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1.
Decontamination of soils with complex pollution using natural strains of microorganisms is a matter of great importance. Here we report that oil-oxidizing bacteria Rhodococcus erythropolis AC-1514D and Rhodococcus ruber AC-1513D can degrade various organophosphorous pesticides (OP). Cell-mediated degradation of five different OP is apparently associated with the presence of N-acylhomoserine lactonase, which is pronouncedly similar (46–50 %) to the well-known enzyme organophosphate hydrolase (OPH), a hydrolysis catalyst for a wide variety of organophosphorous compounds. Additionally, we demonstrated the high lactonase activity of hexahistidine-tagged organophosphate hydrolase (His6-OPH) with respect to various N-acylhomoserine lactones, and we determined the catalytic constants of His6-OPH towards these compounds. These experimental data and theoretical analysis confirmed the hypothesis about the evolutionary proximity of OPH and lactonases. Using Rhodococcus cells, we carried out effective simultaneous biodegradation of pesticide paraoxon (88 mg/kg) and oil hydrocarbon hexadecane (6.3 g/kg) in the soil. Furthermore, the discovered high lactonase activity of His6-OPH offers new possibilities for developing an efficient strategy of combating resistant populations of Gram-negative bacterial cells. 相似文献
2.
Elena Efremenko Ilya Lyagin Yulia Votchitseva Maria Sirotkina Sergey Varfolomeyev 《Biocatalysis and Biotransformation》2013,31(1):103-108
The catalytic and physical–chemical properties of organophosphorus hydrolase (OPH) modified by the addition of an N-terminal dodecahistidine tag (His12-OPH) have been investigated. Introduction of the His12-tag caused a 30- and 74-fold increase in catalytic efficiency of the enzyme with parathion and methyl parathion, respectively, compared to OPH. Concurrently, the His12-OPH had a more alkaline pH-optimum and extended temperature range than OPH and OPH modified with a hexahistidine tag. A study of His12-OPH thermostability showed that the enzyme had a tendency to oligomerise. This resulted in a decrease in the enzymatic activity of His12-OPH at temperatures <50°C, but provided the enzyme with much higher thermostability at temperatures >50°C, compared to OPH. 相似文献
3.
Elena Efremenko Ilya Lyagin Denis Gudkov Sergey Varfolomeyev 《Biocatalysis and Biotransformation》2013,31(2-4):359-364
New biocatalysts were developed using organophosphorus hydrolase (OPH, EC 3.1.8.1) with a polyhistidine tag at the N-terminus of the protein (His6-OPH). The use of His6-OPH together with previously developed approaches for the entrapment of cells into poly(vinyl alcohol) cryogels and covalent immobilization of enzymes into porous fabric materials, impregnated with chemically cross-linked chitosan sulphate gel, enabled dramatic improvement of catalytic characteristics against various organophosphorous compounds (OPCs; Paraoxon, Coumaphos, Methyl parathion, etc.). The polyhistidine tag of OPH was used to create a new immobilized biocatalyst using metal-chelating carriers, such as Ni2+-nitrilotriacetic acid-agarose and Co2+-iminodiacetic acid-polyacrylamide cryogel. The latter biocatalyst had high activity and stability for the continuous hydrolysis of OPCs. 相似文献
4.
Efremenko E Votchitseva Y Plieva F Galaev I Mattiasson B 《Applied microbiology and biotechnology》2006,70(5):558-563
Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His6-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices
[Me2+-iminodiacetic acid (IDA)–polyacrylamide cryogel (PAA) and Me2+-N,N,N’-tris (carboxymethyl) ethylendiamine (TED)–PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography
of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and
sample loading, the use of precolumn with IDA–PAA matrix noncharged with metal ions, and the application of high flow rate
provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA–PAA provided the highest level of selectivity for His6-OPH. Comparative analysis of purification using Co2+-IDA–PAA and Ni-nitrilotriacetic acid–agarose showed obvious advantages of the former in process time, specific activity of
purified enzyme, and simplicity of adsorbent regeneration. 相似文献
5.
6.
J. Chaudhuri T. K. Chakraborti S. Chanda C. N. Pope 《Journal of biochemical and molecular toxicology》1993,8(4):207-216
We previously reported similar levels of brain cholinesterase inhibition but marked differences in toxicity following acute maximum tolerated doses of the organophosphate pesticides parathion and chlorpyrifos. Because extensive acetylcholinesterase inhibition often induces compensatory changes in cholinergic receptor populations, we compared the effects of parathion and chlorpyrifos on brain muscarinic receptors. Adult male rats were treated with vehicle or the maximum tolerated dose of parathion (18 mg/kg, sc) or chlorpyrifos (279 mg/kg, sc) and observed for signs of acute toxicity. Similarly treated animals were sacrificed at 2, 7, or 14 days after treatment for measurement of cholinesterase activity and binding to the nonselective muscarinic antagonist [3H]quinuclidinyl benzilate, the M2-preferential antagonist [3H]AFDX-384, and the high-affinity agonist [3H]cis-methyldioxolane. More acute toxicity was noted after parathion treatment. Both insecticides caused similar levels (> 85%) of maximal cholinesterase inhibition and reductions (up to 55%) in atropine-sensitive quinuclidinyl benzilate binding (i.e., total muscarinic receptors) and [3H]AFDX-384 binding in cortex and striatum. Parathion also reduced, whereas chlorpyrifos increased, total muscarinic receptor binding and [3H]AFDX-384 binding in the cerebellum. When tissues were preincubated with paraoxon (10 μM), radiolabeling of a subset of quinuclidinyl benzilate binding sites was blocked and the apparent densities of these organophosphate-sensitive receptors in all three tissues were decreased (16% maximal) by parathion but increased (up to 37%) by chlorpyrifos. Similarly, parathion decreased whereas chlorpyrifos increased [3H]cis-methyldioxolane binding sites in all three brain regions. We propose that differential modulation of these organophosphate-sensitive muscarinic receptors contributes to differences in acute toxicity following exposure to these pesticides. 相似文献
7.
Young animals are more sensitive than adults to the neurotoxic effects of some organophosphorus insecticides. Many investigators attribute this difference in sensitivity to the immaturity of the detoxification capacity of preweanling rats. Chlorpyrifos [O,O-diethylO-(3,5,6-trichloro-2-pyridyl)phosphorothionate] is an organophosphorus insecticide that demonstrates considerable age-related sensitivity. The carboxylesterases are a group of related enzymes that detoxify organophosphorus insecticides by stoichiometrically binding these molecules before they can inhibit acetylcholinesterase. This study presents in vitro and in vivo evidence demonstrating that the carboxylesterases are critical for explaining the age-related sensitivity of chlorpyrifos. The data show that the fetal rat and the postnatal day 17 (PND17) rat pup have fewer molecules of carboxylesterase (less activity), less sensitive molecules of carboxylesterase, and a larger proportion of chlorpyrifos-insensitive molecules of carboxylesterase. An in vitro mixing experiment, using adult striatum as a source of acetylcholinesterase and liver homogenates as a source of carboxylesterase, demonstrates that the adult liver carboxylesterases are superior to the PND17 liver carboxylesterases for detoxifying chlorpyrifos. In the in vivo experiments the time course profiles of carboxylesterase and cholinesterase activity following a maximum tolerated dose of chlorpyrifos also suggest that the carboxylesterases of the PND17 rat were less capable of detoxifying chlorpyrifos. Carboxylesterase activity in the preweanling rat was not as severely inhibited as in the adult, but decrements in cholinesterase activity as a result of chlorpyrifos treatment were comparable. These in vitro and in vivo findings support the previously proffered postulate that the carboxylesterases are critical for determining the age-related sensitivity of chlorpyrifos. In addition, these detailed experiments allow us to propose that the detoxification potential of these enzymes is multifaceted, and depends on the (1) amount of activity (i.e., number of molecules), (2) affinity for the insecticide or metabolite, and (3) amount of carboxylesterase activity that is refractory to inhibition by the insecticide or metabolite. 相似文献
8.
Mosquitoes represent one of the most significant threats to human and veterinary health throughout the world. Consequently, improving strategies for the control of mosquitoes is essential. In the present study, juvenile Culex pipiens (Diptera: Culicidae), the common house mosquito, are chronically exposed to sublethal concentrations of chlorpyrifos (20% of LC50) and imidacloprid (5% of LC50), both separately and as a mixture. Developmental time, the emergence rate of adults and the expression of five selected genes involved in detoxification and resistance to pesticides are assessed. To assess the effects on oviposition choice, gravid females are forced to oviposit into cups containing water with added chlorpyrifos, imidacloprid or a mixture of both. The time required for the development of second‐ and third‐instar larvae is observed to differ significantly between treatments. Adults of C. pipiens fail to emerge from larvae hatched in both imidacloprid and the binary mixture. The expression of the four quantified detoxification genes differs significantly in third‐larval instars exposed to chlorpyrifos and/or imidacloprid compared with controls. Gravid females also fail to lay eggs on water to which either of the insecticides or the binary mixture is added, although they do lay eggs in cups containing water only. Chronic exposure to sublethal concentrations of chlorpyrifos or imidacloprid has significant adverse effects on development and thus the reproductive fitness of C. pipiens and, accordingly, could be used in the population control of these mosquitoes. 相似文献
9.
Suresh Kumar R Ashraf Ali M Osman H Ismail R Choon TS Yoon YK Wei AC Pandian S Manogaran E 《Bioorganic & medicinal chemistry letters》2011,21(13):3997-4000
Hexacyclic derivatives share vital pharmacological properties, considered useful in Alzheimer’s disease. The aim of this study was synthesis and its evaluation for acetyl cholinesterase inhibitory activity of novel hexacyclic analogues. Compound 4f, showed potent inhibitory activity against acetyl cholinesterase enzyme with IC50 0.72 μmol/L. 相似文献
10.
An enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 °C and withstanding incubation at temperatures up to 70 °C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta, Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 Å. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those factors most important for maintaining the structure and function of the enzyme. 相似文献
11.
A new series of oxopyrrolidines was synthesized and evaluated for their effect on Alzheimer‘s disease by measuring their inhibitory activity against acetyl cholinesterase enzyme and amyloid β 42 protein. Most of the compounds showed good inhibitory activity with ethyl 2-(2-(2, 6-dimethylphenylcarbamoyl)- 5-oxopyrrolidin-1-yl) acetate (V) having the highest activity against acetyl cholinesterase with IC50 value 1.84 ng/g tissue compared to standard donepezil 3.34 ng/g tissue. Furthermore, compound 1-((4-(4-chlorophenyl) piperazin-1-yl) methyl)-N-(2,6-dimethylphenyl)-5- oxopyrrolidine- 2-carboxamide (IIIe) displayed the highest activity against β 42 protein with IC50 value of 11.3 Pg/g tissue compared to 18.4 Pg/g tissue of donepezil. 相似文献
12.
Cholinesterase activities in rat forebrain, erythrocytes, and plasma were assessed after a single oral administration of metrifonate
or dichlorvos. In 3-month-old rats, the dichlorvos (10 mg/kg p.o.)-induced inhibition of cholinesterase reached its peak in
brain after 15–45 min and after 10–30 min in erythrocytes and plasma. Cholinesterase activity recovered rapidly after the
peak of inhibition, but did not reach control values in brain and erythrocytes within 24 h after drug administration. The
recovery of plasma cholinesterase activity, in contrast, was already complete 12 h after dichlorvos treatment. Metrifonate
(100 mg/kg p.o.) had qualitatively similar inhibition kinetics as dichlorvos, albeit with a slightly delayed onset. Peak values
were attained 45–60 min (brain) and 20–45 min (blood), after drug administration. Apparently complete recovery of cholinesterase
activity was noted in both tissues 24 h after treatment. The dose-dependence of drug-induced inhibition of cholinesterase
in rat blood and brain was determined at the time of maximal inhibition, i.e., 30 min after dichlorvos treatment and 45 min
after metrifonate treatment. The oral ED50 values obtained for dichlorvos were 8 mg/kg for brain and 6 mg/kg for both erythrocyte and plasma cholinesterase. The corresponding
oral ED50 values for metrifonate were 10 to 15 times higher, i.e., 90 mg/kg in brain and 80 mg/kg in erythrocytes and plasma. In rats
deprived of food for 18 h before drug treatment, the corresponding ED50 values for metrifonate were 60 and 45 mg/kg, respectively, indicating an about two-fold higher sensitivity of fasted rats
to metrifonate-induced cholinesterase inhibition compared to non-fasted rats. Compared to 3-month-old rats, 19-month-old rats
showed a higher sensitivity towards metrifonate and dichlorvos. At the time of maximal inhibition, there was a strong correlation
between the degree of cholinesterase inhibition in brain and blood. These results demonstrate that single oral administration
of metrifonate and dichlorvos induces an inhibition of blood and brain cholinesterase in the conscious rat in a dose-dependent
and apparently fully reversible manner. While the efficiency of a given dose of inhibitor may vary with the satiety status
or age of the animal, the extent of brain ChE inhibition can be estimated from the level of blood ChE activity. 相似文献
13.
A. Navas Díaz F. García Sanchez J. A. Gonzlez García V. Bracho Del Rio 《Luminescence》1995,10(5):285-289
2-Naphthyl acetate acts as a pro-enhancer of the luminol–H2O2–horseradish peroxidase reaction. Cholinesterase hydrolyses the bound acetyl group and produces 2-naphthol, and this compound is an enhancer of the chemiluminescent reaction. We studied the kinetics of chemiluminescent emission and the influence of 2-naphthyl acetate and cholinesterase enzyme concentration. The cholinesterase concentration versus chemiluminescence intensity maximum was linear for cholinesterase between 0 and 181 μU/mL, with a detection limit of 8 μU/mL and a relative standard deviation of 9.5% (n = 3), for a sample containing 90.67 μU/mL of cholinesterase. 相似文献
14.
15.
Jilin Tang Andreas Ebner Bernhard Kraxberger Michael Leitner Alba Hykollari Christian Kepplinger Christian Grunwald Hermann J. Gruber Robert Tamp Uwe B. Sleytr Nicola Ilk Peter Hinterdorfer 《Journal of structural biology》2009,168(1):217
Crystalline bacterial cell surface layers (S-layers) show the ability to recrystallize into highly regular pattern on solid supports. In this study, the genetically modified S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177, carrying a hexa-histidine tag (His6-tag) at the C-terminus, was used to generate functionalized two-dimensional nanoarrays on a silicon surface. Atomic force microscopy (AFM) was applied to explore the topography and the functionality of the fused His6-tags. The accessibility of the His6-tags was demonstrated by in-situ anti-His-tag antibody binding to the functional S-layer array. The metal binding properties of the His6-tag was investigated by single molecule force microscopy. For this purpose, newly developed tris–NTA was tethered to the AFM tips via a flexible polyethylene glycol (PEG) linker. The functionalized tips showed specific interactions with S-layer containing His6-tags in the presence of nickel ions. Thus the His6-tag is located at the outer surface of the S-layer and can be used for stable but reversible attachment of functional tris–NTA derivatives. 相似文献
16.
Tzu-Fan Wang Min-Guan Lin Huei-Fen Lo Meng-Chun Chi Long-Liu Lin 《Journal of biological physics》2014,40(1):25-40
In the present study, the biophysical properties of His6-tagged Bacillus stearothermophilus aminopeptidase II (His6-tagged BsAmpII) are characterized in detail by gel-filtration, analytical ultracentrifugation, and various spectroscopic techniques. Using size-exclusion chromatography and analytical ultracentrifugation, we demonstrate that His6-tagged BsAmpII exists predominantly as a dimer in solution. The enzyme is active and stable at pHs ranging from 6.5 to 8.5. Far-UV circular dichroism analysis reveals that the secondary structures of His6-tagged BsAmpII are significantly altered in the presence of SDS, whereas the presence of 5–10% acetone and ethanol was harmless to the folding of the enzyme. Thermal unfolding of His6-tagged BsAmpII was found to be irreversible and led to the formation of aggregates. The native enzyme started to unfold beyond 0.6 M guanidine hydrochloride and had a midpoint of denaturation at 1.34 M. This protein remained active at concentrations of urea below 2.7 M but experienced an irreversible unfolding by >5 M denaturant. Taken together, this work lays a foundation for potential biotechnological applications of His6-tagged BsAmpII. 相似文献
17.
Seiya Mochida Satoshi Tsuzuki Makoto Yasumoto Kuniyo Inouye Tohru Fushiki 《Enzyme and microbial technology》2009,45(4):288-294
Matriptase is a transmembrane serine protease expressed in vertebrates. This enzyme is synthesized as a zymogen form and is converted to an active form by cleavage at the N-terminus of the serine protease catalytic domain. In a mammalian cell-based expression system, we have produced pseudozymogen forms of recombinant matriptase (r-matriptase) that are activated by cleavage with a recombinant enterokinase (r-EK) in vitro. In the present study, four different pseudozymogen forms of r-matriptase containing a site for activation by r-EK and a hexahistidine tag (His6-tag) were expressed in and secreted by Pichia pastoris, a methylotrophic yeast. The pseudozymogens with His6-tag at their C-termini formed multimers linked by intermolecular disulfide bonds. After treatment with r-EK, they exhibited no detectable hydrolytic activity toward a chromogenic substrate. A pseudozymogen form of matriptase catalytic domain with N-terminal His6-tag (designated His6t-S-CD) was secreted as a monomer. His6t-S-CD after r-EK treatment exhibited activity comparable to that of the activated form of an r-matriptase expressed in mammalian cells. His6t-S-CD could be purified from culture medium in milligram quantities. The expression in the yeast offers an efficient method of producing larger amounts of r-matriptase. 相似文献
18.
19.
Altering the Substrate Specificity of Organophosphorus Hydrolase for Enhanced Hydrolysis of Chlorpyrifos 总被引:7,自引:0,他引:7
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Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the kcat/Km value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents. 相似文献
20.
Sookwang Lee Torka S. Poet Jordan N. Smith Andrea L. Busby-Hjerpe Charles Timchalk 《Chemico-biological interactions》2010,184(3):449-457
Routine use of tobacco products may modify physiological and metabolic functions, including drug metabolizing enzymes, which may impact the pharmacokinetics of environmental contaminants. Chlorpyrifos is an organophosphorus (OP) insecticide that is bioactivated to chlorpyrifos-oxon, and manifests its neurotoxicity by inhibiting acetylcholinesterase (AChE). The objective of this study was to evaluate the impact of repeated nicotine exposure on the pharmacokinetics of chlorpyrifos (CPF) and its major metabolite, 3,5,6-trichloro-2-pyridinol (TCPy) in blood and urine and also to determine the impact on cholinesterase (ChE) activity in plasma and brain. Animals were exposed to 7-daily doses of either 1 mg nicotine/kg or saline, and to either a single oral dose of 35 mg CPF/kg or a repeated dose of 5 mg CPF/kg/day for 7 days. Groups of rats were then sacrificed at multiple time-points after receiving the last dose of CPF. Repeated nicotine and CPF exposures resulted in enhanced metabolism of CPF to TCPy, as evidenced by increases in the measured TCPy peak concentration and AUC in blood. However, there was no significant difference in the amount of TCPy (free or total) excreted in the urine within the first 24-h post last dose. The extent of brain acetylcholinesterase (AChE) inhibition was reduced due to nicotine co-exposure consistent with an increase in CYP450-mediated dearylation (detoxification) versus desulfuration. It was of interest to note that the impact of nicotine co-exposure was experimentally observed only after repeated CPF doses. A physiologically based pharmacokinetic model for CPF was used to simulate the effect of increasing the dearylation Vmax based upon previously conducted in vitro metabolism studies. Predicted CPF-oxon concentrations in blood and brain were lower following the expected Vmax increase in nicotine treated groups. These model results were consistent with the experimental data. The current study demonstrated that repeated nicotine exposure could alter CPF metabolism in vivo, resulting in altered brain AChE inhibition. 相似文献