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Bioremediation of Organophosphorus Pesticides by Surface-Expressed Carboxylesterase from Mosquito on Escherichia Coli
Abstract
The insecticide resistance-associated esterase, carboxylesterase B1 (CaE B1), from mosquito was used to degrade the organophosphorus compounds. To eradicate the need for enzyme purification and minimize the resistance to mass transport of the substrate and product across the cell membranes, the CaE B1 was displayed on the cell surface of Escherichia coli fused to the C-terminus of the ice nucleation protein (INP). The presence of CaE B1 on the bacterial cell surface was verified by SDS-PAGE, Western blotting analysis, and immunofluorescence microscopy. More than 50% of active CaE B1 is exported across the membrane and anchored onto the cell surface as determined by proteinase accessibility and cell fractionation experiments. In contrast, only a 6% drop in activity for proteinase K-treated cells was detected from E.coli cells containing pET-B1. From the degradation experiment, more than 80% of the malathion was degraded by whole cells containing plasmid pUC-NC-B1. Constitutive expression of CaE B1 on the surface using INPNC resulted in no cell lysis, and the suspended cultures also exhibited good stability. Because of their high biodegradation activity and superior stability, these "live biocatalysts" are promising for detoxification of organophosphorus pesticides.
... Over the past decade, pesticide usage to control pests surpassed 4 million tons annually worldwide [1]. Less than 1% of the applied pesticides can reach to the target pests [2,3]. Thus, the remaining percent of pesticides used end up in the environment, where they are contaminating the nearby water bodies through precipitation, surface runoff, and soil leaching, which will worsen the water quality [4]. ...
... For dimethoate, it is considered as one of the highest water-soluble pesticides (23.8 g/L, Kow =0.7) of the pesticides mixture under study, resulting in difficult removal from water compared to other pesticides, this was noticeable in this study. But with the increase in acidity (3)(4)(5), the ability of CCB to remove dimethoate increases significantly; this can be explained by the increase in the acidity of the solution, the hydroxyl groups attached to the surface increase [43]. Accordingly, increasing the acidity cause attraction of pesticides, including dimethoate. ...
This work aimed to study the isotherm and kinetics of adsorption of a mixture of 10 pesticides (viz., atrazine, chlorfenvinphos, chlorpyrifos, cyprodinil, diazinon, dimethoate, diuron, ethion, malathion, and profenofos) on corn cob biochar (CCB) in an aqueous solution. CCB is a low-cost adsorbent produced from pyrolysis at 500°C for 3 h in an oxygen-limited condition. It was characterized by Brunauer–Emmett–Teller (BET), X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier-transform infrared (FTIR) spectroscopy. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the residual pesticide concentration. Adsorption equilibrium and kinetics were investigated by using batch adsorption experiments. The optimized pH, contact time, adsorbent dose, and initial pesticides concentration for the maximum adsorption rates were found to be 3, 60 min, 10 g/L, and 500 μg/L, respectively. The sorption of all pesticides by CCB was found to enhance by decreasing pH; since the removal percentage of atrazine increased from 57 to 74 % by decreasing the pH from 9 to 3. The Langmuir model represented the adsorption process better than the Freundlich model with R2 values ranging from 0.957 to 0.999. Adsorption kinetic data were fitted well with the pseudo-first order kinetic model. Accordingly, CCB can be used as an effective low-cost agricultural waste that could be applied for removing pesticide residues from water.
... The spots obtained from TLC was purified and fractionated on 10% SDS-PAGE analysis, to quantify the molecular weight of component present in the extract. BSA was used as a standard [12]. ...
... Janarthanan et al. [10] reported cypermethrin resistant Actinobacterium, Streptomyces diastaticus from jasmine garden land in Namakkal district of Tamilnadu. In Flower gardens and BT cotton cultivation, enormous quantity of pesticides are applied to get more yield [12]. Bacteria resistant to these pesticides might be useful for the degradation of ß Cypermethrin pesticide-contaminated soil. ...
The aim of this investigation was to find enzymes and metabolites involved in ß Cypermethrin pesticide degradation. The continuous usage of chemical pesticide for rapid agriculture activities leads to the major cause of soil pollution. In this study, Bacillus cereus, a bacterium isolated from the BT cotton cultivated and pesticide contaminated soil has the efficiency to degrade ß Cypermethrin, a synthetic pyrethroid insecticide used in most crop cultivations. Standard lab scale bioremediation process was followed on mineral salt agar medium (MSM) enriched with ß Cypermethrin. Initially, the fragmentation of ß Cypermethrin was confirmed by Thin-Layer Chromatography (TLC), which revealed the existence of metabolites with Retention Factor (RF) value range of 0.47 and 0.71, corresponding to standard samples of 3-PBA and phenol. Later, SDS-PAGE analysis was carried out to identify enzymes involved in the biodegradation, and a protein of size 56 kDa molecular weight was identified and confirming the presence of Pyrethroid hydrolase. The fragmented ß Cypermethrin metabolite was reconfirmed by GC–MS analysis, which confirmed the degradation of ß Cypermethrin. The peaks of metabolites were recognized as Benzylamine and related components, these are derivatives of ß cypermethrin in degraded by B. cereus. Hence, the study concludes that, the 56 kDa molecular weight Pyrethroid hydrolase synthesized by B. cereus has the efficiency to metabolize the ß Cypermethrin pesticide. Thus, the B. cereus could be most suitable agent for chemical pesticide degradation.
... The results of the degradation experiment revealed that these carboxylesterase enzymes could efficiently degrade more than 80% of malathion. This study concluded that due to their rapid degradation ability, superior stability, and high activity, these enzymes could further degrade other organophosphate pesticides that contaminate the environment (Zhang et al., 2004). ...
Synthetic pesticides are extensively and injudiciously applied to control agriculture and household pests worldwide. Due to their high use, their toxic residues have enormously increased in the agroecosystem in the past several years. They have caused many severe threats to non-target organisms, including humans. Therefore, the complete removal of toxic compounds is gaining wide attention to protect the ecosystem and the diversity of living organisms. Several methods, such as physical, chemical and biological, are applied to degrade compounds, but as compared to other methods, biological methods are considered more efficient, fast, eco-friendly and less expensive. In particular, employing microbial species and their purified enzymes makes the degradation of toxic pollutants more accessible and converts them into non-toxic products by several metabolic pathways. The digestive tract of insects is usually known as a superior organ that provides a nutrient-rich environment to hundreds of microbial species that perform a pivotal role in various physiological and ecological functions. There is a direct relationship between pesticides and insect pests: pesticides reduce the growth of insect species and alter the phyla located in the gut microbiome. In comparison, the insect gut microbiota tries to degrade toxic compounds by changing their toxicity, increasing the production and regulation of a diverse range of enzymes. These enzymes breakdown into their derivatives, and microbial species utilize them as a sole source of carbon, sulfur and energy. The resistance of pesticides (carbamates, pyrethroids, organophosphates, organochlorines, and neonicotinoids) in insect species is developed by metabolic mechanisms, regulation of enzymes and the expression of various microbial detoxifying genes in insect guts. This review summarizes the toxic effects of agrochemicals on humans, animals, birds and beneficial arthropods. It explores the preferential role of insect gut microbial species in the degradation process and the resistance mechanism of several pesticides in insect species. Additionally, various metabolic pathways have been systematically discussed to better understand the degradation of xenobiotics by insect gut microbial species.
... The newly discovered and/or characterized enzymes are mentioned in Table 2. Many microbes were found to express enzymes on the microbial cell surface to rapidly detoxify OPs compounds [30][31][32][33][34]. The degradation of OPs compounds by engineered microorganisms is well documented [35]. ...
Organophosphates (OPs) compounds are universally used as pesticides and maintained as chemical warfare agents by many nations across the globe. These OPs compounds due to their molecular structure are highly persistent in nature, contaminating soil and water equally, thereby adversely affecting terrestrial and aquatic life, and contributing to millions of poisoning cases every year worldwide. Therefore, there are urgent requirements for safe and rapid method/s for environmental restoration and therapeutic detoxications. Organophosphate hydrolyzing enzymes are emerging as an attractive candidate for the degradation of OPs compounds. The biologically driven approach is safe, rapid, and environment-friendly. As genetically modified microbes are not in practice worldwide, scientists are exploring different bioremediation approaches that mainly focus on cell-free biocatalytic systems. In this review, we have discussed the prevalence of OPs hydrolyzing enzymatic systems and the recent advancement of enzyme engineering in enhancing the catalytic activity, substrate specificity, and half-life. It highlights the application in OPs detection, decontamination (environmental bioremediation), and therapeutic detoxification using approaches like immobilization. We have also described the remaining challenges and future prospects.
... The carboxylesterase coding gene, CaE B1, was associated with insecticide resistance in mosquitoes and was expressed in E. coli. Western blotting, a proteinase accessibility assay, and immunofluorescence microscopy were used to determine the expression patterns of CaE B1 on the surface of E. coli cells (Zhang et al., 2004). Green fluorescent protein (GFP) fusion assays were also used to study the organophosphate detoxifying hydrolase gene (Brander et al., 2016;Kang et al., 2002;Lee et al., 2016). ...
Ester-containing organophosphate, carbamate, and pyrethroid (OCP) pesticides are used worldwide to minimize the impact of pests and increase agricultural production. The toxicity of these chemicals to humans and other organisms has been widely reported. Chemically, these pesticides share an ester bond in their parent structures. A particular group of hydrolases, known as esterases, can catalyze the first step in ester-bond hydrolysis, and this initial regulatory metabolic reaction accelerates the degradation of OCP pesticides. Esterases can be naturally found in plants, animals, and microorganisms. Previous research on the esterase enzyme mechanisms revealed that the active sites of esterases contain serine residues that catalyze reactions via a nucleophilic attack on the substrates. In this review, we have compiled the previous research on esterases from different sources to determine and summarize the current knowledge of their properties, classifications, structures, mechanisms, and their applications in the removal of pesticides from the environment. This review will enhance the understanding of the scientific community when studying esterases and their applications for the degradation of broad-spectrum ester-containing pesticides.
... The worldwide pesticide consumption reported by FAOSTAT is 4,116,832 tons of active ingredients in 2016, but less than 1% of the total applied pesticides gets to the target pests [13]. Concerning the type of pesticide used in Ethiopia, there are several disputing reports. ...
... As will be discussed in subsequent sections, several enzymes have been identified in nature capable of degrading OP pesticides and CWAs (Dawson et al., 2008;Yair et al., 2008;Pizzul et al., 2009;Diao et al., 2013;Iyer et al., 2013;Geed et al., 2016;Brar et al., 2017). Several of these enzymes have been localized to the surface of microbes for evolution of substrate promiscuity and direct bioremediation of OP compounds (Richins et al., 1997;Cho et al., 2002;Zhang et al., 2004;Makkar, 2013;Alves et al., 2015Alves et al., , 2016Alves et al., , 2018Bigley et al., 2015;Bigely et al., 2019). Cell-mediated bioremediation and biocatalysis is well-established and documented within the literature, however, application can be limited as the release of genetically-modified organisms is not currently considered a viable approach to bioremediation. ...
Organophosphate compounds are ubiquitously employed as agricultural pesticides and maintained as chemical warfare agents by several nations. These compounds are highly toxic, show environmental persistence and accumulation, and contribute to numerous cases of poisoning and death each year. While their use as weapons of mass destruction is rare, these never fully disappear into obscurity as they continue to be tools of fear and control by governments and terrorist organizations. Beyond weaponization, their wide-scale dissemination as agricultural products has led to environmental accumulation and intoxication of soil and water across the globe. Therefore, there is a dire need for rapid and safe agents for environmental bioremediation, personal decontamination, and as therapeutic detoxicants. Organophosphate hydrolyzing enzymes are emerging as appealing targets to satisfy decontamination needs owing to their ability to hydrolyze both pesticides and nerve agents using biologically-derived materials safe for both the environment and the individual. As the release of genetically modified organisms is not widely accepted practice, researchers are exploring alternative strategies of organophosphate bioremediation that focus on cell-free enzyme systems. In this review, we first discuss several of the more prevalent organophosphorus hydrolyzing enzymes along with research and engineering efforts that have led to an enhancement in their activity, substrate tolerance, and stability. In the later half we focus on advances achieved through research focusing on enhancing the catalytic activity and stability of phosphotriesterase, a model organophosphate hydrolase, using various approaches such as nanoparticle display, DNA scaffolding, and outer membrane vesicle encapsulation.
... When a pesticide is applied, less than 1% reaches the target organism and the remaining fraction is distributed among the plant foliage, soil, atmosphere, biota and aquatic systems and is detected in areas far from the application point (Kuhad et al., 2004;Zhang et al., 2004). Precipitation plays a fundamental role in the input of pesticides to waterbodies, which reach this medium in the dissolved or particulate phase through surface or sub-surface runoff, leaching, drift and/or post-use container cleaning (Arias-Estevez et al., 2008;Bereswill et al., 2013). ...
... Thus, INP is often used as a protein carrier with no influence on the structure of the cell membrane and growth of the cell. Since INP is a large protein, truncated INPs consisting of INP-N alone [26][27][28] or in combination with INP-C (INP-NC) [29,30] are also applied as protein carriers. ...
... It is estimated that less than 1% of the total pesticide sprayed reaches the target, such as pests and weeds (Pimentel, 1983;Zhang et al., 2004). Other authors believe that about 45% of the pesticides reaches the target crop, whereas 30% is lost by drift, 10% is transported (volatilization, leaching and runoff) and 15% reaches the soil (Gavrilescu, 2005). ...
O uso de agrotóxicos na agricultura é praticamente indispensável, contudo, estudos estimam que somente 45% do produto aplicado atinge a cultura, sendo que aproximadamente 30% sofrem deriva, 10% são perdidos por algum processo de transporte (lixiviação, volatilização e escoamento) e 15% atingem o solo. Além disso, do total aplicado, apenas 1% atinge o alvo, ou seja, patógenos, plantas daninhas e insetos. Embora avaliações de riscos de contaminação ambiental de agrotóxicos sejam obrigatórios para o registro e comercialização, frequentemente estudos relatam a presença destes em locais não alvos, sendo os corpos de água os principais prejudicados. Vários processos biológicos, físicos e químicos determinam o destino ambiental e a eficiência desses. Assim, diversos métodos experimentais podem ser utilizados para avaliar o comportamento de agrotóxicos no ambiente. Essa revisão objetiva resumir os métodos mais comumente utilizados para descrever e avaliar os processos nos quais o herbicida está sujeito quando presente no solo, bem como, as vantagens e desvantagens de cada um desses. No geral, em se tratando de destino de agrotóxicos no ambiente, os experimentos em laboratórios fornecem informações mais precisas, pois é possível controlar melhor as condições ambientais e isolar fatores de interesse. Mas é importante salientar que não existe um método mais eficiente, o que existe são métodos que podem fornecer um melhor resultado em determinada situação. Por tanto, a escolha do método vai depender do objetivo do estudo e da estrutura disponível.
... Aujourd'hui, ce sont plus de 500 matières actives différentes qui sont utilisées dans l'environnement et la consommation annuelle est estimée à environ 4 millions tonnes au niveau mondial. Seulement environ 1 % de cette quantité arrive directement sur les parasites cible, tandis que près de 30 à 50 % de la quantité appliquée peut être perdue dans l'air (Gavrilescu, 2005 ;Zhang et al., 2004 ;Gil et Sinfort, 2005). ...
Les principaux processus et facteurs qui influent sur le devenir du glyphosate dans les sols et le risque de contamination de la ressource en eau ont été étudiés. Son adsorption sur les sols est très rapide (Kf compris suivant le sol entre 16,6 à 34,5) et l'effet du pH sur ce processus a été confirmé : l'adsorption diminue quand le pH des sols augmente. Par contre, le glyphosate se désorbe difficilement et sa dégradation en conditions contrôlées ou naturelles est rapide, mais sa dynamique est très variable suivant l'activité biologique des sols. La dégradation conduit à la formation d'un métabolite, l'AMPA qui tend à s'accumuler dans le sol. L'expérimentation en colonnes de sol confirme la faible mobilité du glyphosate et de l'AMPA. Les résidus exportés par les percolats sur une période de 332 jours représentent moins de 0,28 % de la dose appliquée. Les propriétés hydrodynamique du sol et la pluviométrie rencontrée ont un effet important dans le lessivage des résidus
... Different types of adsorbents like plants, activated carbon, carbon cloth, bacteria, straw (Lan et al., 2004) have been used in the case of metribuzin removal. The present study proposes the use of a new biosorbent, Pleurotus mutilus (mycelial basidiomycetes) for the treatment of water charged with metribuzin pesticide. ...
International Biodeterioration & Biodegradation
Available online 16 July 2016
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Adsorption study of metribuzin pesticide on fungus Pleurotus mutilus
Mourad Behloula, c, Hakim Lounicia, , , Nadia Abdia, , Nadjib Drouichea, b, Nabil Mameria
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doi:10.1016/j.ibiod.2016.07.005
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Highlights
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Pleurotus mutilus was used as biosorbent for metribuzin pesticide adsorption.
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Different parameters likely to have an influence on the biosorption of metribuzin were studied.
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The parameters that affect the metribuzin adsorption, have been investigated and optimized.
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Adsorption rate of Metribuzin of 70% were obtained at the optimum conditions.
Abstract
The aim of the present work is the valorization of the biomass Pleurotus mutilus fungal biomass in the biosorption of metribuzin pesticide. The present study constitutes of two principal parts. The first part includes physical pretreatment and structural characterization of the biomass. In the second part, different parameters likely to have an influence on the biosorption capacity of metribuzin such as biomass, particle size, biosorbent content, agitation, temperature, pH and metribuzin concentration were studied. The results of adsorption experiments obtained for synthetic water were convincing, and an adsorption rate of metribuzin of approximately 70% was reached for the following optimum conditions: particle size [250–400 μm], pH = [2–3], biomass content 3 g, agitation of 250 rpm, temperature of 25 °C, and initial concentration of metribuzin = 200 mg L−1.
... Carboxylesterases can degrade OPPs, carbamates, pyrethoid insecticides, and organic chloride pesticides [20], [21]. Usage of significant quantities of these enzymes for the removal of OPPs in waste water treatment had been proposed by several researchers [3], [22]- [25]. ...
Bacterial strains capable of degradation of malathion from the domestic sewage were isolated by an enrichment culture technique. Three bacterial strains were screened and identified as Acinetobacter baumannii (AFA), Pseudomonas aeruginosa (PS1), and Pseudomonas mendocina (PS2) based on morphological, biochemical identification and 16S rRNA sequence analysis. Acinetobacter baumannii AFA was the most efficient malathion degrading bacterium, so used for further biodegradation study. AFA was able to grow in mineral salt medium (MSM) supplemented with malathion (100 mg/l) as a sole carbon source, and within 14 days, 84% of the initial dose was degraded by the isolate measured by high performance liquid chromatography. Strain AFA could also degrade other organophosphorus compounds including diazinon, chlorpyrifos and fenitrothion. The effect of different culture conditions on the degradation of malathion like inoculum density, other carbon or nitrogen sources, temperature and shaking were examined. Degradation of malathion and bacterial cell growth were accelerated when culture media were supplemented with yeast extract, glucose and citrate. The optimum conditions for malathion degradation by strain AFA were; an inoculum density of 1.5x 10 12 CFU/ml at 30°C with shaking. A specific polymerase chain reaction primers were designed manually using multiple sequence alignment of the corresponding carboxylesterase enzymes of Acinetobacter species. Sequencing result of amplified PCR product and phylogenetic analysis showed low degree of homology with the other carboxylesterase enzymes of Acinetobacter strains, so we suggested that this enzyme is a novel esterase enzyme. Isolated bacterial strains may have potential role for use in bioremediation of malathion contaminated.
... Pesticides are applied to crops worldwide at a rate of around four millions tons/year (Zhang et al., 2004). More than 500 different formulations of these compounds are being applied in the environment and create hazards in the environment (Gavrilescu, 2005). ...
... Carboxylesterases (carboxylic ester hydrolase; EC 3.1.1.1) can degrade organophosphorus pesticides, carbamates, pyrethoid insecticides, and organic chloride pesticides (Vontas et al. 2000;Zhang et al. 2004; Barata et al. 2004;Nishi et al. 2006). Carboxylesterases can also catalyze the degradation of malathion to the detoxication products monoacid and diacid derivatives (Yoshii et al. 2007a). ...
A carboxylesterase gene from thermophilic bacterium, Alicyclobacillus tengchongensis, was cloned and expressed in Escherichia coli BL21 (DE3). The gene coded for a 513 amino acid protein with a calculated molecular mass of 57.82 kDa. The deduced amino acid sequence had structural features highly conserved among serine hydrolases, including Ser204, Glu325, and His415 as a catalytic triad, as well as type-B carboxylesterase serine active site (FGGDPENITIGGQSAG) and type-B carboxylesterase signature 2 (EDCLYLNIWTP). The purified enzyme exhibited optimum activity with β-naphthyl acetate at 60 °C and pH 7 as well as stability at 25 °C and pH 7. One unit of the enzyme hydrolyzed 5 mg malathion l(-1) by 50 % within 25 min and 89 % within 100 min. The enzyme strongly degraded malathion and has a potential use for the detoxification of malathion residues.
... It is estimated that four million tons of pesticides are applied to crops annually worldwide for pest control, but less than 1 % of the total applied pesticides gets to the target pests [29,30]. ...
The present paper is an overview of the presence and fate of pesticides as persistent organic pollutants in the environment as well as of the potential for their detoxification, also combined with chemical and physical treatment. It contains information gathered from a range of currently available sources. The fate of pesticides in the environment is analyzed considering the processes that determine their persistence and mobility, grouped into transport, transfer and transformation processes. Few pesticide characteristics such as persistence, mobility and biodegradability are emphasized. The fate of a pesticide and the potential for its persistence and mobility from the site of application are considered to be affected by the chemical and physical properties of the pesticide, site characteristics such as soil and groundwater individuality, climate and local weather conditions, biological population, and the handling practices of the pesticide user. Bioremediation, as one of the most environmentally-sound and cost-effective methods for the decontamination and detoxification of a pesticide-contaminated environment is discussed especially considering the factors affecting the biodegradability of pesticides such as biological factors and the characteristics of the chemical compounds. In situ and ex situ bioremediation as possible types of bioremediation activities are weighted up. Also, the paper includes some considerations for developing strategies regarding the choice of bioremediation technology, as well as advantages and disadvantages of the bioremediation of environmental components polluted with pesticides.
... Today, although most of the registered pesticides are very toxic and prohibited for use, more than 500 different formulations of pesticides are being applied in the environment; agriculture still accounts for the largest single share of pesticide use (Azevedo, 1998). It is estimated that 4 million tons of pesticides are applied to crops annually worldwide with less than 1% of the total applied pesticides getting to intended target species (Pimentel, 1983;Zhang et al., 2004). ...
Pesticides degrade principally through biodegradation processes, whereas antibiotics kill microorganisms or inhibit their growth in soils and thus may affect the fate of pesticides. In this study, the impact of antibiotics on the degradation of atrazine in a sandy soil is investigated in lysimeters over a ninety-day period. Four treatments, monensin, narasin, salinomycin and non-antibiotic, were assigned in triplicate to twelve PVC lysimeters. Both soil and leachate samples were collected and analyzed at predetermined time intervals. In all treatments, atrazine was found to leach down through the soil profiles with the concentration level decreasing with depth, and only trace amounts of atrazine were found in the leachate. However, the statistical analysis of the results showed that all the three antibiotic treatments yielded a significantly slower dissipation of the atrazine level as compared to the non-antibiotic treatment; the mass balance analysis indicated an increased half-life of atrazine in the presence of antibiotics.
... Coumaphos (CM) degrading Nocardioides simplex NRRL B-24074 has been reported to have novel organophosphate-degrading enzyme and gene systems [16]. Expression of opd genes in Escherichia coli has been done to maximize the recombinant protein yield for opd gene [17][18][19][20]. 3,5,6-Trichloro-2-pyridinol (TCP) the main transformation product of CP displays antibacterial activity at higher concentrations [21]. ...
Organophosphates are esters of phosphoric acid and can be hydrolyzed and detoxified by carboxylesterase and phosphotriesterase. In this work esterase enzyme (Est5S) was expressed in yeast to demonstrate the organophosphorus hydrolytic activity from a metagenomic library of cow rumen bacteria. The esterase gene (est5S) is 1098 bp in length, encoding a protein of 366 amino acid residues with a molecular weight of 40 kDa. Est5S enzyme was successfully produced by Pichia pastoris at a high expression level of approximately 4.0 g L−1. With p-nitrophenol butyrate as the substrate, the optimal temperature and pH for enzyme activity were determined to be 40 °C and pH 7.0, respectively. The esterase enzyme was tested for degradation of chlorpyrifos (CP). TLC results obtained inferred that CP could be degraded by esterase enzyme (Est5S) and HPLC results revealed that CP could be efficiently degraded up to 100 ppm. Cadusafos (CS), coumaphos (CM), diazinon (DZ) dyfonate (DF), ethoprophos (EP), fenamiphos (FM), methylparathion (MPT), and parathion (PT) were also degraded up to 68, 60, 80, 40, 45, 60, 95, and 100%, respectively, when used as a substrate with Est5S protein. The results highlight the potential use of this enzyme in the cleanup of contaminated insecticides.
... They show a high degree of sequence similarity and they are believed to be involved in the detoxification of many xenobiotics (Jakoby and Ziegler 1990). In many studies the gene encoding carboxylesterase was cloned and the recombinant protein was expressed (Kim et al. 2003;Zhang et al. 2004;Merone et al. 2005;Kakugawa et al. 2007). ...
Five malathion-degrading bacterial strains were enriched and isolated from soil samples collected from different agricultural sites in Cairo, Egypt. Malathion was used as a sole source of carbon (50 mg/l) to enumerate malathion degraders, which were designated as IS1, IS2, IS3, IS4, and IS5. They were identified, based on their morphological and biochemical characteristics, as Pseudomonas sp., Pseudomonas
putida, Micrococcus lylae, Pseudomonas aureofaciens, and Acetobacter liquefaciens, respectively. IS1 and IS2, which showed the highest degrading activity, were selected for further identification by partial sequence analysis of their 16S rRNA genes. The 16S rRNA gene of IS1 shared 99% similarity with that of Alphaprotoebacterium BAL284, while IS2 scored 100% similarity with that of Pseudomonas
putida 32zhy. Malathion residues almost completely disappeared within 6 days of incubation in IS2 liquid cultures. LC/ESI-MS analysis confirmed the degradation of malathion to malathion monocarboxylic and dicarboxylic acids, which formed as a result of carboxylesterase activity. A carboxylesterase gene (CE) was amplified from the IS2 genome by using specifically designed PCR primers. The sequence analysis showed a significant similarity to a known CE gene in different Pseudomonas sp. We report here the isolation of a new malathion-degrading bacteria from soils in Egypt that may be very well adapted to the climatic and environmental conditions of the country. We also report the partial cloning of a new CE gene. Due to their high biodegradation activity, the bacteria isolated from this work merit further study as potential biological agents for the remediation of soil, water, or crops contaminated with the pesticide malathion.
... Today, although most of the registered pesticides are very toxic and prohibited for use, more than 500 different formulations of pesticides are being applied in the environment; agriculture still accounts for the largest single share of pesticide use (Azevedo, 1998 ). It is estimated that 4 million tons of pesticides are applied to crops annually worldwide with less than 1% of the total applied pesticides getting to intended target species (Pimentel, 1983; Zhang et al., 2004). ...
Antibiotics are widely used in the livestock industry for preventing disease and improving feed efficiency. Many antibiotics are not completely absorbed by animals and they are excreted in urine and feces. When manure containing such antibiotics is used as a fertilizer, it can affect the degradation of pesticides since antibiotics inhibit the action of bacteria in soils or kill the bacteria. A ninety-day field lysimeter study was conducted to assess the effects of antibiotics on the degradation of three herbicides, atrazine, metribuzin and metolachlor, in a sandy soil. Poultry manures, respectively containing monensin, narasin and salinomycin, were used to investigate the effects of antibiotics on the degradation of herbicides, and were compared with a control treatment of manure without antibiotics. Herbicides were applied once to the soil surface of lysimeters at the locally recommended rates, followed by the application of poultry manures. The lysimeters were protected from natural rainfall, and the simulated rainfall was applied seven times. Both soil and leachate samples were collected and analyzed at predetermined time intervals. In the experiment, all the three herbicides were found to leach down through the soil profiles, and their concentrations decreased with soil depth and time. The statistical analysis of the results shows that all the three antibiotic treatments yielded a significantly slower dissipation of herbicide levels compared with the non-antibiotic treatment. The mass balance study reveals that the half lives of the three herbicides were significantly longer in the presence of antibiotics as compared to the control treatment without antibiotics. These results indicate that antibiotics in poultry manures can significantly slow down the degradation of the three herbicides in soil and therefore, increase the threat of herbicide pollution in the environment. Les antibiotiques sont couramment utilisés dans l'industrie du bétail pour prévenir les maladies et améliorer l'efficacité flux. Beaucoup d'antibiotiques ne sont pas complètement absorbés par les animaux et ils sont excrétés dans les urines et fèces. Lorsque le fumier contenant des antibiotiques est utilisé comme un engrais, il peut affecter la dégradation des pesticides puisque les antibiotiques contenus inhiber l'action de bactéries dans le sol ou tuer ces bactéries. Une étude de lysimètre de champ de quatre-vingt-dix-jour a été effectuée pour évaluer les effets des antibiotiques sur la dégradation de trois herbicides, atrazine, métribuzine et métolachlore, dans un sol sableux. Trois traitements des fumiers de volaille contenant monensine, narasine et salinomycine ont été utilisés pour étudier les effets des antibiotiques sur la dégradation des herbicides, et ont été comparés avec un traitement de contrôle de fumier sans antibiotiques. Herbicides étaient appliqués une fois à la surface de sol de lysimètres aux taux recommandés localement, suivie par l'application de fumiers de volaille. Les lysimètres ont été protégés des précipitations naturelles, et les précipitations simulées ont été appliqués sept fois. Des échantillons de sol et des échantillons d'eau ont été recueillis et analysées à intervalles de temps prédéterminés. Dans l'expérience, tous les trois herbicides ont été trouvés à s'infiltrer à travers les profils de sol et leurs concentrations ont diminués avec la profondeur du sol et de temps. L'analyse statistique des résultants ont montré que tous les trois traitements antibiotiques ont donnés une dispersion des niveaux d'herbicide sensiblement plus lent par rapport au traitement nonantibiotic. L'étude du bilan massique a révélé que les demi-vies des herbicides ont été significativement plus long avec l'utilisation d'antibiotiques comme par rapport au ce traitement de contrôle sans antibiotiques. Ces résultats indiquent que les antibiotiques dans les fumiers de volaille peuvent considérablement ralentir la dégradation des herbicides dans les sols et, par conséquent, augmenter la menace de la pollution des herbicides dans l'environnement.
... Compared to InaK anchor, the use of InaV anchor resulted in 5-and 100-fold higher OPH activity in E. coli and Moraxella sp., respectively (Shimazu et al., 2001a,b). We previously displayed CaE B1 (60 kDa) on E. coli surface using the InaV anchor (Zhang et al., 2004). The aim of this study was to explore the feasibility of targeting MPH onto the cell surface using the InaV anchor. ...
Methyl parathion hydrolase (MPH) has been displayed on the surface of microorganisms for the first time using only N- and C-terminal domains of the ice nucleation protein (INPNC) from Pseudomonas syringae INA5 as an anchoring motif. A shuttle vector pINCM coding for INPNC-MPH was constructed and used to target MPH onto the surface of a natural p-nitrophenol (PNP) degrader, Pseudomonas putida JS444, overcoming the potential substrate uptake limitation. Over 90% of the MPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of the INPNC-MPH fusion was further verified by Western blot analysis and immunofluorescence microscopy. The engineered P. putida JS444 degraded organophosphates as well as PNP rapidly without growth inhibition. Compared to organophosphorus hydrolase-displaying systems reported, changes in substrate specificity highlight an important potential use of the engineered strain for the clean-up of specific organophosphate nerve agents.
The environment is polluted with organic contaminants from many sources such as the transportation, chemical industry, and pesticide application in agricultural regions. Pesticides are used in over 500 distinct formulations in the environment today, with agriculture accounting for the majority of pesticide use. Organic (carbon-based) compounds that comprise manufactured molecules have been classified as persistent organic pollutants. These contaminants stay in soils for a long period, where they enter into the food chain directly or seep down to underground water. Their potential as carcinogens, as well as their prevalence in the water, soil, and air, raised concerns about their remediation. Bioremediation is a process which utilizes microbes or microbial enzymes to treat polluted places in order to restore them to their previous state. Microbes either consume the toxins or assimilate all toxic substances from the environment, making the area virtually contaminant-free. Organic molecules are generally eaten up, whereas heavy metals and pesticides are digested within the system. In this chapter, various microbes and recent advance tools for enhanced efficiency of pesticides bioremediation have been discussed in detail.KeywordsBioremediationMicrobesEnzymesPesticidePollution
Organophosphorus compounds are widely used in pesticides, insecticides in agriculture and as nervous chemical agents. These chemicals inhibit the acetylcholinesterase enzyme activity that is responsible for the nervous impulse in organisms. This effect leads to an increase in acetylcholine level and finally neuronal complications. Many methods are used to degrade and decontaminate these compounds, such as: the use of chemicals, burial of toxins, burning and biodegradation. The chemical and physical methods are often toxic, allergic, corrosive and nonspecific and harmful for the environment and are not usable in war spaces. Biodegradation is an effective and safe method that is performed under controlled conditions for the decomposition of various constituents, including organophosphorus compounds, by biological agents. Biodegradation is performed using microbes to detoxify and decompose contaminants. These strains contain broad substrate-degrading enzymes. Although the use of natural strains as vital catalysts is an interesting method for the treatment of organophosphorus compounds, the inability of organophosphorus compounds to cross the membrane width reduces the total catalytic power, so the use of recombinant enzymes for the decomposition of organophosphorus compounds can be of great help in removing contaminants, especially in war environments. Many enzymes have been identified and used for this purpose, but most notably include: Diisopropyl-fluorophosphatase (DFPasae), Organophosphorus acid anhydrolase (OPAA) and Organophosphorus Hydrolase (OPH). In this review, in addition to describing the organophosphorus compounds and their effects, biodegradation especially by use of enzymes was considered. The understanding and mastering of this knowledge could help researchers in the use of chemical degrading enzymes, especially organophosphates, in spray and enzymatic ointments in military environments.
Malathion organophosphates considered as the major constituent of herbicides, pesticides and insecticides. Extensively used in agricultural, horticultures and for numerous household applications contributes to precedence organic pollutants leading antagonistic effects on human health and environment. Therefore detoxification of malathion from contaminated site is of general interest. Simultaneously it is very emerging to isolated novel indigenous microbial strains from contaminated site with a record of pesticide application. In this study Escherichia coli IES-02 isolated from malathion contaminant effluent and the strain showed maximum efficiency in malathion degradation that utilized it as the sole source of carbon. Carboxylesterase (33.0, 30.0, 28.0 kDa) were purified (1685.71 U/mg) from Escherichia coli IES-02 showed significant results in malathion degradation approximately 81% within 20 min as compared with Escherichia coli IES-02 cells within 4 h (99.0 to 95.0%) into monocarboxylic acid and diacid derivatives. The generation time of Escherichia coli was also observed at 60 min with 0.1 ppm, 68 min with 0.5 ppm, 74.5 min with 2.0 ppm and 91.37 min with 50 ppm of malathion. The degradation rate and transformation metabolites were estimated by Gas Chromatography-Mass Spectrometry respectively. Malathion metabolites pathway proposed in this study which revealed the potential application against lethal environmental pollution.
Pesticides are among the most widespread organic contaminants in aquatic environments. In this work, a new green fluorescence application was proposed for the simultaneous determination of four widely employed pesticides in environmental water samples. To overcome the highly overlapped spectra within the analytes, and with the tissue matrix interferences in complex solutions, we have used the multivariate calibration methods such as parallel factor analysis (PARAFAC) and unfolded partial least squares coupled to residual bilinearization (U-PLS/RBL). These four pesticides can be identified simultaneously, and the correlation coefficients between resolved and actual spectra are all above 0.95. The second-order advantage allowed the determination of four pesticides at the ng mL-1 level, even in the presence of humic acid (HA). The best results were obtained with the limits of detection of 1.72-18.69 for Carbendazim (CBZ), 0.30-5.19 for carbaryl (CAR), 0.35-6.32 for chlorothalonil (CHL), and 4.92-29.96 for tsumacide (TSU) (ng mL-1), which can fully meet the quantitative detection and analysis requirements of trace pesticides in water samples. The real water sample of Bohai Seawater was used to check the performance of this approach in practical applications, which have achieved good prediction results of U-PLS/RBL. This study demonstrated the proposed method is rapid, accurate, sensitive, low detection limit, and environmentally friendly to determinate multi-pesticide residues in environmental water samples.
A field-scale experiment with randomized block design of three blocks was performed to study the degradation of Malathion, an organophosphate pesticide (commercial grade). Each block was further divided in to four plots of 3 square meters. Malathion was applied in two different concentrations (1000 mg/Kg in blocks A and B and 1500 mg/Kg in C). Twenty bacterial strains previously isolated from pesticides contaminated soils were applied in the form of consortium. One block was taken as without inoculums for the activity of indigenous microbes. The application rate was based on the soil weight, present in an area of 3 square meters with 6 inches depth. Sodium nitrate and potassium phosphate were applied as micronutrients for microbial growth. Soil samples were taken with interval of one week. Pesticides were extracted from soil samples with acetone. Extracts were centrifuged and analyzed by HPLC. Malathion was biodegraded up to 96% in a period of one week in each plot except plot A-l and C-l. Negative effect of increasing micronutrients was observed in plot C-1 with indigenous microbes. Microbial counts in the field plots were continuously increasing and no decline observed in growth even after six weeks.
Studies of microbial cell envelopes and particularly cell surface proteins and mechanisms of their localization brought about new biotechnological applications of the gained knowledge in surface display of homologous and heterologous proteins. By fusing surface
proteins or their anchoring domains with different proteins of interest, their so-called genetic immobilization is achieved. Hybrid proteins are engineered in a way that they are expressed in the host cells, secreted to the cell surface and incorporated into the wall/
envelope moiety. In this way, laborious and often detrimental procedure of chemical immobilization of the protein is avoided by letting the cells do the whole procedure. Both bacterial and yeast cells have been used for this purpose and a number of potential biotechnological applications of surface-displayed proteins have been reported. Among the most frequently used passenger proteins are lipolytic enzymes, due to their great technological significance and numerous important applications. In this review, our current knowledge on mechanisms and molecular systems for surface display of lipolytic enzymes on bacterial and yeast cell surfaces is summarized.
IntroductionSystems for Gram-Negative BacteriaSystems for Gram-Positive BacteriaSummaryAcknowledgmentsReferences
Carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranoyl N-methylcarbamate) was first introduced into China in 1979 as a pesticide and is extensively used as a soil-incorporated N-methylcarbamate insecticide to control a variety of insect pests that infest many kinds of crops, such as corn and potato. Carbofuran is of a concern due to its relatively high mammalian toxicity and relatively high mobility in soil. Some N-methylcarbamate compounds and their metabolites can be sufficiently mobile in certain soil, thus considered as potential pollutants of ground and surface waters. One carbofuran-degrading strain named CFDS-1 was isolated from soil polluted by carbofuran for a long time. According to its physiological & biochemical characters, and the homology analysis of its 16S rDNA sequence (GenBank accession No. AY702969), CFDS-1, was primarily identified as Sphingomonas sp. CFDS-1 was able to completely degrade carbofuran in medium within 48 hours at the initial concentration of 100 mg L-1. CFDS-1 could well degrade carbofuran at concentrations from 20 mg L -1 to 300 mg L-1. The degradation speed was related positively to initial inoculation amount. The optimal pH and aeration for the degradation were 8.0-9.0 and 100∼250 mL, respectively. The temperature (between 25 °C and 42 °C) had no notable effect on the degradation of carbofuran by CFDS-1. CFDS-1 could well degrade carbofuran not only in medium, but also in soil.
The use of genetically modified or native microorganisms and plants to degrade or remove pollutants has emerged as a powerful technology for in situ remediation. An understanding of the genetic basis of the mechanisms of how microorganisms and plants biodegrade pollutants and how they interact with the environment is important for successful implementation of this technology. Recent studies have demonstrated that microbes and transgenic plants produce pesticide-degradaing enzymes that can mineralize different groups of pesticides and their metabolites with greater efficiency. This review describes the most recent progress in biotechnological approaches for enhancing the capability of microorganisms and plants through the characterization and transfer of pesticide-degrading genes, induction of catabolic pathways, and display of cell surface enzymes.
Biosorption of metal ions is a metabolism-independent metal uptake event at the cell wall polysaccharides, associated molecules,
and functional groups. It involves mainly the ion-exchange, chemisorption, adsorption, and, in some cases, also the inorganic
microprecipitation of certain heavy metal species. In the search for strategies allowing for enhancements of the biosorption
capacity for a specific metal ion, display of particular amino acid sequences with the capacity to form coordination centers
for the metal ions at the microbial cell wall has proved to be a promising approach. To anchor particular metal-binding moiety
by means of recombinant DNA technology, a wide range of cell-surface display systems is available for both Gram-negative and
Gram-positive bacteria. These involve outer membrane proteins, autotransporters, lipoproteins, cell-wall associated and covalently
bound proteins, S-layer proteins and subunits of surface appendages. Surface displays of metal-binding oligopeptides, metallothioneins,
or metalloproteins has been shown to improve the natural biosorption capacity or even selectivity for particular metal ion
in model cells Escherichia coli. Furthemore, this approach was successfully extended to environmentally robust Pseudomonas, Cupravidus or Moraxella species. This chapter provides overview of available surface display systems and summarizes evidence supporting suitability
of cell-surface-display technology in tailoring microbial biosorbents.
KeywordsBioremediation-Biosorption-Cell-surface display-Metal-binding peptide-Heterologous gene expression
Chemical control of pests is still necessary in agriculture, despite the growing efforts to introduce biocontrol-based strategies. Many studies have evidenced the harm of pesticide side-effects on natural populations of pest enemies and other non-target organisms. Moreover, pesticide-contaminated soils can act as a secondary pollution source causing contamination in environmental compartments of critical concern to public health (water resource). These environmental risks need to be assessed and monitored for decision-making related to the post-authorization management of pesticides. Under these considerations, earthworm esterases can be a suitable tool for these regulatory and environmental purposes. Herein, it is suggested the use of earthworm esterases as biomarkers to be included in a field toxicity test. Furthermore, the potential role of gut carboxylesterases (CEs) in the modulation of pesticide toxicity is discussed in view of their contribution to the natural tolerance of earthworms to pesticides, and consequently the appropriate selection of earthworm species for regulatory toxicity testing. Finally, it is postulated that CE secretion into the earthworm gut could be an environmental friendly methodology in the enzymatic bioremediation of pesticide-contaminated soils. (C) Pesticide Science Society of Japan
Bacterial surface display entails the presentation of recombinant proteins or peptides on the surface of bacterial cells. Escherichia coli is the most frequently used bacterial host for surface display and, as such, a variety of E. coli display systems have been described that primarily promote the surface exposure of peptides and small proteins. By contrast, display systems based on autotransporter proteins (ATs) and ice nucleation protein (INP) are excellent systems for the display of large and complex proteins, and are therefore of considerable biotechnological relevance. Here, we review recent advances in AT and INP-mediated display and their biotechnological applications. Additionally, we discuss several promising alternative display methods, as well as novel bacterial host organisms.
Carboxylesterases (CbEs) are key enzymes in pesticide detoxification. These esterases are involved in the biochemical mechanism for pesticide resistance in some pest species, and further they are considered an efficient protective mechanism against acute toxicity by organophosphate (OP) pesticides in mammals. To gain knowledge on the role of CbEs in pesticide toxicity and natural tolerance in earthworms, we performed an enzyme kinetic analysis to investigate whether these annelids are able to secrete them into their gut lumen. We determined levels of CbE activity and isozyme abundance in the gut wall and ingested soil collected from different portions of the gastrointestinal tract of Lumbricus terrestris. Moreover, modulation of enzyme activity by selected substrates (alpha-naphthyl acetate [alpha-NA], 4-nitrophenyl valerate [4-NPV] and 4-nitrophenyl acetate [4-NPA]) and OP pesticides was examined to compare the response between tissue and soil CbEs. We found a high CbE activity in the ingested soil extracts from the crop/gizzard (alpha-NA-CbE=8.43+/-2.76U mg(-1) protein and 4-NPA-CbE=5.98+/-2.11U mg(-1) protein) compared to the gut wall. Three lines of evidences suggest that the gut epithelium is the main source of this luminal CbE activity. First, the effect of substrate concentrations on CbE activity from both the ingested soil extracts and gut tissues resulted in similar apparent K(m) and V(max) values. Second, native PAGE gels revealed that some of the CbE isozymes in the gut tissue were also present in the soil extracts. Third, tissue and soil CbEs showed the same sensitivity to inhibition by OPs. The concentrations of insecticide causing 50% of esterase inhibition (IC(50)) was comparable between tissue (IC(50)s range=4.01-9.67nM dichlorvos and 8480-6880nM paraoxon) and soil (IC(50)s range=6.01-11.5nM dichlorvos and 8400-7260nM paraoxon). Our results suggest a set of (eco)toxicological implications and environmental applications derived from the ability of earthworms to secrete these pesticide-detoxifying enzymes.
The kinetics of nucleophilic dephosphorylation of p-nitrophenyl diphenyl phosphate by hydroxamate ions (R'(C=O)N(RO-)) have been investigated in aqueous cationic micellar media at pH 9.12 and 27 degrees C. The pseudo-first-order rate constant-surfactant profiles show micelle-assisted bimolecular reactions involving interfacial ion exchange between bulk aqueous media and micellar pseudophase. N-Substituted hydroxamate ion shows higher reactivity over the unsubstituted hydroxamate ions in cationic micellar media. The kinetic data are discussed in terms of the pseudophase ion exchange model.
Genetically engineered Escherichia coli, expressing the fusion protein of enhanced green fluorescent protein (EGFP) and carboxylesterase B1 (CarE B1), was successfully constructed by cloning the genes into the pET-28b vector and then transforming E. coli BL21 (DE3). Expression of the fusion protein was induced in E. coli BL21 (DE3) which could then degrade environmental pesticides and could be easily detected using fluorescence spectrophotometry or by the naked eye in daylight.
The display of heterologous proteins on the microbial cell surface by means of recombinant DNA biotechnologies has emerged as a novel approach for bioremediation of contaminated sites. Both bacteria and yeasts have been investigated for this purpose. Cell surface expression of specific proteins allows the engineered microorganisms to transport, bio-accumulate and/or detoxify heavy metals as well as to degrade xenobiotics. These otherwise would not be taken up and transformed by the microbial cell. This review focuses on the application of cell surface displays for the enhanced bio-accumulation of heavy metals by metal binding proteins. It also reviews the biodegradation of xenobiotics by enzymes/proteins expressed on microbial cell surfaces.
Surface display is a powerful technique that uses natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications, ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production.
In order to develop a rapid and specific detection test for bacteria in soil, we improved a method based on the polymerase chain reaction (PCR). Each step of the protocol, including direct lysis of cells, DNA purification, and PCR amplification, was optimized. To increase the efficiency of lysis, a step particularly critical for some microorganisms which resist classical techniques, we used small soil samples (100 mg) and various lytic treatments, including sonication, microwave heating, and thermal shocks. Purification of nucleic acids was achieved by passage through up to three Elutip d columns. Finally, PCR amplifications were optimized via biphasic protocols using booster conditions, lower denaturation temperatures, and addition of formamide. Two microorganisms were used as models: Agrobacterium tumefaciens, which is naturally absent from the soil used and was inoculated to calibrate the validity of the protocol, and Frankia spp., an actinomycete indigenous to the soil used. Specific primers were characterized either in the plasmid-borne vir genes for A. tumefaciens or in the variable regions of the 16S ribosomal gene for Frankia spp. Specific detection of the inoculated A. tumefaciens strain was routinely obtained when inocula ranged from 10(7) to 10(3) cells. Moreover, the strong correlation we observed between the size of the inocula and the results of the PCR reactions permitted assessment of the validity of the protocol in enumerating the number of microbial cells present in a soil sample. This allowed us to estimate the indigenous population of Frankia spp. at 0.2 x 10(5) genomes (i.e., amplifiable target sequences) per g of soil.
The ice-nucleation protein (Inp) is a glycosyl phosphatidylinositol-anchored outer membrane protein found in some Gram-negative bacteria. Using Pseudomonas syringae inp as an anchoring motif, we investigated the functional display of a foreign protein, Zymomonas mobilis levansucrase (LevU), on the surface of Escherichia coli. The cells expressing Inp-LevU were found to retain both the ice-nucleation and whole-cell levansucrase enzyme activities, indicating the functional expression of Inp-LevU hybrid protein on the cell surface. The surface localization was further verified by immunofluorescence microscopy, fluorescence-activated cell sorting flow cytometry and immunogold electron microscopical examination. No growth inhibition or changes in the outer membrane integrity were observed upon the induction of fusion protein synthesis. Viability of the cells was also maintained over 48 hours in the stationary phase. Surface-displayed levansucrases were found to be resistant to the externally added proteases unless the cells were treated with EDTA. When the levansucrase-displayed cells were used as the enzyme source, levan (44 g/L) was efficiently synthesized from sucrose (130 g/L) with 34% (wt/wt) conversion yield, generating glucose (65 g/L) as a by-product.
The bacterial surface display method was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growth rates. This technique will provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases.
In most lineages of the subgenus Sophophora esterase-6 is a homodimeric haemolymph protein. In the melanogaster subgroup of species it has become a monomer which is mainly expressed in the male sperm ejaculatory duct. Our analyses of esterase-6 sequences from three melanogaster subgroup species and two close relatives reveal a brief period of accelerated amino acid sequence change during the transition between the ancestral and derived states. In this period of 2-6Myr the ratio of replacement to silent site substitutions (0.51) is about three times higher than the values in other lineages of the phylogeny. There are about 50 more replacements in this period than would be predicted from the ratios of replacement to silent site substitutions found elsewhere in the phylogeny. Modelling on the known structure of a related acetylcholinesterase suggests that an unusually high proportion of the replacements in the transitional branch are non-conservative changes on the protein surface. Up to half the accelerated replacement rate can be accounted for by clusters of changes to the face of the molecule containing the opening of the active site gorge. This includes changes in and around regions homologous to peripheral substrate binding sites in acetylcholinesterase. There are also three changes in glycosylation status. One region predicted to lie on the protein surface which becomes markedly more hydrophilic is proposed to be the ancestral dimerisation site that is lost in the transitional branch.
The paper includes a brief overview of the approaches and advantages of bioremediation along with the rationale of using insect genes in recombinant microbial and plant systems for bioremediation. Insecticide resistance- associated esterases and phosphotriesterases have been purified and characterized from several insect species. These esterases are responsible for high levels of insecticide resistance and bioaccumulate/metabolize a wide variety of toxic chemicals. A new family of cytochrome P450 (CYP9A1) apparently associated with insect insecticide resistance in the tobacco budworm was discovered. This P450 has been engineered into bacteria, tobacco and baculovirus for evaluation in bioremediation. Two epoxide hydrolases from the fat body and gut of the cabbage looper were cloned and sequenced. The fat body epoxide hydrolase was expressed in an insect cell line using baculovirus and is involved in the metabolism of the C10,11 epoxide of juvenile hormone. These and other insect enzymes are being investigated for possible practical uses in bioremediation.
Esterases in organophosphate susceptible and resistant houseflies were studied by means of sensitive Gomori method. In susceptible flies the esterase activity to α-naphthylacetate was found to be mainly due to the cholinesterase and an ali-esterase identical with that responsible for most of the activity to methylbutyrate. The latter enzyme is much less active to β-naphthylacetate than to α-naphthylacetate. The Km values of the cholinesterase for α- and β-naphthylacetate were 1·0 × 10-4 and 2·3 × 10-4 (M) respectively. The ali-esterase had a Km of approximately 10-4 (M) for α-naphthylacetate. The activity to α-naphthylacetate of the cholinesterase was strongly, that of the ali-esterase only weakly pH-dependent in the range from pH 6 to 8. Both enzymes were more active at higher pH. Eserine and diazoxon were used in inhibition experiments, acetylcholine and methylbutyrate in experiments on substrate competition. The Km value of the cholinesterase for acetylcholine was calculated as approximately 10-5 (M). The addition of heat-inactivated homogenate strongly enhanced the ali-esterase activity to α-naphthylacetate. It did so much more at low than at high concentrations of the active homogenate and thus caused the disappearance of the disproportionality initially observed between enzyme concentration and activity. This activation phenomenon was, to a lesser extent, also observed with β-naphthylacetate. The ali-esterase activity to α-naphthylacetate in homogenates of organophosphate resistant strains was only about 15 per cent of that found in homogenates of organophosphate susceptible strains. No significant differences between the activities in susceptible and resistant strains were found if β-naphthylacetate and indophenylacetate were used as substrates. Agar-gel electrophoresis of the supernatants obtained by high-speed centrifugation of homogenates proved the presence of about seven electrophoretically different esterases that occurred in more or less strain-specific patterns.
We constructed bacterial strains capable of efficient degradation of chlorinated environmental pollutants. The biphenyl dioxygenase (BP Dox) of Pseudomonas pseudoalcaligenes KF707, which is a multicomponent enzyme, was engineered in vitro and in vivo to degrade a wide range of polychlorinated biphenyls (PCB) and chloroethenes, such as trichloroethylene (TCE). One such PCB-degrading strain expressed a chimeric BP Dox and degraded PCB congeners via 2,3-dioxygenation and/or 3,4-dioxygenation, depending on the chlorine substitution. TCE-degrading Pseudomonas strains expressed hybrid dioxygenases in which todCl (encoding a large subunit of toluene dioxygenase of Pseudomonas putida F1) was substituted with bphA1 (encoding a large subunit of BP Dox) within chromosomal bph operons, and degraded TCE and cis-1,2- dichloroethylene very efficiently.
The use of synthetic pesticides has become an indispensable tool in agriculture for the control of pests. Therefore, the search for remedies and techniques for decontamination and detoxification of a pesticide-contaminated environment has become an important part of the research. Currently, bioremediation seems to be one of the most environmentally safe and cost-effective methods. In nature, the existence of abundant material resources can be used to degrade the environmental pesticide pollutants. At present, a number of microorganisms, capable of degrading pesticides, have been isolated and characterised. For insects, insecticide resistance-associated esterases have been purified and characterised from several insect species, and a new family of cytochrome P450 apparently associated with insecticide resistance in the tobacco budworm, was discovered. Generally, two bioremediation approaches have been used one directly based on microorganisms, and the other involved in isolated enzymes. For the former, in addition to using natural microorganism strains, with genetic techniques certain desirable biodegradation pathways from different organisms are brought together in a single host. However, because of their own limits, especially problems associated with releasing genetically altered microorganisms into the environment, the strategy based on enzymes seems more feasible. In the long term, collaborations between microbiologists, biochemists, and engineers will become increasingly important to efficiently dispose of the pesticide pollutants.
In this paper, the microstructure of Mg–10Zn–4Al–0.15Ca (wt-%) (ZAC104015) magnesium alloy was characterised in the as cast condition. Thermal analysis was performed to determine the liquidus, solidus, and intermediate reactions that occur upon cooling. The results show that this alloy is composed of α-Mg and two intermetallic phases containing calcium τ'1 and τ'2. These two intermetallic phases have the same type of crystal structure as that of phase τ (Mg32(Al,Zn)49). The thermal stability of the phases τ, τ'1, and τ'2, were also evaluated. This experimental study indicates that the phase τ'2 is more stable than τ and τ'1 at elevated temperature and directly related to creep resistance improvement.
Heliothis virescens (F.), tobacco budworms eggs were collected from 3 adjacent counties in North Carolina. A laboratory strain (YDK) was established from these eggs using precautions to avoid loss of genetic diversity. A subset of this laboratory strain (YHD2) was selected on artificial diet containing the Bacillus thuringiensis (Berliner) toxin, CryIA(c). In the, first 12 episodes of selection only moderate resistance (≍7- to 8-fold) was found. However, after 19 episodes of selection the strain had developed >500-fold resistance to the CryIA(c) toxin. Further selection lead to higher levels of resistance with the greatest resistance ratio recorded being ≍10,000-fold. The YHD2 strain was cross-resistant to CryIA(a), CryIA(b) and CryIF. We also found some resistance to CryIB, CryIC and CryIA, but the level of resistance to these toxins was more moderate. Reciprocal genetic crosses between the resistant and control strains indicated that resistance to CryIA(c) and to CryIA(b) was partially recessive, but that the low level of resistance to CryIA was more dominant. Progeny from backcrosses of F1 larvae to the resistant parent were placed on artificial diet containing a concentration of CryIA(b) that had previously been found to slow the growth of F1 larvae. When these larvae were weighed after 10 d, two clearly demarcated size classes were found in ≍1:1 ratios as is expected in backcrosses when a single locus (or a set of tightly linked loci) is coding for a major component of a recessive trait. Adults that developed from the larger size class of larvae were mated and their offspring inherited the ability to grow well on CryIA(b). As expected from the single locus model, one quarter of the offspring from matings of the small backcross larvae grew well on CryIA(b). Results of this selection experiment indicate that the initial frequency of this resistance trait could be approximately 10-3, but field tests will be needed to confirm this rough estimate.
Abstract We inserted the mosquito esterase B1 gene into the expression vector pRL-439, which possesses the strong promoter PpsbA. The recombinant plasmid pRL-Bl was used to transform E. coli HBlOl, and the positive clones were screened on LB medium plate containing 100 mg/mL ampicillin. The results of dot blotting and Southern hybridization demonstrated that these positive clones were transformed bacteria. Western blotting indicated that esterase B1 gene had been successfully expressed under the control of the PpsbA promoter in E. coli.
A shuttle verruction-B1 (pDGBl) was constructed by inserting B1-cDNA from pRL-Bl into polycloning site of plasmid pDc8. PDGBl was transferred into Synechoccus sp. PCC7942 through triparental conjugal transfer. Transformed single Synechococcus sp. PCC 7942 clone was obtained by neomycin screening, and large-scale culture in liquid medium was carried out. Results of Southern blotting proved that pWB1 was transferred into Synechococcus sp. PCC 7942.
Two strains of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), were found to be resistant to carbofuran and azinphosmethyl when compared to a susceptible strain in bioassays using a discriminating insecticide dose. The percentage of mortalities for carbofuran were 115.1- and 70.8-fold lower in the resistant New York (NY) and Hot Spot (HS) strains, respectively, than in susceptible beetles. When treated with azinphosmethyl, the percentages of mortalities for the NY and HS populations were 65.1- and 4.4-fold lower, respectively, than the susceptible strain. The synergist S,S,S-tributyl phosphorotrithioate (DEF) increased azinphosmethyl toxicity, suggesting esterase involvement in azinphosmethyl resistance. Although 1-naphthyl acetate esterase activities in individual fourth instars of resistant strains were not significantly different, a novel resistance-associated esterase (RAE) (pI = 6.23) was discovered in the first and fourth instars and adults of NY and HS CPBs, but not in the susceptible strain. The RAE was purified from fourth stadium NY CPBs by Rotofor followed by polyacrylamide gel isoelectric focusing. Methyl paraoxon was the most potent inhibitor of the purified RAE with an I50 of 0.1 μM. The esterase inhibitors octylihio-1,1,1-trifluoropropan-2-one (OTFP), DEF, and azinphosmethyl had I50s of 1.1, 5.5, and 9.8 μM, respectively. Carbofuran and eserine hemisulfate were poor inhibitors, with I50s greater than 100 μM. In substrate competition assays with the RAE, methyl paraoxon and OTFP were competitive inhibitors.
Esterases in organophosphate susceptible and resistant houseflies were studied by means of sensitive Gomori method. In susceptible flies the esterase activity to α-naphthylacetate was found to be mainly due to the cholinesterase and an ali-esterase identical with that responsible for most of the activity to methylbutyrate. The latter enzyme is much less active to β-naphthylacetate than to α-naphthylacetate.The Km values of the cholinesterase for α- and β-naphthylacetate were 1·0 × 10−4 and 2·3 × 10−4 (M) respectively. The ali-esterase had a Km of approximately 10−4 (M) for α-naphthylacetate.The activity to α-naphthylacetate of the cholinesterase was strongly, that of the ali-esterase only weakly pH-dependent in the range from pH 6 to 8. Both enzymes were more active at higher pH.Eserine and diazoxon were used in inhibition experiments, acetylcholine and methylbutyrate in experiments on substrate competition. The Km value of the cholinesterase for acetylcholine was calculated as approximately 10−5 (M).The addition of heat-inactivated homogenate strongly enhanced the ali-esterase activity to α-naphthylacetate. It did so much more at low than at high concentrations of the active homogenate and thus caused the disappearance of the disproportionality initially observed between enzyme concentration and activity. This activation phenomenon was, to a lesser extent, also observed with β-naphthylacetate.The ali-esterase activity to α-naphthylacetate in homogenates of organophosphate resistant strains was only about 15 per cent of that found in homogenates of organophosphate susceptible strains. No significant differences between the activities in susceptible and resistant strains were found if β-naphthylacetate and indophenylacetate were used as substrates.Agar-gel electrophoresis of the supernatants obtained by high-speed centrifugation of homogenates proved the presence of about seven electrophoretically different esterases that occurred in more or less strain-specific patterns.
Ice-nucleation protein (INP), an outer membrane protein from Pseudomonas syringae, is able to catalyze the ice crystal formation of supercooled water. It was exploited for anchoring of Bacillus subtilis carboxymethylcellulase (CMCase) on the surface of Escherichia coli. A surface anchoring vector, pGINP21M, was created that contains the multicloning sites including BamHI, SmaI and EcoRI at the end of the 3' flanking region encoding the C-terminus of INP instead of the stop codon for subcloning the foreign genes. The CMCase gene was in-frame subcloned for making INP-CMCase fusion proteins. The ability of this vector for directing the actual synthesis of INP-CMCase fusion proteins was confirmed by Western blotting analysis. CMCase targeted on the surface of cells was verified by measuring whole cell CMCase activity and ice-nucleation activity. CMCase activity was mainly detected on the cell surface whereas no enzyme activity was detected in the culture supernatant. Ice-nucleation activity was also maintained even if an INP-CMCase hybrid was made. This means that the fusion protein is functionally expressed and has its biological conformation on the surface. INP-CMCase fusion proteins were stable in the stationary phase. INP deleted of the repeating domain, thus producing no ice-nucleation activity, could also direct CMCase on the cell surface. This suggests that it has the secretion and targeting signal to the outer membrane.
The outer membrane of Gram-negative bacteria presents an effective barrier that restricts the release of proteins from the cell. Virtually all extracellular proteins of Gram-negative bacteria are exported by specialized systems requiring the action of several gene products. We have constructed a tripartite fusion consisting of (i) the signal sequence and first nine N-terminal amino acids of the mature major Escherichia coli lipoprotein, (ii) amino acids 46-159 of the outer membrane protein OmpA, and (iii) the complete mature beta-lactamase (EC 3.5.2.6) sequence. This protein had an enzymatically active beta-lactamase and was found predominantly in the outer membrane. Immunofluorescence microscopy, the accessibility of the fusion protein to externally added proteases, and the rates of hydrolysis of nitrocefin and penicillin G by whole cells demonstrated that a substantial fraction (20-30%) of the beta-lactamase domain of the fusion protein was exposed on the external surface of E. coli. In cells grown at 24 degrees C the localization of beta-lactamase on the cell surface was almost quantitative (greater than 80% of the enzymatically active protein was exposed to the extracellular fluid) as determined by nitrocefin and penicillin G hydrolysis and trypsin accessibility. These results demonstrated that a soluble protein, beta-lactamase, can be transported through--and become anchored on--the outer membrane by fusion to the proper targeting and localization signals.
Polymerase chain reaction was used to amplify the low copy number of two 16S ribosomal gene fragments from soil and sediment extracts. Total DNA for polymerase chain reaction was extracted from 1 g of seeded or unseeded samples by a rapid freeze-and-thaw method. Amplified DNA fragments can be detected in DNA fractions isolated from seeded soil containing less than 3 Escherichia coli cells and from seeded sediments containing less than 10 cells. This research demonstrated that coupling polymerase chain reaction to direct DNA extraction improves sensitivity by 1 and 2 orders of magnitude for sediments and soils, respectively. This technique could become a powerful tool for genetic ecology studies.
A class of membrane molecules has been identified whose primary translation product includes a COOH-terminal protein sequence that signals attachment of a glycosyl-phosphatidylinositol anchor at a COOH-terminal residue that is newly formed by cleavage of the signaling sequence. This class includes a wide diversity of protein types from eukaryotes at many stages of evolution. The structures of the glycosyl-phosphatidylinositol anchors are being resolved, but their functions aside from membrane attachment and dynamics remain to be determined.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The effect of phosphotriesterase (PTE) on cholinesterase (ChE) activities was studied with exposures to different organophosphates
in mice. Paraoxon (PO) (1.0 mg/kg, ip) almost totally inhibited serum ChE activity. This activity, however, recovered to the
normal level within 24 hr. The PTE pretreatment (16.8 U/animal, 2.5 μg/10 g body wt, iv 10 min before the organophosphate)
accelerated this reactivation. The same phenomenon was also seen in vitro. In vitro with human serum, there was only minimal reactivation of the inhibited ChE. PTE, however, reactivated it significantly. The
PTE-pretreated mice (168 U/animal, 30 μg/10 g body wt, iv) tolerated even 50 mg/kg of PO without showing any remarkable signs
of intoxication. In PTE-untreated animals, however, PO doses as low as 1.0 and 1.5 mg/kg caused severe signs of poisoning.
PTE (16.8 U/animal, 4 μg/10 g body wt, iv) reduced the inhibition of brain and serum ChE activities after PO and diisopropyl
fluorophosphate exposure. In sarin and soman intoxications, PTE decreased only slightly the inhibition of ChE activities.
The results indicate that PTE pretreatment given iv prevents the inhibition of ChE activities after certain organophosphates
and it also hastens the recovery of activities after PO poisoning.
residues at a given location does not mean the end of the problem. Pesticides can be translocated, bioconcentrated or converted into more dangerous chemicals (Matsumura 1985). The organophosphorus compounds are widely used because of their rapid biodegradability and nonpersistent nature. It has been shown that these compounds acts on various enzyme systems (Nitiforo and Stein 1967; Abou-Donia 1978). Malathion is one of the most selective organophosphorus insecticides used in Turkey for the control of pests on vegetables, field crops, fruits and domestic animals.Malathion is known to have potent insecticidal activity and low mammalian toxicity (Martin and Worthing 1977). In B6531 mice fed malathion, however, statistically significant dose responsive increases in liver carcinomas and neoplastic nodules occured. In the F344 rat, there were increases in thyroid and benign mammary tumors in rats fed malaoxon, the more toxic metabolite/breakdown product of malathion (US-EPA 1988). This conversion is catalyzed by mixed-functio n oxidases in mammals and insects (Reuber 1985). Malathion is metabolized rapidly by the soil fungus Trichoderma viride and the bacterium Pseudomonas sp. (Matsumura 1966). These microorganisms are capable of utilizing malathion as a sole source of carbon. In this study the effects of orally administered pesticide, both in the form of commercial and microorganism-degraded solutions, on albino mice were investigated. MATERIALS AND METHODS
Organophosphorus hydrolase (OPH) was displayed and anchored onto the surface of Escherichia coli using an Lpp-OmpA fusion system. Production of the fusion proteins in membrane fractions was verified by immunoblotting with OmpA antisera. Inclusion of the organophosphorus hydrolase signal sequence was necessary for achieving enzymatic activity on the surface. More than 80% of the OPH activity was located on the cell surface as determined by protease accessibility experiments. Whole cells expressing OPH on the cell surface degraded parathion and paraoxon very effectively without any diffusional limitation, resulting in sevenfold higher rates of parathion degradation compared with whole cells with similar levels of intracellular OPH. Immobilization of these live biocatalysts onto solid supports could provide an attractive means for pesticide detoxification in place of immobilized enzymes, affording a reduced diffusional barrier.
The ice nucleation protein (INP) is a glycosyl phosphatidylinositol anchored outer membrane protein found in certain Gram-negative bacteria. In this study, the INP from Pseudomonas syringae was applied as a fusion partner with the single chain antibody fragment (ScFv) against the human oncoprotein c-myc. Two new plasmids pNinaZ-myc and pNinaZScFv-BsaA1 were constructed and cloned into Escherichia coli JM109. The expression of the fusion protein was successfully demonstrated in the cloned cells. The fusion proteins had no effect on the viability of the host cells. Ice nucleation activity measurements and flow cytometry studies were followed to investigate the membrane expression of the fusion protein.
Cell surface display on Escherichia coli using ice nucleation protein was performed in order to develop a new expression system for recombinant eukaryotic proteins. Salmobin, the thrombin-like enzyme obtained from Korean snake (Agkistrodon halys) venom was displayed on the surface of Escherichia coli fused to the C-terminus of the ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. The thrombin cleavage site was inserted between salmobin and INP. The presence of salmobin on the bacterial cell surface was verified by SDS-PAGE, Western blotting, whole cell ELISA, and immunofluorescence microscopy. After thrombin cleavage the thrombin-like enzyme activity of recombinant salmobin was tested and verified. We concluded that INP-based cell surface display can be used as a novel expression system for eukaryotic proteins.
A new anchor system based on the ice nucleation protein (InaV) from Pseudomonas syringae INA5 was developed for cell surface display of functional organophosphorus hydrolase (OPH). The activity and stability of cells expressing the truncated InaV (INPNC)-OPH fusions were compared to cells with surface-expressed OPH using two other fusion anchors based on Lpp-OmpA and the truncated InaK protein. Whole cell activity was as much as 5-fold higher using the InaV anchor. Majority of the OPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of OPH was further verified by immunofluorescence microscopy. Constitutive expression of OPH on the surface using the InaV anchor resulted in no cell lysis or growth inhibition, in contrast to the Lpp-OmpA anchor. Suspended cultures also exhibited good stability, retaining almost 100% activity over a period of 3 weeks. Therefore, the InaV anchor system offers an attractive alternative to the currently available surface anchors, providing high-level expression and superior stability.
In insects, esterases play an important role in the degradation of organophosphate (OP) and Carbamate (CB) insecticides. The ability of esterase B1 to degrading OP and CB insecticides opens broad prospect of using it in programs to the insecticide pollution. OPs can be detoxified by hydrolysis of their phosphoester bonds, pyrethroids and some OPs like malathion, by hydrolysis of their carboxylester bonds, and diverse carbamate insecticides, herbicides and fungicides by hydrolysis of amide or other similar bonds. Most of the candidate bioremediation enzymes identified to date have been hydrolases. In recent years, we have focused our work on the transfer of a detoxifying esterase B1 gene from mosquito into E. coli and explored the possibility of using the detoxified enzymes in environmental protection. 5' initial of esterase B1 cDNA was cloned by RT-PCR and sequenced consequently. After the combination of 5' initial and 3' terminal fragment of esterase B1 cDNA, the recombinant vector pET-ESTB1 was constructed and transformed into E. coli BL21. A 60 kD protein was induced by IPTG and its expression was temperature-dependent. After 12 h induction, the target protein occupied 27% of the total protein. A pure recombinant protein was obtained by purification, and was detected with 10% SDS-PAGE. The results showed that 22.1% of malathion was degraded by crude detoxification enzyme in 15 mins, demonstrated a high degradation property. This research provides a novel approach, which takes the advantages of eucaryotes for bioremediation of pesticide pollutions.
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.
Effects of pesticides on the environment
- D Pimentel
Pimentel, D. Effects of pesticides on the environment. In 10th International Congress on Plant Protection; Crydon, UK, 1983; Vol. 2, pp 685-691.