Purification and biochemical characterization of the carbamate hydrolase from Pseudomonas sp. 50432

Abstract

A soluble carbamate hydrolase that had a wide specificity was purified 2032-fold from Pseudomonas sp. 50432. This was achieved using a combination of anion-exchange, gel-filtration and hydrophobic-interaction- chromatography techniques. Carbamate hydrolase cleaved the ester linkage of the N-methylcarbamates. The native enzyme was a monomer with a molecular mass of 88 kDa. The optimum pH and temperature of the enzyme activity were 8.5 and 37 degrees C respectively. The tested cations or EDTA did not affect the enzyme activity. However, 2-mercaptoethanol reversibly inhibited the enzyme activity. The enzyme showed the K(m) values of 16 and 12 microM for carbofuran and carbaryl respectively. The purified enzyme did not hydrolyse o-nitrophenyl dimethylcarbamate but hydrolysed several N-methylcarbamates and 1-naphthyl acetate.

Metabolism of Carbaryl via 1,2-Dihydroxynaphthalene by Soil Isolates Pseudomonas sp. Strains C4, C5, and C6

“…Widespread and repeated use leads to pollution of soil and groundwater (5). The ester bond between N-methylcarbamic acid and 1-naphthol is responsible for carbaryl toxicity.…”

“…Bacteria capable of degrading carbamate pesticides have been isolated from the soil (3,6,7,13,16,25). The first step in degradation is the hydrolysis of carbaryl to 1-naphthol by carbaryl hydrolase, which has been purified and characterized from various organisms (3,5,10,11,20,24). Depending on the strain, 1-naphthol is metabolized via salicylate to either gentisate or catechol (4,7,9,16,17).…”

Characterization and Malathion Degradability of Carboxylesterase in Wheat Kernels

“…Furthermore, it is reported in bacterial esterases that the disulfide linkages around the active sites are reductively cleaved by sulfhydryl compounds and the enzymatic activities are inhibited. 26,27) Both enzyme activities were classified by Aldridge's method for mammalian esterases, 12) which divides esterases into the three classes, CE, cholinesterase and arylesterase based on the influence profile of various inhibitors against the enzyme activities. In this classification method, neither CE nor cholinesterase is inhibited by sulfhydryl compounds or EDTA, but by serine-esterase inhibitors.…”

“…The inhibitors examined were paraoxon, DFP and PMSF (serine esterase inhibitors), [19][20][21] eserine (cholinesterase inhibitor), 22,23) ME, DTT and GSH (sulfhydryl compounds) [24][25][26][27] or EDTA (chelating agent for bivalent metal ions). [28][29][30] Figure 1 shows the inhibitory extent by esterase inhibitors against hydrolyzing activity of malathion or p-nitrophenyl acetate in wheat kernels after incubation of wheat kernel extract with 1.2 mM esterase inhibitor for 10 min and after removal of the inhibitor by a NAP10 gel filtration column.…”

Induction of carbofuran oxidation to 4-hydroxycarbofuran by Pseudomonas sp. 50432

“…Microorganisms in soil are known to degrade carbamate pesticides via hydrolysis or oxidation [2^8]. Microbial metabolites of carbofuran have been reported previously [3,9,10] and include 7-phenol (2,3-dihydro-2,2-dimethyl-7-hydroxybenzofuran), 3-hydroxycarbofuran (2,3-dihydro-2,2-dimethyl-3-hydroxybenzofuran-7-yl methylcarbamate), 3-hydroxycarbofuran 7-phenol (2,3-dihydro-2,2-dimethyl-3,7-dihydroxybenzofuran), 3-ketocarbofuran (2,3-dihydro-2,2-dimethyl-3-keto-benzofuran-7-yl methylcarbamate), 3-ketocarbofuran 7-phenol (2,3-dihydro-2,2-dimethyl-3-keto-7-hydroxybenzofuran) [3,8] and 5-hydroxycarbofuran (2,3-dihydro-2, 2-dimethyl-5-hydroxybenzofuran-7-yl methylcarbamate) [22]. As in the case of aldicarb [11], the oxidative metabolites of carbofuran could be more toxic than that of hydrolytic products.…”