the technical mixture of nonylphenol was first assessed. Then the radioactive and non-labelled form of these diastereomers were both synthesised. The radioactive isomers were synthesised using [ring-U-14C]-labelled phenol and 3,5-dimethyl-3-heptanol by Friedel and Crafts alkylation. The time-course of degradation was performed with and without 14C-p353NP; balancing of radioactivity was calculated from different soluble fractions (organic, aqueous), bacterial biomass, and 14CO2 evolved as mineralization product. The noticeable portion of 14C bound to biomass showed that at least the aromatic ring of 14C-p353NP was degraded and served as energy source and probably as carbon source for bacterial growth. In addition, the appearance of 3,5-dimethyl-3-heptanol, the nonanol corresponding with the side-chain of p353NP, was demonstrated in the bacterial media, and its concentration determined during the course of fermentation. Besides the parent 14C-p353NP, no other radioactive compounds, i.e. metabolites of 14C-p353NP were detected in the media.     

The specificities and configurations of ternary complexes of yeast and liver alcohol dehydrogenases

1. Some aspects of the substrate specificities of liver and yeast alcohol dehydrogenases have been investigated with pentan-3-ol, heptan-4-ol, (+)-butan-2-ol, (+/-)-butan-2-ol, (+/-)-hexan-3-ol and (+/-)-octan-2-ol as potential substrates. The liver enzyme is active with all substrates tested, including both isomers of each optically active alcohol. In contrast, the yeast enzyme is completely inactive towards those secondary alcohols where both alkyl groups are larger than methyl and active with only the (+)-isomers of butan-2-ol and octan-2-ol. 2. The absence of stereospecificity of liver alcohol dehydrogenase towards optically active secondary alcohols and its broad specificity towards secondary alcohols in general are explained in terms of an alkyl-binding site that will react with a variety of alkyl groups and the ability of the enzyme to accommodate a fairly large unbound alkyl group in an active enzyme-NAD(+)-secondary alcohol ternary complex. The absolute optical specificity of the yeast enzyme towards n-alkylmethyl carbinols and its unreactivity towards pentan-3-ol, hexan-3-ol and heptan-4-ol are explained by its inability to accept alkyl groups larger than methyl in the unbound position in a viable ternary complex. 3. Comparison of the known configurations of the n-alkylmethyl carbinols and [1-(2)H]ethanol and [1-(3)H]geraniol, which have been used in stereospecificity studies with these enzymes by other workers, provides strong evidence for which alkyl group of the substrate is bound to the enzyme in the oxidation of n-alkylmethyl carbinols. The conclusions reached are, for butan-2-ol oxidation with the liver enzyme, confirmed by deductions from kinetic data obtained with (+)-butan-2-ol and a sample of butan-2-ol containing 66% of (-)-butan-2-ol. 4. Initial-rate parameters for the oxidations of (+)-butan-2-ol, 66% (-)-butan-2-ol and pentan-3-ol by NAD with liver alcohol dehydrogenase are presented. The data are completely consistent with a general mechanism of catalysis previously proposed for this enzyme.     

The effect of concentration of phenols on their batch methanogenesis

The biodegradability of phenol and six other phenolic compounds (o-, m-, and p-cresol, 2-, 3-, and 4-ethylphenol) was examined in batch methanogenic cultures. The effect of concentration of these alkyl phenols on the anaerobic biodegradation of phenol was also evaluated. The inoculum used in this study was cultivated in a continuous flow laboratory fermenter with phenol as the primary substrate. Phenol, at initial concentrations as high to 1400 mg/L was completely degraded to methane and carbondioxide after 350 hours incubation. Complete degradation of m- and p-cresol was also observed while the ethylphenols and o-cresol were not significantly degraded.At initial concentrations exceeding 600 mg/L, phenol inhibited the phenol-degrading microorganisms but not the methanogens. At about 600 mg/L, cresols reduced the rate of phenol degradation to 50% of that observed in a control culture containing only 200 mg/L phenol. Ethylphenols were more inhibitory than cresols. Phenol degrading microorganisms were more susceptible to inhibition by cresols and ethylphenols than were the methanogens. The inhibitory effects of the three isomers of cresol and ethylphenol did not vary with the isomer but rather with the substituted functional group.     

Long-chain and very long-chain methyl-branched alcohols and their acetate esters in pupae of the tobacco hornworm,Manduca sexta

Four homologous series of very long-chain methyl-branched alcohols (VLMA, C₃₈ to >C₄₄) were found in the internal lipids of developing male pupae of the tobacco hornworm, Manduca sexta, both as free alcohols and as acetate esters. The four major homologous series, with carbon chain backbones of 36–44 carbon atoms, consisted of a monomethyl, two dimethyls and a trimethyl-branched alcohol series. The major alcohol of each homologous series (with the corresponding alkane obtained by reduction in parentheses) was identified as 24-methyltetracontan-1-ol (17-methyltetracontane), 24,28-dimethyltetracontan-1-ol (13,17-dimethyltetracontane), 22,34-dimethyloctatriacontan-1-ol (5,17-dimethyloctatriacontane) and 22,26,34-trimethyloctatriacontan-1-ol (5,13,17-trimethyloctatriacontane). The minor components of the VLMA had backbones with an odd number of carbon atoms (37, 39, 41 and 43). Methyl branches of the minor components were identified on the 18- and 14,18-positions when numbered from the alkyl end of the chain. Also identified were minor amounts of long-chain methyl-branched alcohols (LMA, C₂₅ to C₃₂). The major components in the “wax ester” TLC fraction were acetate esters of the LMA and VLMA.     

Responses of individual antennal olfactory cells of tsetse flies (Glossina m. morsitans) to phenols from cattle urine

Abstract. Action potentials from olfactory cells in antennae (funiculi) of living tsetse flies, Glossina morsitans morsitans Westwood, were recorded using a surface-contact technique. Stimuli were the vapours of the seven alkylphenols identified in cattle urine: phenol, 3-methyl-, 3-ethyl-, 3- n -propyl-, 4-methyl-, 4-ethyl-, and 4- n -propylphenol. In addition, responses to the vapours of 1-octen-3-ol, acetone and dichloromethane were recorded. The phenol-sensitive cells could be grouped into four subclasses. Subclass, 1, 2 and 3 cells responded to the phenols only, cells of subclass 1 and 2 being activated by these substances, those of subclass 3 inhibited. Cells of subclass 4 were activated to a similar degree by all phenols and by one or more of the other chemicals tested. Subclass 1 cells were strongly activated by the 3-alkylphenols, whereas subclass 2 cells were most sensitive to 4-methylphenol. Subclass 3 cells were most strongly inhibited by phenol, and 3- and 4-methylphenol. The results suggest that though individual phenols may be attractive to G. m. morsitans , preference for certain blends of phenols may exist; for example, blends composed of moderate doses of 4-methylphenol and 3-methyl-, 3-ethyl- or 3- n -propylphenol.     

Degradation of 4-<Emphasis Type="Italic">n</Emphasis>-nonylphenol under nitrate reducing conditions

Nonylphenol (NP) is an endocrine disruptor present as a pollutant in river sediment. Biodegradation of NP can reduce its toxicological risk. As sediments are mainly anaerobic, degradation of linear (4-n-NP) and branched nonylphenol (tNP) was studied under methanogenic, sulphate reducing and denitrifying conditions in NP polluted river sediment. Anaerobic bioconversion was observed only for linear NP under denitrifying conditions. The microbial population involved herein was further studied by enrichment and molecular characterization. The largest change in diversity was observed between the enrichments of the third and fourth generation, and further enrichment did not affect the diversity. This implies that different microorganisms are involved in the degradation of 4-n-NP in the sediment. The major degrading bacteria were most closely related to denitrifying hexadecane degraders and linear alkyl benzene sulphonate (LAS) degraders. The molecular structures of alkanes and LAS are similar to the linear chain of 4-n-NP, this might indicate that the biodegradation of linear NP under denitrifying conditions starts at the nonyl chain. Initiation of anaerobic NP degradation was further tested using phenol as a structure analogue. Phenol was chosen instead of an aliphatic analogue, because phenol is the common structure present in all NP isomers while the structure of the aliphatic chain differs per isomer. Phenol was degraded in all cases, but did not affect the linear NP degradation under denitrifying conditions and did not initiate the degradation of tNP and linear NP under the other tested conditions.     

Inactivation of bovine thrombin by water-soluble carbodiimides: the essential carboxyl group has a pKa of 5.51

Bovine thrombin is rapidly and completely (greater than 99%) inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in a pseudo-first-order process. A plot of the pseudo-first-order rate constant for inactivation by 20 mM EDC at different pH values from pH 4.0 to 7.7 at 25 degrees C shows that inactivation is critically dependent on the protonated form of an acidic side chain with a pKa of 5.51. Significant protection against inactivation is provided by the competitive inhibitor dansyl-L-arginine N-(3-ethyl-1,5-pentanediyl)amide, suggesting that the essential carboxyl group may be involved in substrate binding. 1-Ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) inactivates thrombin much more rapidly than EDC under the same conditions.     

Optimized trap lure for male Melolontha cockchafers

Abstract:  Melolontha cockchafer males search for mates using green leaf volatiles (GLV), released by host plants after female feeding. Thus, the feeding-induced plant volatiles act as sexual kairomones. Males of both Melontha hippocastani and Melontha melolontha are strongly attracted by the GLV ( Z )-3-hexen-1-ol ( Z -3-ol). Sex pheromones enhance the attractiveness of Z -3-ol and have been identified as toluquinone (TQ) in M. melolontha , and 1,4-benzoquinone (BQ) in M. hippocastani . Additionally, phenol acts as a male attractant in both species. From the perspective of potential application, we investigated by field experiments with volatile-baited traps the ways of enhancing the number of captured males by the use of specific binary or ternary blends of Z -3-ol with phenol, and TQ or BQ respectively. The data show that in both species binary lures containing Z -3-ol combined with TQ or BQ at a ratio of 10 : 1 are the most potent male attractants.