Publications

• Purification and Characterization of Carbaryl Hydrolase from Blastobacter sp. Strain M501.

  Hayatsu M., Nagata T.

  A bacterium capable of hydrolyzing carbaryl (1-naphthyl-N-methylcarbamate) was isolated from a soil enrichment. This bacterium was characterized taxonomically as a Blastobacter sp. and designated strain M501. A carbaryl hydrolase present in this strain was purified to homogeneity by protamine sulf ... >> More

  A bacterium capable of hydrolyzing carbaryl (1-naphthyl-N-methylcarbamate) was isolated from a soil enrichment. This bacterium was characterized taxonomically as a Blastobacter sp. and designated strain M501. A carbaryl hydrolase present in this strain was purified to homogeneity by protamine sulfate treatment, ammonium sulfate precipitation, and hydrophobic, anion-exchange, gel filtration, and hydroxylapatite chromatographies. The native enzyme had a molecular mass of 166,000 Da and was composed of two subunits with molecular masses of 84,000 Da. The optimum pH and temperature of the enzyme activity were 9.0 and 45 degrees C, respectively. The enzyme was not stable at temperatures above 40 degrees C. The purified enzyme hydrolyzed seven N-methylcarbamate insecticides and also exhibited activity against 1-naphthyl acetate and 4-nitrophenyl acetate. << Less

  Appl Environ Microbiol 59:2121-2125(1993) [PubMed] [EuropePMC]

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

  Zhang Q., Liu Y., Liu Y.H.

  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 ... >> More

  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. << Less

  FEMS Microbiol Lett 228:39-44(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Nucleotide sequence and genetic structure of a novel carbaryl hydrolase gene (cehA) from Rhizobium sp. strain AC100.

  Hashimoto M., Fukui M., Hayano K., Hayatsu M.

  Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequ ... >> More

  Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon. << Less

  Appl Environ Microbiol 68:1220-1227(2002) [PubMed] [EuropePMC]

• Purification and characterization of carbaryl hydrolase from Arthrobacter sp. RC100.

  Hayatsu M., Mizutani A., Hashimoto M., Sato K., Hayano K.

  A carbaryl hydrolase was purified to homogeneity from Arthrobacter sp. strain RC100 by protamine sulfate treatment, ammonium sulfate precipitation, and hydrophobic, anion-exchange, and gel filtration chromatographies. The native enzyme had a molecular mass of 100 kDa and was composed of two identi ... >> More

  A carbaryl hydrolase was purified to homogeneity from Arthrobacter sp. strain RC100 by protamine sulfate treatment, ammonium sulfate precipitation, and hydrophobic, anion-exchange, and gel filtration chromatographies. The native enzyme had a molecular mass of 100 kDa and was composed of two identical subunits with molecular masses of 51 kDa. The hydrolase activity was strongly inhibited by DIFP, PMSF, Hg(2+) and paraoxon but not by EDTA. The optimum pH and temperature for the enzyme activity were 9.0 and 50 degrees C, respectively. The enzyme hydrolyzed four N-methylcarbamate insecticides (carbaryl, xylylcarb, metolcarb and XMC), but was not able to hydrolyze fenobucarb, propoxur, and isoprocarb. << Less

  FEMS Microbiol Lett 201:99-103(2001) [PubMed] [EuropePMC]

• Expanded insecticide catabolic activity gained by a single nucleotide substitution in a bacterial carbamate hydrolase gene.

  Ozturk B., Ghequire M., Nguyen T.P., De Mot R., Wattiez R., Springael D.

  Carbofuran-mineralizing strain Novosphingobium sp. KN65.2 produces the CfdJ enzyme that converts the N-methylcarbamate insecticide to carbofuran phenol. Purified CfdJ shows a remarkably low K_M towards carbofuran. Together with the carbaryl hydrolase CehA of Rhizobium sp. strain AC100, C ... >> More

  Carbofuran-mineralizing strain Novosphingobium sp. KN65.2 produces the CfdJ enzyme that converts the N-methylcarbamate insecticide to carbofuran phenol. Purified CfdJ shows a remarkably low K_M towards carbofuran. Together with the carbaryl hydrolase CehA of Rhizobium sp. strain AC100, CfdJ represents a new protein family with several uncharacterized bacterial members outside the proteobacteria. Although both enzymes differ by only four amino acids, CehA does not recognize carbofuran as a substrate whereas CfdJ also hydrolyzes carbaryl. None of the CfdJ amino acids that differ from CehA were shown to be silent regarding carbofuran hydrolytic activity but one particular amino acid substitution, i.e., L152 to F152, proved crucial. CfdJ is more efficient in degrading methylcarbamate pesticides with an aromatic side chain whereas CehA is more efficient in degrading the oxime carbamate nematicide oxamyl. The presence of common flanking sequences suggest that the cfdJ gene is located on a remnant of the mobile genetic element Tnceh carrying cehA. Our results suggest that these enzymes can be acquired through horizontal gene transfer and can evolve to degrade new carbamate substrates by limited amino acid substitutions. We demonstrate that a carbaryl hydrolase can gain the additional capacity to degrade carbofuran by a single nucleotide transversion. << Less

  Environ Microbiol 18:4878-4887(2016) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Compartmentalization of the Carbaryl Degradation Pathway: Molecular Characterization of Inducible Periplasmic Carbaryl Hydrolase from Pseudomonas spp.

  Kamini, Shetty D., Trivedi V.D., Varunjikar M., Phale P.S.

  <i>Pseudomonas</i> sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of <i>mcbA</i>, encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a sig ... >> More

  <i>Pseudomonas</i> sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of <i>mcbA</i>, encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4-to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm of <i>Escherichia coli</i> The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis of <i>E. coli</i> expressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparent <i>K_m</i> and <i>V</i>_max in the range of 98 to 111 μM and 69 to 73 μmol · min^-1 · mg^-1, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm of <i>Pseudomonas</i> spp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol.<b>IMPORTANCE</b> Proteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm of <i>Pseudomonas</i> spp. Predicted transmembrane domain and signal peptide sequences of <i>Pseudomonas</i> were found to be functional in <i>Escherichia coli</i> and to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds. << Less

  Appl Environ Microbiol 84:e02115-17(2018) [PubMed] [EuropePMC]

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

  Mulbry W.W., Eaton R.W.

  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 phenylcar ... >> More

  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. << Less

  Appl Environ Microbiol 57:3679-3682(1991) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Isolation of a constitutively expressed enzyme for hydrolysis of carbaryl in Pseudomonas aeruginosa.

  Chapalmadugu S., Chaudhry G.R.

  A hydrolase constitutively expressed in Pseudomonas aeruginosa which converts carbaryl to 1-naphthol was purified 1,767-fold by using a combination of anion-exchange, hydroxylapatite, gel filtration, and hydrophobic interaction chromatography techniques. The presence of Triton X-100 in buffers was ... >> More

  A hydrolase constitutively expressed in Pseudomonas aeruginosa which converts carbaryl to 1-naphthol was purified 1,767-fold by using a combination of anion-exchange, hydroxylapatite, gel filtration, and hydrophobic interaction chromatography techniques. The presence of Triton X-100 in buffers was necessary for deaggregation and purification of the hydrolase. This is the first membrane-bound hydrolase involved in the hydrolysis of any methylcarbamate pesticide purified from a bacterial source to date. The enzyme exhibited a unique specificity of hydrolyzing only carbaryl (1-naphthyl N-methylcarbamate) but not any other methylcarbamates. The purified enzyme was a monomer with a molecular mass of 65,000 Da. The pH and temperature optima for the enzyme activity were 8.5 and 45 degrees C, respectively. No cofactor requirement for the hydrolase activity could be demonstrated, and none of the divalent cations studied affected the activity of the enzyme. Also, the enzyme activity was not affected by the thiols: dithioerythritol, dithiothreitol, and 2-mercaptoethanol. The Km and Vmax values for carbaryl were 9 microM and 7.9 mumol/min/mg of protein, respectively. << Less

  J Bacteriol 175:6711-6716(1993) [PubMed] [EuropePMC]