Biodegradation of cyanide compounds by a Pseudomonas species (S1).

A Pseudomonas sp. (S1), isolated from soil by an enrichment technique was tested for its potential to degrade different cyanide compounds. Further, biodegradation/biotransformation of binary mixtures of the cyanide compounds by the culture was also studied. The results indicated that the culture could grow on the following nitriles by using them as carbon and nitrogen sources: acetonitrile, butyronitrile, acrylonitrile, adiponitrile, benzonitrile, glutaronitrile, phenylacetonitrile, and succinonitrile. Studies on the biodegradation of these cyanide compounds in binary mixtures showed that the presence of acrylonitrile or KCN delayed the degradation of acetonitrile in a mixture, while none of the other cyanide compounds affected the degradation of one another. The transformation products of the nitriles were their corresponding acids, and similarly, KCN was also directly transformed to formic acid. Studies on the transformation of these cyanide compounds showed that the rate of transformation of nitriles to their corresponding carboxylic acids was acrylonitrile > acetonitrile > adiponitrile > benzonitrile > KCN. This culture has the unique characteristic of transforming representatives of saturated aliphatic, aliphatic olefinic, aromatic, and aralkyl nitriles, as well as alkali cyanide, to their corresponding carboxylic acids.     

Properties and biotechnological applications of halohydrin dehalogenases: current state and future perspectives

Halohydrin dehalogenases (HHDHs) are lyases that catalyze the cleavage of carbon–halogen bond of halohydrins. They also can catalyze the reverse reaction in the presence of nucleophiles such as cyanide, azide, and nitrite ions. HHDHs have been recognized as the ideal tools for the degradation of various halogenated environmental pollutants. Moreover, they can be used as biocatalysts for the kinetic resolution of halohydrins and epoxides, and for the preparation of various substituted alcohols. This review is mainly focused on the current status of research on HHDHs, highlighting the production, characterization, structures and mechanism, protein engineering, and biotechnological applications of HHDHs.     

Effect of energy metabolism inhibitors on the morphometric parameters and 3-dimensional structure of L-cell mitochondria

It has been shown by stereological analysis that the earlier discovered changes in the structure of mitochondria in cyanide treated L-cells (decrease in numerical density of mitochondria, increase in volume density of mitochondria, and surface density of mitochondrial membranes) are prevented by oligomycin, and they do not occur in the presence of oligomycin and protonophorous uncoupler carbonylcyanide m-chlorphenyl hydrazone applied separately. Proceeding from three-dimension reconstructed mitochondrial models it has been shown that cyanide treatment of L-cells for 23 hours causes a transformation of mitochondria as discrete column-like structures into a network of mitochondrial reticulum oriented from the nucleus to the periphery of the cell. After the treatment of L-cells with cyanide together with oligomycin, or with oligomycin and protonophore applied separately, the mitochondria retain the structure of discrete column-like for mations characteristic of the control cells. It is assumed that the functioning ATP-system is a physiological prerequisite of the formation of mitochondrial reticulum under conditions of the inhibited respiratory energy metabolism in the cell.     

The Effects of Selected Chemical Agents on the Amoeba-Flagellate Transformation in Naegleria gruberi*

SYNOPSIS. Amoeboid Naegleria gruberi grown on an agar surface were induced to transform synchronously into flagellates by changing the pH of the environment from 3.8 to 7.2. Flagellates started to appear 70 min after stimulation, reached the 90% level within the next 30–40 min, and gradually reverted to the amoeboid form in the next several hrs. The presence of previously induced cells did not influence normal induction of transformation, indicating that no interaction took place during transformation. Inhibitors of oxidative phosphorylation (DNP and cyanide), of protein synthesis (puromycin and cycloheximide), and of RNA synthesis (actinomycin D), delayed or blocked the transformation, suggesting that RNA and protein synthesis are required. Because the flagellated stage is shortened by puromycin treatment, protein synthesis appears to be linked to the duration of the flagellated stage.     

Redox reactions in hydrocortisone transformation by Arthrobacter globiformis cells

Hydrocortisone and prednisolone transformation by Arthrobacter globiformis cells in aerobic and anaerobic conditions was studied. 3-Ketosteroid-1-en-dehydrogenase activity was shown to be the major factor regulating the direction of transformation. When it is high (aerobic conditions), the end products of hydrocortisone transformation are prednisolone or its 20 beta-hydroxy derivative. The latter is produced via 1-en-dehydrogenation, which is not a limiting stage of the process. Low 3-ketosteroid-1-en-dehydrogenase activity (in the presence of cyanide) or its complete inhibition (strictly anaerobic conditions) result in the direct reduction of 20-keto group of hydrocortisone.     

Anaerobic biodegradation of cyanide under methanogenic conditions

Upflow, anaerobic, fixed-bed, activated charcoal biotreatment columns capable of operating at free cyanide concentrations of greater than 100 mg liter-1 with a hydraulic retention time of less than 48 h were developed. Methanogenesis was maintained under a variety of feed medium conditions which included ethanol, phenol, or methanol as the primary reduced carbon source. Under optimal conditions, greater than 70% of the inflow free cyanide was removed in the first 30% of the column height. Strongly complexed cyanides were resistant to removal. Ammonia was the nitrogen end product of cyanide transformation. In cell material removed from the charcoal columns, [14C]bicarbonate was the major carbon end product of [14C]cyanide transformation.     

The CO and CN(-) ligands to the active site Fe in [NiFe]-hydrogenase of Escherichia coli have different metabolic origins

The Fe atom in the bimetallic active site of [NiFe]-hydrogenases has one CO and two cyanide ligands. To determine their metabolic origin, [NiFe]-hydrogenase-2 was isolated from Escherichia coli grown in the presence of L-[ureido-(13)C]citrulline, purified and analyzed by infrared spectroscopy. The spectra indicate incorporation of (13)C only into the cyanide ligands and not into the CO, showing that cyanide and CO have different metabolic origins. After growth of E. coli in the presence of (13)CO only the CO ligand was labelled with (13)C. Labelling did not result from an exchange of the intrinsic CO ligand with the exogenous CO.     

Anaerobic biodegradation of cyanide under methanogenic conditions.

Upflow, anaerobic, fixed-bed, activated charcoal biotreatment columns capable of operating at free cyanide concentrations of greater than 100 mg liter-1 with a hydraulic retention time of less than 48 h were developed. Methanogenesis was maintained under a variety of feed medium conditions which included ethanol, phenol, or methanol as the primary reduced carbon source. Under optimal conditions, greater than 70% of the inflow free cyanide was removed in the first 30% of the column height. Strongly complexed cyanides were resistant to removal. Ammonia was the nitrogen end product of cyanide transformation. In cell material removed from the charcoal columns, [14C]bicarbonate was the major carbon end product of [14C]cyanide transformation.     

Evidence of hydrolytic route for anaerobic cyanide degradation.

Products observed during anaerobic cyanide transformation are consistent with a hydrolytic pathway (HCN + H2O <--> HCONH2 + H2O <--> HCOOH + NH3). Formate, the most frequently observed product, was generally converted to bicarbonate. Formamide was rapidly hydrolyzed to formate upon exposure to the anaerobic consortium but was not detected as an intermediate of cyanide transformation.     

Natural attenuation potential of cyanide via microbial activity in mine tailings

Biological removal by indigenous microflora of cyanide, contained in old (6-9 years) and fresh tailings (3 months), was studied in order to assess its natural attenuation potential via biodegradation. To investigate the presence of indigenous microflora in tailings, total heterotrophic and cyanide resistant bacteria were counted using the spread-plate method. The free cyanide mineralization potential was estimated using K14CN in the presence of various unlabeled cyanide concentrations (0, 5, and 10 mg CN/kg). The biodegradation of cyanide contained initially in the samples was also investigated by monitoring formate, formamide, ammonia and total cyanide (CNT) concentrations over 111 days. The enumeration of total heterotrophic and cyanide-resistant bacteria in old tailings showed an average population of 105 cfu/g. However, no growth was detected in fresh tailings. Nevertheless, cyanide mineralization tests indicated the presence, in both old and fresh tailings, of a cyanide-degrading microflora. In old tailings, maximum mineralization percentages of free cyanide ranging from 85% to 100% were obtained after 65 days at all concentrations tested. A mineralization percentage of 83% after 170 days was also observed in fresh tailings. No decrease of total cyanide concentration in old tailings was observed when the biodegradation of endogenous cyanide was tested whereas a significant decrease was recorded in fresh tailings after 96 days. The presence of strong metal-cyanide complexes resistant to biodegradation could explain the absence of biodegradation in old tailings. This study demonstrated the presence of an indigenous free cyanide-degrading microflora in both old and fresh tailings, and suggests that natural attenuation of cyanide in gold mine tailings is likely to occur via microbial activity.     

Chemical and biological removal of cyanides from aqueous and soil-containing systems

A study was conducted to determine the effect of various factors on the rate and extent of potassium cyanide and potassium hexacyanoferrate (II, complex form) removal from aqueous and soil-containing systems. In a sterile aqueous system at neutral pH, the concentration of free cyanide was reduced by 42% in 334 h as a result of the protonation of CN^– and the volatilization of the HCN formed. In the presence of aerobic mixed consortium of the Institute of Gas Technology and a methylotrophic culture, Isolate 3, the concentration of free cyanide was reduced by 59% and 66% in 357 h, respectively, as a result of combined chemical conversion and microbial degradation. In the sterile aqueous system amended initially with the complex form of cyanide, a less-than-20% reduction in cyanide occured. The sorption equilibria for free and complex cyanides in slurries of the topsoil and manufactured gas plant (MGP) soil was reached in less than 22 and 4 days, respectively. The extent of desorption of cyanides from topsoil and MGP soil into water decreased with time. In sterile systems containing topsoil and MGP soil that were previously equilibrated to cyanides, only a 2% reduction in cyanide concentration occurred in 336 h due to chemical conversion. In the presence of microbial cultures, the concentration of cyanide was reduced by less than 15% and 7% in the slurries of topsoil and MGP soil, respectively. The comparison of the rate and extent of cyanide removal from the aqueous and soil-containing systems in the presence of micro-organisms suggests that cyanides were retained by the solid phase of the soil-containing systems and therefore were less available for biodegradation.     

Archean geochemistry of formaldehyde and cyanide and the oligomerization of cyanohydrin

The sources and speciation of reduced carbon and nitrogen inferred for the early Archean are reviewed in terms of current observations and models, and known chemical reactions. Within this framework hydrogen cyanide and cyanide ion in significant concentration would have been eliminated by reaction with excess formaldehyde to form cyanohydrin (glycolonitrile), and with ferrous ion to form ferrocyanide. Natural reactions of these molecules would under such conditions deserve special consideration in modeling of primordial organochemical processes.As a step in this direction, transformation reactions have been investigated involving glycolonitrile in the presence of water. We find that glycolonitrile, formed from formaldehyde and hydrogen cyanide or cyanide ion, spontaneously cyclodimerizes to 4-amino-2-hydroxymethyloxazole. The crystalline dimer is the major product at low temperature (0 °C); the yield diminishes with increasing temperature at the expense of polymerization and hydrolysis products. Hydrolysis of glycolonitrile and of oxazole yields a number of simpler organic molecules, including ammonia and glycolamide. The spontaneous polymerization of glycolonitrile and its dimer gives rise to soluble, cationic oligomers of as yet unknown structure, and, unless arrested, to a viscous liquid, insoluble in water.A loss of cyanide by reaction with formaldehyde, inferred for the early terrestrial hydrosphere and cryosphere would present a dilemma for hypotheses invoking cyanide and related compounds as concentrated reactants capable of forming biomolecular precursor species. Attempts to escape from its horns may take advantage of the efficient concentration and separation of cyanide as solid ferriferrocyanide, and most directly of reactions of glycolonitrile and its derivatives.     

Activation by reduction of the resting form of cytochrome c oxidase: tests of different models and evidence for the involvement of CuB

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2.10(4) M-1.s-1. In the absence of cyanide, ferrocytochrome a3 appears at a rate (kobs) of 0.016 s-1. Ferricytochrome a3 maintains its 418 nm Soret maximum until reduced. The rate of a3 reduction is independent of dithionite concentration over a range 0.9 mM-131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a3/cyanide complex occurs at the same rate as a3 reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a3 with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a3 shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a3. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow (kobs approximately 0.02 s) entry of a third electron.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of the human soluble epoxide hydrolase gene (EPHX2) to chromosomal region 8p21-p12

Epoxide hydrolases have an important function in organisms in that they catalyze the transformation of potentially toxic or carcinogenic epoxides into the corresponding diols. In this study, the chromosomal localization was determined for the human gene encoding soluble epoxide hydrolase. A polymerase chain reaction fragment corresponding to the C-terminal region of the mouse protein was used to isolate a cosmid clone from a human genomic library. By fluorescence in situ hybridization to metaphase chromosomes, the soluble epoxide hydrolase gene was then localized to chromosomal region 8p21-p12.     

Dérivés C-glycosyliques : Partie XII. Élaboration de cycles furoxannes,isoxazoles et 2-isoxazoline par l'intermédiaire d'oxydes de nitrile dérivés de sucres

Treatment with a base of hydroxymoyl chlorides derived from sugars led to the corresponding unstable nitrile oxides which, in the absence of nucleophilic or dipolarophilic reagents, dimerize into furoxans. When nitrile oxides are formed in the presence of an excess of acetylenic (or olefinic) dipolarophilic reagents, they give 1,3-dipolar cycloaddition reactions with the formation of the corresponding 3-glycosylisoxazoles (or -2-isoxazolines). The action of nucleophilic reagents such as the alkaline cyanides or ethylmagnesium bromide allows the elongation of the sugar chain by one or two carbon atoms, respectively. The action of ethynylmagnesium bromide on these nitrile oxides gave a mixture of two compounds: an α-ethynyloxime and a 3-glycosylisoxazole; in alkaline medium the former compound can undergo cyclization and transformation into the latter, thus allowing deuterium labeling of that compound.     

Cyanide catabolizing enzymes in Trichoderma spp.

An investigation was made into the occurrence and distribution of the enzymes involved in HCN catabolism in different strains of the fungus Trichoderma. Three enzymes, cyanide hydratase, rhodanese and β-cyanoalanine synthase were studied. All the strains showed a high capacity to degrade cyanide via both the cyanide hydratase and rhodanese pathways. β-Cyanoalanine synthase, however, was not observed in any of the strains. The enzyme activities were found in varying levels in each of the Trichoderma strains. Experiments conducted with cyanide addition to the medium to assess whether the enzymes were induced in the presence of cyanide failed to show any statistically significant increase. This suggests a constitutive nature of both the enzymes in all the selected strains of Trichoderma used in this study.     

Evidence of hydrolytic route for anaerobic cyanide degradation.

Products observed during anaerobic cyanide transformation are consistent with a hydrolytic pathway (HCN + H2O <--> HCONH2 + H2O <--> HCOOH + NH3). Formate, the most frequently observed product, was generally converted to bicarbonate. Formamide was rapidly hydrolyzed to formate upon exposure to the anaerobic consortium but was not detected as an intermediate of cyanide transformation.     

Mutagenicity of Epoxides of Polycyclic Hydrocarbons correlates with Carcinogenicity of Parent Hydrocarbons

MANY chemical carcinogens are mutagenic¹ and some non-mutagenic carcinogens are metabolized to mutagenic derivatives^2,3. Recent work^4–6 has confirmed that epoxides are intermediates in the metabolism of the aromatic double bonds of carcinogenic polycyclic hydrocarbons to hydroxylated derivatives, as Boyland suggested⁷. In addition to chemical reactions with nucleic acids and histone⁸, epoxides derived from polycyclic hydrocarbons bind more extensively to the nucleic acids of cells in culture than the parent hydrocarbons⁹. Hydrocarbon epoxides are also more active in inducing malignant transformation in vitro of hamster embryo and mouse prostate cells¹⁰ although, in whole animals, they were less potent carcinogens than the hydrocarbons themselves^11–13. As potential mutagens, polycyclic hydrocarbon epoxides are therefore of particular interest, mainly because of the support positive results would give to the somatic mutation theory of carcinogenesis. In the work described here we have tested K-region epoxides of hydrocarbons for their ability to cause host range mutations of T₂h⁺ bacteriophage, specifically because there is no possibility, in this test system, of the epoxides being further metabolized. The epoxides tested were phenanthrene 9,10-oxide (Ph-E), benz(a)anthracene 5,6-oxide (BA-E), dibenz(a,h)anthracene 5,6-oxide (DBA-E), 7-methylbenz(a)anthracene 5,6-oxide (7-MeBA-E), 3-methylcholanthrene 11,12-oxide (MCA-E) and chrysene 5,6-oxide (Ch-E). Ethylene oxide and propylene oxide were used as examples of aliphatic epoxides which do not increase the frequency of host range mutants of T₂ bacteriophage and ethyl methanesulphonate (EMS) was used as a known mutagen¹⁴.     

Metabolism of urea in Chlorella ellipsoidea

The effect of cyanide on ammonia and urea metabolism was studiedwith intact cells of Chlorella ellipsoidea Gerneck, a greenalga which apparently lacks urease. Ammonia uptake was inhibited more readily by cyanide than wasurea uptake. Urea uptake was stimulated by lower concentrationsof cyanide. The addition of cyanide caused the formation ofammonia from some cellular nitrogenous compounds. In the presenceof exogenously added urea, the molar ratio of ammonia accumulatedin the medium to urea taken up exceeded 2.0 as the cyanide concentrationincreased. However, the molar ratio of ammonia actually producedfrom urea nitrogen to urea taken up was less than 1.35 at anyconcentration of cyanide tested. In the presence of higher concentrationsof cyanide, the rate of incorporation of ¹⁵N into amino acidsfrom ¹⁵N-urea was higher than that from ¹⁵N-ammonium sulfate. The results suggest that Chlorella ellipsoidea possesses a pathwaythrough which urea nitrogen is assimilated directly withouta preliminary breakdown to ammonia. (Received October 18, 1976; )     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The preparation and metabolism of epoxides related to 7,12-dimethylbenz[a]-anthracene

The syntheses of 7,12-dimethylbenz[a]anthracene 5,6-oxide, 7-acetoxymethyl-12-methylbenz[a]anthracene 5,6-oxide and a product that appears to be mainly 7-hydroxymethyl-12-methylbenz[a]anthracene 5,6-oxide are described. The compounds readily rearranged to phenols in the presence of mineral acid, and 7,12-dimethylbenz[a]anthracene 5,6-oxide and its 7-hydroxymethyl derivative reacted slowly with water to yield trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a] anthracene and trans-5,6-dihydro-5,6-dihydroxy-7-hydroxymethyl-12-methylbenz [a]anthracene respectively. Both epoxides were converted enzymically by rat liver microsomal fractions and homogenates into the related trans-dihydrodiols. The epoxides reacted chemically with GSH to form conjugates that were identical with the conjugates formed when the epoxides were incubated with rat liver homogenates. The GSH conjugates were more stable to acid than conjugates derived from other arene oxides. In the alkylation of 4-(p-nitrobenzyl)pyridine, 7,12-dimethyl-benz[a]anthracene 5,6-oxide was more active than the 5,6-oxides of 7-methylbenz[a]-anthracene and benz[a]anthracene.