Purification and characterization of the N-methylcarbamate hydrolase from Pseudomonas strain CRL-OK.

A unique cytosolic enzyme that hydrolyzes the carbamate linkage of the insecticide carbaryl (1-naphthyl N-methylcarbamate) was purified from extracts of Pseudomonas sp. strain CRL-OK. Substrates of the hydrolase include the N-methylcarbamate pesticides carbofuran and aldicarb but not the phenylcarbamate isopropyl m-chlorocarbanilate, the thiocarbamate S-ethyl N,N-dipropylthiocarbamate, or the dimethylcarbamate o-nitrophenyldimethylcarbamate.     

The kinetics of inhibition of erythrocyte cholinesterase by monomethylcarbamates

1. The kinetics of the interaction of erythrocyte cholinesterase with 1-naphthyl N-methylcarbamate, 2-isopropoxyphenyl N-methylcarbamate and phenyl N-methylcarbamate were studied. Rate constants for inhibition and rate constants for spontaneous reactivation were determined. The calculated rate constants for spontaneous reactivation agreed well with those obtained experimentally. 2. The degree of inhibition obtained after preincubation of enzyme and inhibitor was found to be independent of both the substrate concentration and the dilution of the inhibited enzyme. 3. The reaction between the enzyme and the inhibitor was consistent with carbamates being regarded as poor substrates of cholinesterases. There was no evidence for the formation of a reversible complex between the enzyme and the carbamate.     

Metabolism of 3,4-Dimethylphenyl N-Methylcarbamate in White Rats

Orally administered 3,4-dimethylphenyl N-methylcarbamate labelled with carbon-14 at 4-CH₃ was easily absorbed from the gastrointestinal tract of male Wistar rats and distributed into the tissues. Elimination of the radioactivity was rapid and essentially complete; namely during 48 hr approximately 92% and 5% of the total radioactivity were excreted respectively into urine and feces. The content of the intact carbamate compound in the urine was less than 0.5%. Major degradation products were identified as 3-methyl-4-carboxyphenyl N-methylcarbamate, its N-hydroxymethyl analog and its component phenol. Much less amount of direct hydrolysis product of the original carbamate, 3,4-dimethylphenol and its conjugated forms was demonstrated. 3,4-Dimethylphenyl N-methylcarbamate is presumed to undergo biodegradation through oxidative pathways.     

Involvement of two plasmids in the degradation of carbaryl by Arthrobacter sp. strain RC100

A bacterium capable of utilizing carbaryl (1-naphthyl N-methylcarbamate) as the sole carbon source was isolated from carbaryl-treated soil. This bacterium was characterized taxonomically as Arthrobacter and was designated strain RC100. RC100 hydrolyzes the N-methylcarbamate linkage to 1-naphthol, which was further metabolized via salicylate and gentisate. Strain RC100 harbored three plasmids (designated pRC1, pRC2, and pRC3). Mutants unable to degrade carbaryl arose at a high frequency after treating the culture with mitomycin C. All carbaryl-hydrolysis-deficient mutants (Cah-) lacked pRC1, and all 1-naphthol-utilization-deficient mutants (Nat-) lacked pRC2. The plasmid-free strain RC107 grew on gentisate as a carbon source. These two plasmids could be transferred to Cah- mutants or Nat- mutants by conjugation, resulting in the restoration of the Cah and Nah phenotypes.     

The metabolism of 3,5-di-tert.-butylphenyl N-methylcarbamate in insects and by mouse liver enzymes

The oxidation of 3,5-di-tert.-butylphenyl N-methylcarbamate (Butacarb) has been studied in the flies Musca domestica and Lucilia sericata, grass grubs Costelytra zealandica and the mouse. In all species eleven oxidation products, which were formed by hydroxylation of the tert.-butyl groups and the N-methyl group, were detected.     

Cloning of a carbofuran hydrolase gene from Achromobacter sp. strain WM111 and its expression in gram-negative bacteria.

A 14-kilobase-pair (kbp) EcoRI DNA fragment that encodes an enzyme capable of rapid hydrolysis of N-methylcarbamate insecticides (carbofuran hydrolase) was cloned from carbofuran-degrading Achromobacter sp. strain WM111. When used to probe Southern blots containing plasmid and total DNAs from WM111, this 14-kbp fragment hybridized strongly to a 14-kbp EcoRI fragment from the greater than 100-kbp plasmid harbored by this strain but weakly to EcoRI-digested total DNA from Achromobacter sp. strain WM111, indicating that the gene for N-methylcarbamate degradation (mcd) is plasmid encoded. Further subcloning localized the mcd gene on a 3-kbp ScaI-ClaI fragment. There was little or no expression of this gene in the alternative gram-negative hosts Pseudomonas putida, Alcaligenes eutrophus, Acinetobacter calcoaceticus, and Achromobacter pestifer. Western blotting (immunoblotting) of the protein products produced by low-level expression in P. putida confirmed that this 3-kbp fragment encodes the two 70+-kilodalton protein products seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified carbofuran hydrolase.     

Rates of dissolution and biodegradation of water-insoluble organic compounds.

We conducted a study of the relationship between the dissolution rates of organic compounds that are sparingly soluble in water and the biodegradation of these compounds by mixed cultures of bacteria. The rates of dissolution of naphthalene and 4-chlorobiphenyl were directly related to their surface areas. The bacteria caused a decline in the concentration of the soluble substrate. The rate of bacterial growth fell abruptly when 4-chlorobiphenyl or naphthalene was no longer detectable in solution. The population continued to increase in media with different surface areas of insoluble 4-chlorobiphenyl, but the final counts were higher in media in which the surface areas of the substrate were larger. The rates of dissolution of palmitic acid, octadecane, di(2-ethylhexyl) phthalate, and 1-naphthyl N-methylcarbamate were determined in the absence of microorganisms. A mixed culture of microorganisms mineralized palmitic acid, di(2-ethylhexyl) phthalate, and Sevin (1-naphthyl N-methylcarbamate) at a logarithmic rate, but octadecane mineralization was linear. The rates of mineralization at the end of the active phase of the biodegradation were lower than the rate of dissolution of palmitic acid but higher than the rate of dissolution of octadecane in the uninoculated medium. We suggest that spontaneous dissolution rates are only one of the factors that govern the rates of biodegradation.     

Rates of dissolution and biodegradation of water-insoluble organic compounds

We conducted a study of the relationship between the dissolution rates of organic compounds that are sparingly soluble in water and the biodegradation of these compounds by mixed cultures of bacteria. The rates of dissolution of naphthalene and 4-chlorobiphenyl were directly related to their surface areas. The bacteria caused a decline in the concentration of the soluble substrate. The rate of bacterial growth fell abruptly when 4-chlorobiphenyl or naphthalene was no longer detectable in solution. The population continued to increase in media with different surface areas of insoluble 4-chlorobiphenyl, but the final counts were higher in media in which the surface areas of the substrate were larger. The rates of dissolution of palmitic acid, octadecane, di(2-ethylhexyl) phthalate, and 1-naphthyl N-methylcarbamate were determined in the absence of microorganisms. A mixed culture of microorganisms mineralized palmitic acid, di(2-ethylhexyl) phthalate, and Sevin (1-naphthyl N-methylcarbamate) at a logarithmic rate, but octadecane mineralization was linear. The rates of mineralization at the end of the active phase of the biodegradation were lower than the rate of dissolution of palmitic acid but higher than the rate of dissolution of octadecane in the uninoculated medium. We suggest that spontaneous dissolution rates are only one of the factors that govern the rates of biodegradation.     

Water-soluble metabolites of p-nitrophenol and 1-naphthyl N-methylcarbamate in flies and grass grubs. Formation of glucose phosphate and phosphate conjugates

Metabolites isolated from houseflies dosed with 1-napththol or p-nitrophenol were identified as the phosphate and glucose phosphate conjugates of these phenols by titrations, hydrolysis, ionophoresis, i.r. spectra and mixed melting point. [(3)H]Carbaryl (1-naphthyl N-methylcarbamate) was metabolized by houseflies, blowflies and grass grubs to water-soluble metabolites which had chromatographic and ionophoretic behaviour similar to those of the conjugates of 1-naphthol with glucose, sulphate, phosphate and glucose 6-phosphate.     

Colorimetric Determination of 2-Chlorophenyl N-Methylcarbamate

2-Chlorophenyl N-methylcarbamate was determined colorimetrically. It was hydrolyzed with a veronal buffer solution (pH 8.0) to give corresponding 2-chlorophenol, which was coupled 4-nitrobenzenediazonium fluoroborate to produce a color having a maximum absorption at 520 mμ. The developed color was very stable. On the other hand, contaminated 2-chlorophenol was adsorbed in treated alumina before being hydrolyzed. Other contaminated impurities gave little influences. Technical materials and some formulated products were determined and good results were obtained.     

微生物降解呋喃丹的研究进展

呋喃丹(2,3-二氢-2,2-二甲基-7-苯并呋喃基甲氨基甲酸酯)是一种曾被广泛应用于农业生产的氨基甲酸酯类杀虫剂。虽然呋喃丹现在是我国的限制使用农药品种,但是它的残留仍然破坏生态环境和威胁人体健康。微生物降解是消除环境中呋喃丹污染的有效手段,但是目前对微生物降解呋喃丹的分子机制还未阐明。本文从微生物菌株资源、降解途径、关键酶和基因等几个方面综合阐述了国内外对微生物降解呋喃丹的最新研究进展,为呋喃丹污染环境的生物修复提供参考。     

Metabolism of m-tert.-butylphenyl N-methylcarbamate in insects and mice

The metabolism of m-tert.-butylphenyl N-methylcarbamate was studied in mice and five species of insects. Both the tert.-butyl group and the N-methyl group were hydroxylated. The major phenolic metabolite was m-(beta-hydroxy-tert.-butyl)phenol, which was identified by mass spectroscopy. Significant amounts of dihydroxy compounds were formed at a constant rate from the start of the enzymic oxidation process. The considerable species variation in the yields of the different types of oxidation products suggests that N-demethylation and oxidation of the tert.-butyl groups were catalysed by different enzymes. A microsomal NADPH-dependent enzyme also catalysed the splitting of the ester link in the insecticide.     

Structure-activity relationships of the N-methylcarbamate series in Salmonella typhimurium

Aromatic hydrocarbons of low molecular weight, hydroxy and N-methylcarbamate derivatives were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA98 and TA1535 in the presence of a rat-liver 9000 X g supernatant fraction. The presence of 2 or 3 aromatic rings resulted in a weak increase in revertants. Hydroxylation and carbamylation of aromatic rings increased the mutagenic activity of these aromatic compounds. In order to evaluate the structure-activity relationship, the specific molecular connectivity indices were calculated. A significant inverse relationship exists between mutagenicity and zero- and second-order specific molecular connectivity indices. Only compounds with second-order specific molecular connectivity indices lower than 0.300 increased mutagenic activity.     

The potential of carbaryl as a treatment for sea lice infestations of farmed Atlantic salmon, Salmo salar L.

The treatment of sea lice, Lepeophtheirus salmonis infestations on farmed Atlantic salmon, Salmo salar requires regular bathing of the fish in diachlorvos (DDVP), 2,2-dichlorovinyl dimethyl phosphate, with subsequent release to the marine environment of spent pesticide. The toxicity of a possible alternative compound, carbaryl, l-naphthyl N-methylcarbamate) to kill sea lice and Atlantic salmon was examined. Preliminary studies with carbaryl indicate the potential advantage of a reduced treatment dose was outweighed by the greater persistence of both the parent compound and of its toxic degradation product. Hence this carbamate insecticide is unlikely to be considered by regulatory authorities as an alternative treatment for sea lice infestations.     

Purification and characterization of a novel carbaryl hydrolase from Aspergillus niger PY168

A fungus capable of using carbaryl as the sole source of carbon and energy was isolated from a soil enrichment, and characterized as Aspergillus niger and designated strain PY168. A novel carbaryl hydrolase from cell extract was purified 262-fold to apparent homogeneity with 13.6% overall recovery. It had a monomeric structure with a molecular mass of 50,000 Da and a pI of 4.6, and the enzyme activity was optimal at 45 degrees C and pH 7.5, The activities were strongly inhibited by Hg(2+), Ag+, rho-chloromercuribenzoate, iodoacetic acid, diisofluorophosphate and phenylmethylsulfonyl fluoride but not EDTA and phenanthroline. The purified enzyme hydrolyzed various N-methylcarbamate insecticides. Carbaryl is the preferred substrate.     

Methylcarbamate effects on Meckel's cartilages in the chick embryo

On the basis of previous observations on the teratogenic effects of a variety of organophosphorus and methylcarbamate compounds on the avian skeletal apparatus, the Meckel's cartilage shape and structure were analyzed in carbaryl (1-naphthyl N-methylcarbamate) treated and control chick embryos of 9, 10, 12 days of incubation. The results indicate that both during normal development and under experimental conditions these cartilages undergo similar deformities, apparently subsequent to chondroblast death and regressive processes in the extracellular matrix. Since the macro- and microscopical cartilage alterations are significantly more frequent in the treated embryos than in the controls, a hypothesis is advanced that the methylcarbamate may increase the spontaneous tendency of the above mentioned cartilaginous anlagen to be affected by degenerative processes during embryogenesis.     

Substrate and dilution effects on the inhibition of acetylcholinesterase by carbamates

1. The kinetics of acetylcholinesterase (EC 3.1.1.7) activity and its inhibition by eserine or by Sevin (1-naphthyl N-methylcarbamate) have been studied over the substrate concentration range 5x10(-8) to 2.5x10(-2)m. 2. Equations are given for inhibition as a function of time, substrate and inhibitor concentrations, and the relevant parameters determined at 25 degrees and 37 degrees . 3. The observed and calculated effects of time, dilution, substrate addition and enzyme concentration were in good agreement and consistent with a steady-state carbamylation by eserine or by Sevin in the presence of excess of inhibitor. 4. The quantitative destruction of either inhibitor at high enzyme concentrations implied by the carbamylation hypothesis has been confirmed experimentally. 5. The importance and possibility of allowing quantitatively for dilution and substrate effects when estimating carbamate inhibition are demonstrated.     

Design, synthesis, and antiviral evaluation of some 3'-carboxymethyl-3'-deoxyadenosine derivatives

3'-Carboxymethyl-3'-deoxyadenosine derivatives were prepared from 2'-O-TBDMS-3'-[(ethoxycarbonyl)methyl]-3'-deoxyadenosine (1) via simple and efficient procedures. Conversion of 1 to its 5'-azido-5'-deoxy derivative 5 was accomplished via a novel one-pot method employing 5'-activation (TosCl) followed by efficient nucleophilic displacement with tetramethylguanidinium azide. Compound 5 was converted to 5'-[(N-methylcarbamoyl)amino] derivative 8 via one-pot reduction/acylation employing H(2)/Pd-C followed by treatment with p-nitrophenyl N-methylcarbamate. N(6)-phenylcarbamoyl groups were introduced by treatment with phenylisocyanate, and an efficient new method for lactonization of 2'-O-TBDMS-3'-[(ethoxycarbonyl)methyl]-3'-deoxyadenosines to give corresponding 2',3'-lactones was also developed. Target compounds were evaluated for anti-HIV and anti-HIV integrase activities, but were not active at the concentrations tested.     

Hydrolysis of carbaryl by a Pseudomonas sp. and construction of a microbial consortium that completely metabolizes carbaryl.

Two Pseudomonas spp. (isolates 50552 and 50581) isolated from soil degraded 1-naphthol and carbaryl, an N-methylcarbamate pesticide, respectively. They utilized these compounds as a sole source of carbon. 1-Naphthol was completely metabolized to CO2 by the isolate 50552, while the carbaryl was first hydrolyzed to 1-naphthol and then converted into a brown-colored compound by the isolate 50581. The colored metabolite was not degraded, but 1-naphthol produced by the isolate 50581 during the exponential phase of growth was metabolized by the isolate 50552. The two isolates were used to construct a bacterial consortium which completely catabolized carbaryl to CO2. No metabolite was detected in the cell cultures of the consortium. The isolate 50581 harbored a 50-kb plasmid pCD1, while no plasmid was detected in the isolate 50552. The isolated bacteria individually or as a consortium may be used for detoxification of certain industrial and agricultural wastes.     

Hydrolysis of carbaryl by a Pseudomonas sp. and construction of a microbial consortium that completely metabolizes carbaryl

Two Pseudomonas spp. (isolates 50552 and 50581) isolated from soil degraded 1-naphthol and carbaryl, an N-methylcarbamate pesticide, respectively. They utilized these compounds as a sole source of carbon. 1-Naphthol was completely metabolized to CO2 by the isolate 50552, while the carbaryl was first hydrolyzed to 1-naphthol and then converted into a brown-colored compound by the isolate 50581. The colored metabolite was not degraded, but 1-naphthol produced by the isolate 50581 during the exponential phase of growth was metabolized by the isolate 50552. The two isolates were used to construct a bacterial consortium which completely catabolized carbaryl to CO2. No metabolite was detected in the cell cultures of the consortium. The isolate 50581 harbored a 50-kb plasmid pCD1, while no plasmid was detected in the isolate 50552. The isolated bacteria individually or as a consortium may be used for detoxification of certain industrial and agricultural wastes.