Immobilization of microbial cells on inner epidermis of onion bulb scale for biosensor application

Inner epidermis of onion bulb scales was used as a natural support for immobilization of microbial cells for biosensor application. A bacterium Sphingomonas sp. that hydrolyzes methyl parathion into a chromophoric product, p-nitrophenol (PNP), has been isolated and identified in our laboratory. PNP can be detected by electrochemical and colorimetric methods. Whole cells of Sphingomonas sp. were immobilized on inner epidermis of onion bulb scale by adsorption followed by cross-linking methods. Cells immobilized onion membrane was directly placed in the wells of microplate and associated with the optical transducer. Methyl parathion is an organophosphorus pesticide that has been widely used in the field of agriculture for insect pest control. This pesticide causes environmental pollution and ecological problem. A detection range 4-80 μM of methyl parathion was estimated from the linear range of calibration plot of enzymatic assay. A single membrane was reused for 52 reactions and was found to be stable for 32 days with 90% of its initial hydrolytic activity. The applicability of the cells immobilized onion membrane was also demonstrated with spiked samples.     

Optical microbial biosensor for detection of methyl parathion pesticide using Flavobacterium sp. whole cells adsorbed on glass fiber filters as disposable biocomponent

An optical microbial biosensor was described for the detection of methyl parathion pesticide. Whole cells of Flavobacterium sp. were immobilized by trapping in glass fiber filter and were used as biocomponent along with optic fiber system. Flavobacterium sp. has the organophosphorus hydrolase enzyme, which hydrolyzes the methyl parathion into detectable product p-nitrophenol. The immobilized microbial biocomponent was disposable, cost-effective and showed high reproducibility and uniformity. The detection of methyl parathion by the use of disposable microbial biocomponent with optical biosensor was simple, single step and direct measurement of very low quantity of the sample. The home made reaction vessel was small and needed only 75 microl of sample. A lower detection limit 0.3 microM methyl parathion was estimated from the linear range (4-80 microM) of calibration plot of organophosphorus hydrolase enzymatic assay. The applicability to synthetic methyl parathion spiked samples was demonstrated.     

Bacterial cell surface display of organophosphorus hydrolase for selective screening of improved hydrolysis of organophosphate nerve agents

Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.     

有机磷农药在水生态系中生物净化机理研究——1.对硫磷的酶解

从氧化塘系统中分离出能降解对硫磷的细菌Pseudomonas sp.代号CTP-01,能将对硫磷分解成对硝基酚和二乙基硫代磷酸酯,并进一步分解对硝基酚。在有Cu++存在的情况下,酶比活可以达到1×104毫微克分子/毫克蛋白/分钟,Cu++对酶有激活作用,并对温度和pH影响有保护作用。对硫磷水解酶反应最适温度为40—50℃,超过50℃活性急剧降低,80℃完全失活。 CTP-01的对硫磷水解酶大部分是同膜片结合状态存在,超声破碎的无细胞酶制剂中,只有37.2%的活力存在于可溶性蛋白部分。       

对硝基苯酚降解菌P3的分离、降解特性及基因工程菌的构建

分离到一株假单胞菌 (Pseudomonassp .)P3 ,该菌能够以对硝基苯酚为唯一碳源和氮源进行生长。在有外加氮源的条件下 ,P3降解对硝基苯酚并在培养液中积累亚硝酸根。P3有比较广泛的底物适应性 ,对多种芳香族化合物都有降解能力。不同金属离子对P3降解对硝基苯酚有不同的作用。葡萄糖的存在对P3降解对硝基苯酚无明显促进作用 ,而微量酵母粉可以大大促进P3对硝基苯酚的降解。以P3为受体菌 ,通过接合转移的手段将甲基对硫磷水解酶基因mpd克隆至P3菌中 ,获得了表达甲基对硫磷水解酶活性的基因工程菌PM ,PM能够以甲基对硫磷为唯一碳源进行生长。工程菌PM具有较高的甲基对硫磷降解活性及稳定性     

Simultaneous degradation of organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp. with surface-expressed organophosphorus hydrolase.

Moraxella sp., a native soil organism that grows on p-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of organophosphorus (OP) pesticides and p-nitrophenol (PNP). The truncated ice nucleation protein (INPNC) anchor was used to target the pesticide-hydrolyzing enzyme, organophosphorus hydrolase (OPH), onto the surface of Moraxella sp., alleviating the potential substrate uptake limitation. A shuttle vector, pPNCO33, coding for INPNC-OPH was constructed and the translocation, surface display, and functionality of OPH were demonstrated in both E. coli and Moraxella sp. However, whole cell activity was 70-fold higher in Moraxella sp. than E. coli. The resulting Moraxella sp. degraded organophosphates as well as PNP rapidly, all within 10 h. The initial hydrolysis rate was 0.6 micromol/h/mg dry weight, 1.5 micromol/h/mg dry weight, and 9.0 micromol/h/mg dry weight for methyl parathion, parathion, and paraoxon, respectively. The possibility of rapidly degrading OP pesticides and their byproducts should open up new opportunities for improved remediation of OP nerve agents in the future.     

Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase.

An amperometric microbial biosensor for the direct measurement of organophosphate nerve agents is described. The sensor is based on a carbon paste electrode containing genetically engineered cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, parathion and methyl parathion to p-nitrophenol. The later is detected anodically at the carbon transducer with the oxidation current being proportional to the nerve-agent concentration. The sensor sensitivity was optimized with respect to the buffer pH and loading of cells immobilized using paraoxon as substrate. The best sensitivity was obtained using a sensor constructed with 10 mg of wet cell weight per 100 mg of carbon paste and operating in pH 8.5 buffer. Using these conditions, the biosensor was used to measure as low as 0.2 microM paraoxon and 1 microM methyl parathion with very good sensitivity, excellent selectivity and reproducibility. The microbial biosensor had excellent storage stability, retaining 100% of its original activity when stored at 4 degrees C for up to 45 days.     

Isolation of methyl parathion-degrading strain M6 and cloning of the methyl parathion hydrolase gene

A degradative bacterium, M6, was isolated and presumptively identified as Plesiomonas sp. strain M6 was able to hydrolyze methyl parathion to p-nitrophenol. A novel organophosphate hydrolase gene designated mpd was selected from its genomic library prepared by shotgun cloning. The nucleotide sequence of the mpd gene was determined. The gene could be effectively expressed in Escherichia coli.     

A constructed microbial consortium for biodegradation of the organophosphorus insecticide parathion

A consortium comprised of two engineered microorganisms was assembled for biodegradation of the organophosphate insecticide parathion. Escherichia coli SD2 harbored two plasmids, one encoding a gene for parathion hydrolase and a second carrying a green fluorescent protein marker. Pseudomonas putida KT2440 pSB337 contained a p-nitrophenol-inducible plasmid-borne operon encoding the genes for p-nitrophenol mineralization. The co-culture effectively hydrolyzed 500 microM parathion (146 mg l(-1)) and prevented the accumulation of p-nitrophenol in suspended culture. Kinetic analyses were conducted to characterize the growth and substrate utilization of the consortium members. Parathion hydrolysis by E. coli SD2 followed Michaelis-Menten kinetics. p-Nitrophenol mineralization by P. putida KT2440 pSB337 exhibited substrate-inhibition kinetics. The growth of both strains was inhibited by increasing concentrations of p-nitrophenol, with E. coli SD2 completely inhibited by 600 microM p-nitrophenol (83 mg l(-1)) and P. putida KT2440 pSB337 inhibited by 1,000 microM p-nitrophenol (139 mg l(-1)). Cultivation of the consortium as a biofilm indicated that the two species could cohabit as a population of attached cells. Analysis by confocal microscopy showed that the biofilm was predominantly comprised of P. putida KT2440 pSB337 and that the distribution of E. coli SD2 within the biofilm was heterogeneous. The use of biofilms for the construction of degradative consortia may prove beneficial.     

Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp

Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon. Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation. One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth. The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis. Maximum enzymatic activity occurred between 35 and 40 degrees C. The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3. Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite. Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium. The hybridization data showed that the DNAs from a Pseudomonas sp. and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp. The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.     

Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp.

Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon. Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation. One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth. The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis. Maximum enzymatic activity occurred between 35 and 40 degrees C. The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3. Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite. Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium. The hybridization data showed that the DNAs from a Pseudomonas sp. and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp. The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.     

Studies on the metabolism of diethyl 4-nitrophenyl phosphorothionate (parathion) in vitro

1. The metabolism of the phosphorothionate parathion in vitro was examined by using [(32)P]parathion and microsomes isolated from the livers of various animal species. 2. The major metabolic products of parathion in this system in vitro were identified as diethyl 4-nitrophenyl phosphate (paraoxon), diethyl hydrogen phosphate, diethyl hydrogen phosphorothionate and p-nitrophenol. 3. The reaction leading to the formation of diethyl hydrogen phosphorothionate and p-nitrophenol requires the same cofactors (NADPH and oxygen) required for metabolism of parathion to its active anti-acetylcholinesterase paraoxon. 4. The enzyme activity towards parathion per unit weight of liver is increased some 65-130% by pretreatment of male rats with phenobarbital and 3,4-benzopyrene. 5. The metabolism of parathion is inhibited by incubation in a nitrogen atmosphere and in an atmosphere containing carbon monoxide. Pure oxygen is also inhibitory. These results are discussed in terms of a deficiency of oxygen for maximal activity as well as the lability of some component of the system to oxidation.     

Kinetics of succinic dehydrogenase inhibition by methyl parathion and its recovery by oximes and L-cysteine in hepatopancreas of Lamellidens marginalis

Effects in vitro of methyl parathion on some kinetic constants of succinic dehydrogenase (SDH) in hepatopancreas of freshwater mussel, L. marginalis were studied. Altered pH vs. specific activity curves for SDH demonstrated significant inhibition by methyl parathion in buffered acidic, neutral and alkaline ranges. At high pH ranges IC50 (12.5 microM) of methyl parathion did not cause 50% inhibition enzyme as it did at neutral and acidic pHs. Activation energies (delta E) were found to be increased suggesting decreased efficiency of enzyme in presence of methyl parathion. Non-competitive inhibition with respect to activation by succinate was indicated by decreased maximal velocity (V) without change in Michaelis Menten constant (Km). Pyridine-2-aldoxime (25 microM), pyridine-4-aldoxime (15 microM) and L-cysteine (40 microM) neutralized the inhibition of SDH by methyl parathion (12.5 microM). The kinetic data suggests that inhibition of SDH by methyl parathion was pH and temperature independent.     

Methyl parathion hydrolase based nanocomposite biosensors for highly sensitive and selective determination of methyl parathion

This article reports the fabrication of a nanocomposite biosensor for the sensitive and specific detection of methyl parathion. The nanocomposite sensing film was prepared via the formation of gold nanoparticles on silica particles, mixing with multiwall carbon nanotubes and subsequent covalent immobilization of methyl parathion hydrolase. The composite of the individual materials was finely tuned to offer the sensing film with high specific surface area and high conductivity. A significant synergistic effect of nanocomposites on the biosensor performance was observed in biosensing methyl parathion. The square wave voltammetric responses displayed well defined peaks, linearly proportional to the concentrations of methyl parathion in the range from 0.001 μg mL?1 to 5.0 μg mL?1 with a detection limit of 0.3 ng mL?1. The application of this biosensor in the analysis of spiked garlic samples was also evaluated. The proposed protocol can be used as a platform for the simple and fast construction of biosensors with good performance for the determination of enzyme-specific electroactive species.     

Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta

A purified phosphotriesterase was successfully immobilized onto trityl agarose in a fixed bed reactor. A total of up to 9200 units of enzyme activity was immobilized onto 2.0 mL of trityl agarose (65 mumol trityl groups/mL agarose), where one unit is the amount of enzyme required to catalyze the hydrolysis of one micromole of paraoxon in one min. The immobilized enzyme was shown to behave chemically and kinetically similar to the free enzyme when paraoxon was utilized as a substrate. Several organophosphate pesticides, methyl parathion, ethyl parathion, diazinon, and coumaphos were also hydrolyzed by the immobilized phosphotriesterase. However, all substrates exhibited an affinity for the trityl agarose matrix. For increased solubility and reduction in the affinity of these pesticides for the trityl agarose matrix, methanol/water mixtures were utilized. The effect of methanol was not deleterious when concentrations of less than 20% were present. However, higher concentrations resulted in elution of enzyme from the reactor. With a 10-unit reactor, a 1.0 mM paraoxon solution was hydrolyzed completely at a flow rate of 45 mL/h. Kinetic parameters were measured with a 0.1-unit reactor with paraoxon as a substrate at a flow rate of 22 mL/h. The apparent K(m) for the immobilized enzyme was 3-4 times greater than the K(m) (0.1 mM) for the soluble enzyme. Immobilization limited the maximum rate of substrate hydrolysis to 40% of the value observed for the soluble enzyme. The pH-rate profiles of the soluble and immobilized enzymes were very similar. The immobilization of phosphotriesterase onto trityl agarose provides an effective method esterase onto trityl agarose provides an effective method for hydrolyzing and thus detoxifyuing organophosphate pesticides and mammalian acetylcholinesterase inhinbitors.     

Detoxication of paraoxon by rat liver homogenate and serum carboxylesterases and A-esterases.

Paraoxon, the active metabolite of parathion, can be detoxified through a noncatalytic pathway by carboxylesterases and a catalytic pathway by calcium-dependent A-esterases, producing p-nitrophenol as a common metabolite. The detoxication patterns of carboxylesterases and A-esterases were investigated in vitro in the present study with a high tissue concentration (75 mg/mL rat liver homogenate or 50% rat serum solution) to more closely reflect enzyme concentrations in intact tissues. A final paraoxon concentration of 3.75 microM was used to incubate with liver homogenates or serum solutions for 5 seconds or 3, 5, 15, or 25 minutes; also 0.625, 1.25, 2.5, 3.125, 3.75, or 5.0 microM paraoxon (final concentration) was incubated with liver homogenates or serum solutions for 15 minutes. Phenyl saligenin cyclic phosphate and EDTA were used to inhibit carboxylesterases and A-esterases, respectively. Significant amounts of p-nitrophenol were generated with or without either inhibitor during a 15 minute incubation with paraoxon from low (0.625 microM) to high (5.0 microM) concentrations. The amount of p-nitrophenol generated via carboxylesterase phosphorylation was greater than via A-esterase-mediated hydrolysis in the initial period of incubation or when incubating with a low concentration of paraoxon. Plateau shape curves of p-nitrophenol concentration versus time or paraoxon concentration indicated that carboxylesterase phosphorylation was saturable. When incubated for long time intervals or with high concentrations of paraoxon, more p-nitrophenol was generated via A-esterase-mediated hydrolysis than from carboxylesterase phosphorylation. The ratio of paraoxon concentration to tissue amount used in in vitro assays of this study was equivalent to dosing a rat with toxicologically relevant dosages. These in vitro data suggest that both carboxylesterases and A-esterases detoxify paraoxon in vivo; carboxylesterases may be an important mode of paraoxon detoxication in initial exposures to paraoxon or parathion before they become saturated, whereas A-esterases may contribute to paraoxon detoxication in repeated exposures to paraoxon or parathion because they will not become inhibited and will remain catalytically active unlike the carboxylesterases. The importance of carboxylesterases in detoxication of paraoxon was verified by an in vivo study. In rats pretreated with tri-o-tolyl phosphate, an in vivo carboxylesterase inhibitor, brain acetylcholinesterase was significantly inhibited after intravenous exposure to parathion. No significant inhibition of brain acetylcholinesterase was observed in rats pretreated with corn oil.     

有机磷农药在水生态系中生物净化机理研究 4. Pseudomonas sp. CTP-01 的对硫磷水解酶诱导合成性质

Pseudomonas sp.CTP-01的对硫磷水解酶具有底物诱导合成性质。停滞生长期的细胞接触底物半小时即产生相应酶的合成,而指数生长期的细胞接触底物48小时后才发生酶的合成。甲基对硫磷及对硝基酚也具有诱导作用,可见合成对硫磷水解酶的诱导特异基团可能与对硝基酚及其苯环上的取代基有密切关系。       

Electrochemical sensing of trimethylamine based on polypyrrole-flavin-containing monooxygenase (FMO3) and ferrocene as redox probe for evaluation of fish freshness

Amperometric and impedimetric biosensor for detecting trimethylamine (TMA) which represents good parameters for estimating fish freshness has been developed. The biosensor is based on a conducting polypyrrole substituted with ferrocenyl, where flavin-containing monooxygenase 3 (FMO3) enzyme was immobilised by covalent bonding. FMO3 catalyzes the monooxygenation TMA to trimethylamine N-oxide (TMO). For catalysis FMO require flavin adenine (FAD) as a prosthetic group, NADPH as a cofactor and molecular oxygen as cosubstrate. Ferrocenyl group substituted on the polypyrrole matrix will serve as redox probe for monitoring the response of the biosensor to TMA. The construction of the biosensor was characterized by FT-IR, cyclic voltammetry and impedance measurements. Detection is done through the analysis of the current of oxidation signal of the ferrocenyl groups and compared to the measurement of impedance related to the electrical properties of the layers. Amperometric and impedimetric response were measured as a function of TMA concentration in range of 0.4 μgm L(-1)-80 μgm L(-1) (6.5 μmol L(-1)-1.5 mmol L(-1)). Amperometric measurements show a decrease in current response which is in correlation with the increase of the charge transfer resistance demonstrated by impedance. Calibration curve obtained by impedance spectroscopy shows a high sensitivity with a dynamic range from (0.4 μgm L(-1) to 80 μgm L(-1)). We demonstrated, using ferrocene as redox probe for catalytic reaction of FMO3, that high sensitivity and dynamic range was obtained. The biosensor was stable during 16 days. The biosensor shows high selectivity and its sensitivity to TMA in real samples was evaluated using fish extract after deterioration during storage.     

Parathion utilization by bacterial symbionts in a chemostat.

A continuous-culture device was used to select and enrich for microorganisms, from sewage and agricultural runoff, that were capable of using the organophosphorus insecticide parathion as a sole growth substrate. Parathion was dissimilated by the highly acclimated symbiotic activities of Pseudomonas stutzeri, which non-oxidatively and cometabolically hydrolyzed the parathion to ionic diethyl thiophosphate and p-nitrophenol, and P. aeruginosa, which utilized the p-nitrophenol as a sole carbon and energy source. Ionic diethyl thiophosphate was found to be inert to any transformations. Methyl parathion was dissimilated in an analogous way. The device functioned as a chemostat with parathion as the growth-limiting nutrient, and extraordinarily high dissimilation rates were attained for parathion (8 g/liter per day) and for p-nitrophenol (7 g/liter per day). This is the first report of parathion utilization by a defined microbial culture and by symbiotic microbial attack and of dissimilation of an organophosphorus pesticide in a chemostat.     

Degradation of Parathion by Bacteria Isolated from Flooded Soil

Two bacteria, Bacillus sp. and Pseudomonas sp., were isolated from parathionamended flooded alluvial soil which exhibited parathion-hydrolyzing ability. Bacillus sp. readily liberated nitrite from the hydrolysis product, p-nitrophenol, but not from intact parathion. Pseudomonas sp. hydrolyzed parathion and then released nitrite from p-nitrophenol. These studies establish bacterial degradation of parathion past the p-nitrophenol stage to the end product, nitrite.