Biodegradation of atrazine and nicosulfuron by Streptomyces nigra LM01: Performance, degradative pathway, and possible genes involved.

Microbial herbicide degradation is an efficient bioremediation method. In this study, a strain of Streptomyces nigra, LM01, which efficiently degrades atrazine and nicosulfuron, was isolated from a corn field using a direct isolation method. The degradation effects of the identified strain on two herbicides were investigated and optimized using an artificial neural network. The maximum degradation rates of S. nigra LM01 were 58.09 % and 42.97 % for atrazine and nicosulfuron, respectively. The degradation rate of atrazine in the soil reached 67.94 % when the concentration was 108 CFU/g after 5 d and was less effective than that of nicosulfuron. Whole genome sequencing of strain LM01 helped elucidate the possible degradation pathways of atrazine and nicosulfuron. The protein sequences of strain LM01 were aligned with the sequences of the degraded proteins of the two herbicides by using the National Center for Biotechnology Information platform. The sequence (GE005358, GE001556, GE004212, GE005218, GE004846, GE002487) with the highest query cover was retained and docked with the small-molecule ligands of the herbicides. The results revealed a binding energy of - 6.23 kcal/mol between GE005358 and the atrazine ligand and - 6.66 kcal/mol between GE002487 and the nicosulfuron ligand.

Kj-mhpC Enzyme in Klebsiella jilinsis 2N3 Is Involved in the Degradation of Chlorimuron-Ethyl via De-Esterification.

Microbial degradation is a highly efficient and reliable approach for mitigating the contamination of sulfonylurea herbicides, such as chlorimuron-ethyl, in soil and water. In this study, we aimed to assess whether Kj-mhpC plays a pivotal role in the degradation of chlorimuron-ethyl. Kj-mhpC enzyme purified via prokaryotic expression exhibited the highest catalytic activity for chlorimuron-ethyl at 35 °C and pH 7. Bioinformatic analysis and three-dimensional homologous modeling of Kj-mhpC were conducted. Additionally, the presence of Mg+ and Cu2+ ions partially inhibited but Pb2+ ions completely inhibited the enzymatic activity of Kj-mhpC. LC/MS revealed that Kj-mhpC hydrolyzes the ester bond of chlorimuron-ethyl, resulting in the formation of 2-(4-chloro-6-methoxypyrimidine-2-amidoformamidesulfonyl) benzoic acid. Furthermore, the point mutation of serine at position 67 (Ser67) confirmed that it is the key amino acid at the active site for degrading chlorimuron-ethyl. This study enhanced the understanding of how chlorimuron-ethyl is degraded by microorganisms and provided a reference for bioremediation of the environment polluted with chlorimuron-ethyl.

Microbial degradation as a powerful weapon in the removal of sulfonylurea herbicides.

Sulfonylurea herbicides have been widely used worldwide and play a significant role in modern agricultural production. However, these herbicides have adverse biological effects that can damage the ecosystems and harm human health. As such, rapid and effective techniques that remove sulfonylurea residues from the environment are urgently required. Attempts have been made to remove sulfonylurea residues from environment using various techniques such as incineration, adsorption, photolysis, ozonation, and microbial degradation. Among them, biodegradation is regarded as a practical and environmentally responsible way to eliminate pesticide residues. Microbial strains such as Talaromyces flavus LZM1, Methylopila sp. SD-1, Ochrobactrum sp. ZWS16, Staphylococcus cohnii ZWS13, Enterobacter ludwigii sp. CE-1, Phlebia sp. 606, and Bacillus subtilis LXL-7 can almost completely degrade sulfonylureas. The degradation mechanism of the strains is such that sulfonylureas can be catalyzed by bridge hydrolysis to produce sulfonamides and heterocyclic compounds, which deactivate sulfonylureas. The molecular mechanisms associated with microbial degradation of sulfonylureas are relatively poorly studied, with hydrolase, oxidase, dehydrogenase and esterase currently known to play a pivotal role in the catabolic pathways of sulfonylureas. Till date, there are no reports specifically on the microbial degrading species and biochemical mechanisms of sulfonylureas. Hence, in this article, the degradation strains, metabolic pathways, and biochemical mechanisms of sulfonylurea biodegradation, along with its toxic effects on aquatic and terrestrial animals, are discussed in depth in order to provide new ideas for remediation of soil and sediments polluted by sulfonylurea herbicides.

Characterization of a new chlorimuron-ethyl-degrading strain Cedecea sp. LAM2020 and biodegradation pathway revealed by multiomics analysis.

The widespread use of the herbicide chlorimuron-methyl is hazard to rotational crops and causes soil degradation problems. Biodegradation is considered a promising way for removing herbicide residues from the environment. Here, a new isolated strain, Cedecea sp. LAM2020, enabled complete degradation of 100 mg/L chlorimuron-methyl within five days. Transcriptome analysis revealed that ABC transporters, atrazine degradation and purine metabolism were enriched in the KEGG pathway. Integrating GO and KEGG classification with related reports, we predict that carboxylesterases are involved in the biodegradation of chlorimuron-methyl by LAM2020. Heterologous expression of the carboxylesterase gene carH showed 26.67% degradation of 50 mg/L chlorimuron-methyl within 6 h. The intracellular potential biological response and extracellular degradation process of chlorimuron-ethyl were analyzed by the nontarget metabolomic and mass spectrometry respectively, and the biodegradation characteristics and complete mineralization pathway was revealed. The cleavage of the sulfonylurea bridge and the ester bond achieved the first step in the degradation of chlorimuron-methyl. Together, these results reveal the presence of acidolysis and enzymatic degradation of chlorimuron-methyl by strain LAM2020. Hydroponic corn experiment showed that the addition of strain LAM2020 alleviated the toxic effects of chlorimuron-ethyl on the plants. Collectively, strain LAM2020 may be a promising microbial agent for plants chlorimuron-ethyl detoxification and soil biofertilizer.

Citric acid modified ultrasmall copper peroxide nanozyme for in situ remediation of environmental sulfonylurea herbicide contamination.

Herbicide residues in the environment threaten high-quality agriculture and human health. Consequently, in situ remediation of herbicide contamination is vital. We synthesized a novel self-catalyzed nanozyme, ultrasmall (2-3 nm) copper peroxide nanodots modified by citric acid (CP@CA) for this purpose, which can break down into H2O2 and Cu2+ in water or soil. Ubiquitous glutathione reduces Cu2+ into Cu+, which promotes the decomposition of H2O2 into •OH through a Fenton-like reaction under mild acid conditions created by the presence of citric acid. The generated •OH efficiently degrade nicosulfuron in water and soil, and the maximum degradation efficiency could be achieved at 97.58% in water at 56 min. The possible degradation mechanisms of nicosulfuron were proposed through the 25 intermediates detected. The overall ecotoxicity of the nicosulfuron system was significantly reduced after CP@CA treatment. Furthermore, CP@CA had little impact on active components of soil bacterial community. Moreover, CP@CA nanozyme could effectively remove seven other sulfonylurea herbicides from the water. In this paper, a high-efficiency method for herbicide degradation was proposed, which provides a new reference for the in situ remediation of herbicide pollution.

A Novel Pathway of Chlorimuron-Ethyl Biodegradation by Chenggangzhangella methanolivorans Strain CHL1 and Its Molecular Mechanisms.

Chlorimuron-ethyl is a widely used herbicide in agriculture. However, uncontrolled chlorimuron-ethyl application causes serious environmental problems. Chlorimuron-ethyl can be effectively degraded by microbes, but the underlying molecular mechanisms are not fully understood. In this study, we identified the possible pathways and key genes involved in chlorimuron-ethyl degradation by the Chenggangzhangella methanolivorans strain CHL1, a Methylocystaceae strain with the ability to degrade sulfonylurea herbicides. Using a metabolomics method, eight intermediate degradation products were identified, and three pathways, including a novel pyrimidine-ring-opening pathway, were found to be involved in chlorimuron-ethyl degradation by strain CHL1. Transcriptome sequencing indicated that three genes (atzF, atzD, and cysJ) are involved in chlorimuron-ethyl degradation by strain CHL1. The gene knock-out and complementation techniques allowed for the functions of the three genes to be identified, and the enzymes involved in the different steps of chlorimuron-ethyl degradation pathways were preliminary predicted. The results reveal a previously unreported pathway and the key genes of chlorimuron-ethyl degradation by strain CHL1, which have implications for attempts to enrich the biodegradation mechanism of sulfonylurea herbicides and to construct engineered bacteria in order to remove sulfonylurea herbicide residues from environmental media.

Transcriptomic response of Pseudomonas nicosulfuronedens LAM1902 to the sulfonylurea herbicide nicosulfuron.

The overuse of the herbicide nicosulfuron has become a global environmental concern. As a potential bioremediation technology, the microbial degradation of nicosulfuron shows much promise; however, the mechanism by which microorganisms respond to nicosulfuron exposure requires further study. An isolated soil-borne bacteria Pseudomonas nicosulfuronedens LAM1902 displaying nicosulfuron, chlorimuron-ethyl, and cinosulfuron degradabilities in the presence of glucose, was used to determine the transcriptional responses to nicosulfuron exposure. RNA-Seq results indicated that 1102 differentially expressed genes (DEGs) were up-regulated and 702 down-regulated under nicosulfuron stress. DEGs were significantly enriched in "ABC transporters", "sulfur metabolism", and "ribosome" pathways (p ≤ 0.05). Several pathways (glycolysis and pentose phosphate pathways, a two-component regulation system, as well as in bacterial chemotaxis metabolisms) were affected by nicosulfuron exposure. Surprisingly, nicosulfuron exposure showed positive effects on the production of oxalic acid that is synthesized by genes encoding glycolate oxidase through the glyoxylate cycle pathway. The results suggest that P. nicosulfuronedens LAM1902 adopt acid metabolites production strategies in response to nicosulfuron, with concomitant nicosulfuron degradation. Data indicates that glucose metabolism is required during the degradation and adaptation of strain LAM1902 to nicosulfuron stress. The present studies provide a glimpse at the molecular response of microorganisms to sulfonylurea pesticide toxicity and a potential framework for future mechanistic studies.

Effects of long-term exposure to the herbicide nicosulfuron on the bacterial community structure in a factory field.

This study aims to investigate the effects of long-term nicosulfuron residue on an herbicide factory ecosystem. High-throughput sequencing was used to investigate the environmental microbial community structure and interactions. The results showed that the main contributor to the differences in the microbial community structure was the sample type, followed by oxygen content, pH and nicosulfuron residue concentration. Regardless of the presence or absence of nicosulfuron, soil, sludge, and sewage were dominated by groups of Bacteroidetes, Actinobacteria, and Proteobacteria. Long-term exposure to nicosulfuron increased alpha diversity of bacteria and archaea but significantly decreased the abundance of Bacteroidetes and Acidobateria compared to soils without nicosulfuron residue. A total of 81 possible nicosulfuron-degrading bacterial genera, e.g., Rhodococcus, Chryseobacterium, Thermomonas, Stenotrophomonas, and Bacillus, were isolated from the nicosulfuron factory environmental samples through culturomics. The co-occurrence network analysis indicated that the keystone taxa were Rhodococcus, Stenotrophomonas, Nitrospira, Terrimonas, and Nitrosomonadaceae_MND1. The strong ecological relationship between microorganisms with the same network module was related to anaerobic respiration, the carbon and nitrogen cycle, and the degradation of environmental contaminants. Synthetic community (SynCom), which provides an effective top-down approach for the critical degradation strains obtained, enhanced the degradation efficiency of nicosulfuron. The results indicated that Rhodococcus sp. was the key genus in the environment of long-term nicosulfuron exposure.

Nicosulfuron, a sulfonylurea herbicide, alters embryonic development and oxidative status of hatchlings at environmental concentrations in an amphibian species.

The widespread use of agrochemicals for controlling pests and diseases of crops is recognized as a main threat to biodiversity. Sulfonylurea herbicides are being increasingly used and display low levels of degradation in water which suggest that they might affect non-target organisms. In a common garden experiment, eggs of a widespread amphibian (Bufo spinosus) were exposed to sublethal environmentally relevant concentrations of a widely used sulfonylurea herbicide, nicosulfuron, during the whole embryonic development. We assessed development-related traits (i.e., development duration, hatching success, hatchling size and occurrence of malformation) as well as antioxidant markers in response to contamination (i.e., SOD, GPx, catalase, thiols and relevant ratios thereof). We found that sublethal concentrations of nicosulfuron increased embryonic development duration, increased hatchling size and tended to increase malformations. Embryos exposed to nicosulfuron displayed decreased thiols and increased catalase activity suggesting alteration of oxidative status. We did not find any effect of nicosulfuron on SOD and GPx levels. Interestingly, higher catalase activity was linked to higher proportion of malformed individuals, suggesting that exposure to nicosulfuron induced teratogenic effects. Our results suggest that alteration of antioxidant levels might be one physiological mechanism through which nicosulfuron might cause detrimental effects on amphibian embryos. Sublethal effects of pesticides at environmentally relevant concentrations have been overlooked and require further investigations, especially in non-target taxa occurring in agricultural landscapes.

Genetically Engineered Methanotroph as a Platform for Bioaugmentation of Chemical Pesticide Contaminated Soil.

Bioaugmentation is a promising alternative in soil remediation. One challenge of bioaugmentation is that exogenous pollutant-degrading microbes added to soil cannot establish enough biomass to eliminate pollutants. Considering that methanotrophs have a growth advantage in the presence of methane, we hypothesize that genetically engineered methanotrophs could degrade contaminants efficiently in soil with methane. Here, methanotroph Methylomonas sp. LW13, herbicide bensulfuron-methyl (BSM), and two kinds of soil were chosen to confirm this hypothesis. The unmarked gene knock-in method was first developed for strain LW13. Then, BSM hydrolase encoding gene sulE was inserted into the chromosome of strain LW13, conferring it BSM-degrading ability. After inoculation, the cell amount of strain LW13-sulE in soil raised considerably (over 100 fold in 9 days) with methane provision; meanwhile, >90% of BSM in soil was degraded. This study provides a proof of the concept that genetically engineered methanotroph is a potential platform for soil remediation.

Pseudomonas nicosulfuronedens sp. nov., a nicosulfuron degrading bacterium, isolated from a microbial consortium.

A Gram-stain-negative, aerobic, motile, short-rod-shaped bacterium with nicosulfuron-degrading ability, designated strain LAM1902T, was isolated from a microbial consortium enriched with nicosulfuron as a sole nitrogen and energy source. The optimal temperature and pH for growth of strain LAM1902T were 30 °C and pH 6.0, respectively. Strain LAM1902T could grow in the presence of NaCl with concentration up to 4.0 % (w/v). Comparative analysis of 16S rRNA gene sequences revealed that LAM1902T was closely related to the members of the family Pseudomonadaceae to the genus Pseudomonas, with the highest similarity to Pseudomonas nitroreducens DSM 14399T (99.6 %), Pseudomonas nitritireducens WZBFD3-5A2T (99.3 %) and Pseudomonas panipatensis Esp-1T (98.8 %). Multi-locus sequence analysis based on both concatenated sequences of the 16S rRNA gene and three housekeeping genes (gyrB, rpoB and rpoD) further confirmed the intrageneric phylogenetic position of strain LAM1902T. The genomic DNA G+C content of LAM1902T was 64.8 mol%. The low values of in silico DNA-DNA hybridization (less than 43.7 %) and average nucleotide identity (less than 90.9 %) also showed that the strain was distinctly different from known species of the genus Pseudomonas. The major fatty acids were C16 : 0, C17 : 0 cyclo and anteiso C15 : 0. Ubiquinone Q-9 was detected as the predorminant respiratory quinone. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and aminophospholipid. Based on phylogenetic, phenotypic and chemotaxonomic analyses and genome comparisons, we conclude that strain LAM1902T represents a novel species, for which the name Pseudomonas nicosulfuronedens sp. nov. is proposed. The type strain is LAM1902T (=JCM 33860T=KCTC 72830T).

Study on the characteristics and mechanisms of nicosulfuron biodegradation by Bacillus velezensis CF57.

Nicosulfuron is one of the main sulfonylurea herbicides that have been widely used to protect maize crops. A total of 10 nicosulfuron-degrading strains were isolated from the intestine tract of earthworm Eisenia foetida. Among them, Bacillus velezensis CF57 with the highest degradation efficiency was selected and studied in detail. The degradation characteristics of CF57 showed that it was able to effectively degrade nicosulfuron in a wide range of temperature, pH, and a low inoculation amount, and the response surface analysis revealed that the optimum degradation conditions were 30.8 °C, pH 6.31, and inoculation amount 3.04%. Meanwhile, CF57 could degrade high-concentration nicosulfuron efficiently and posed a broad degradation spectrum of other sulfonylurea herbicides. Furthermore, the localization of degradation enzyme indicated that the nicosulfuron-degrading enzyme was an extracellular fraction. By analyzing the metabolites of nicosulfuron, it could be further determined that the degradation of nicosulfuron by strain CF57 was mainly through the extracellular enzyme, and its possible degradation pathway was mainly derived from the cleavage of the C-N bond of the sulfonylurea bridge. These results may provide new insights into bioremediation of nicosulfuron-contaminated environments and enrich the resources of degrading bacteria of sulfonylurea herbicides.

Phytoextraction of diuron, hexazinone, and sulfometuron-methyl from the soil by green manure species.

The herbicides diuron, hexazinone, and sulfometuron-methyl present a potential risk of environmental contamination and are widely used for weed control in sugarcane cultivation. Our objectives were to measure the tolerance of Canavalia ensiformes (L.) DC., Stilizobium aterrimum L., Raphanus sativus L., Crotalaria spectabilis Röth, Lupinus albus L., and Pennisetum glaucum (L.) R. Br. To the herbicides diuron, hexazinone, and sulfometuron-methyl to assess the capacity of these species to extract and accumulate the herbicides in their tissues. Before sowing the green manure species, the soils were individually contaminated with the three 14C-radiolabeled herbicides. 14C-diuron and 14C-sulfometuron-methyl showed higher values remaining in the soil (>90%) for all species of green manure compared to hexazinone (<80%). The green manure species analyzed showed greater potential to remedy soils contaminated with hexazinone than the other herbicides. C. ensiformes showed high phytoextraction of hexazinone when compared to the other species, removing 11.2% of the pollutant from the soil, followed by L. albus (8.6%), S. aterrimum (7.3%), R. sativus (4.8%), C. spectabilis (2.5%), and P. glaucum (1.1%). The results indicate that the phytoextraction of diuron, hexazinone and sulfometuron-methyl is dependent on the species of green manure and can be an important tool for the decontamination of areas polluted by these herbicides.

Preparation and characterization of glimepiride eutectic mixture with l-arginine for improvement of dissolution rate.

In this study, glimepiride and l-arginine (GA) binary mixtures at various molar ratios were prepared to evaluate whether they could improve the poor water solubility and dissolution characteristics of glimepiride. It was shown that glimepiride and arginine form a eutectic mixture, a type of crystalline solid dispersions, at a 1:1 M ratio and eutectic temperature of 426.9 K using a phase diagram constructed using differential scanning calorimetry (DSC) and thermo-microscopy. The preserved characteristic powder X-ray diffraction (PXRD) patterns and infrared (IR) spectra of each material in those of GA binary mixtures confirmed the formation of eutectic mixture without molecular interaction in solid state. The formation of GA eutectic mixture (GAEM) resulted in the improvement of solubility through pH modification and the intermolecular interaction of glimepiride and l-arginine in aqueous mediums, thereby wettability and dissolution rate of glimepiride were also enhanced. The intermolecular interaction between glimepiride and l-arginine at a 1:1 stoichiometry of the complex in solution state was identified by phase solubility, stoichiometric determination, and solution state nuclear magnetic resonance (NMR) spectroscopy. Specific molecular interactions such as hydrogen bonding and hydrophobic interaction were suggested as main mechanisms of GA complexation in solution. Therefore, this study concludes that the GAEM could be an effective way to improve the solubility and dissolution rate of glimepiride.

[Isolation and identification of a chlorimuron-ethyl-degrading bacterium and optimization of its degradation conditions].

In order to solve the problem of soil, water pollution and sensitive crop drug damage caused by chlorosulfuron residue, and to provide degradation strain resources for microbial remediation of contaminated soil, a chlorimuron-ethyl-degrading strain T9DB-01 was isolated from chlorosulfuron contaminated soil by the method of enrichment culture and step by step domestication. Strain T9DB-01 was identified as Pseudomonas sp. by morphological characteristics, physiological and biochemical analysis and 16S rDNA gene sequence analysis. The effects of temperature, pH value, substrate concentration, medium volume, and inoculation volume on the degradation of chlorsulfuron-methyl by strain T9DB-01 were investigated by single factor experiment. The degradation conditions of chlorosulfuron by strain T9DB-01 were optimized by orthogonal test and verification. Results show that 30 °C, pH 8.0, inoculum 4%, liquid volume 100 mL/250 mL, substrate concentration of 200 mg/L, cultured for 5 d, the strain degraded 93.7% chlorsulfuron-methyl. The degrading strain has certain application potential for bioremediation of chlorsulfuron-contaminated soil.

ISCU interacts with NFU1, and ISCU[4Fe-4S] transfers its Fe-S cluster to NFU1 leading to the production of holo-NFU1.

NFU1 is a late-acting factor in the biogenesis of human mitochondrial iron-sulfur proteins. Mutations in NFU1 are associated with genetic diseases such as multiple mitochondrial dysfunctions syndrome 1 (MMDS1) that involve defects in mitochondrial [4Fe-4S] proteins. We present results from NMR spectroscopy, small angle X-ray scattering, size exclusion chromatography, and isothermal titration calorimetry showing that the structured conformer of human ISCU binds human NFU1. The dissociation constant determined by ITC is Kd = 1.1 ± 0.2 µM. NMR and SAXS studies led to a structural model for the complex in which the cluster binding region of ISCU interacts with two α-helices in the C-terminal domain of NFU1. In vitro experiments demonstrate that ISCU[4Fe-4S] transfers its Fe-S cluster to apo-NFU1, in the absence of a chaperone, leading to the assembly of holo-NFU1. By contrast, the cluster of ISCU[2Fe-2S] remains bound to ISCU in the presence of apo-NFU1.

Application of Methylopila sp. DKT for Bensulfuron-methyl Degradation and Peanut Growth Promotion.

Bensulfuron-methyl is an herbicide widely used for weed control although its residues cause damage to other crops during crop rotations. In this study, the biodegrading activity of bensulfuron-methyl by a plant growth-promoting bacterial strain was carried out. Methylopila sp. DKT isolated from soil was determined for bensulfuron-methyl degradation and phosphate solubilization in the liquid media and soil. Moreover, the effects of the herbicide on peanut development and the role of Methylopila sp. DKT on the growth promotion of peanut were investigated. The results showed that the isolate effectively utilized the compound as a sole carbon source and solubilized low soluble inorganic phosphates. Methylopila sp. DKT also utilized 2-amino-4,6-dimethoxypyrimidine, a metabolite of bensulfuron-methyl degradation, as a sole carbon and energy source, and released ammonium and nitrate. The supplementation with Methylopila sp. DKT in soil increased the peanut biomass and the phosphorus content in the plant. In addition, the inoculation with Methylopila sp. DKT in soil and peanut cultivation increased the bensulfuron-methyl degradation by 57.7% for 1 month, which suggests that both plants and the bacterial isolate play a key role in herbicide degradation. These results indicate that the studied strain has a high potential for soil remediation and peanut growth promotion.

Insight into the Characteristics and New Mechanism of Nicosulfuron Biodegradation by a Pseudomonas sp. LAM1902.

A total of five strains of nicosulfuron-degrading bacteria were isolated from a continuously cultivated microbial consortium using culturomics. Among them, a novel Pseudomonas strain, LAM1902, with the highest degradation efficiency was investigated in detail. The characteristics of nicosulfuron-degradation by LAM1902 were investigated and optimized by response surface analysis. Furthermore, non-targeted metabolomic analysis of extracellular and intracellular biodegradation of nicosulfuron by LAM1902 was carried out by liquid chromatography/mass spectroscopy (LC-MS) and gas chromatography-time-of-flight/mass spectroscopy (GC-TOF/MS). It was found that nicosulfuron was degraded by LAM1902 mainly via breaking the sulfonylurea bridge, and this degradation might be attributed to oxalate accumulation. The results of GC-TOF/MS also showed that the intracellular degradation of nicosulfuron did not occur. However, nicosulfuron exerted a significant influence on the metabolism of inositol phosphate, pyrimidine, arginine/proline, glyoxylate, and dicarboxylate metabolism and streptomycin biosynthesis. The changes of myo-inositol, trehalose, and 3-aminoisobutanoic acid were proposed as a mechanism of self-protection against nicosulfuron stress.

Interaction of Organic Anion Transporter 3-Mediated Uptake of Steviol Acyl Glucuronide, a Major Metabolite of Rebaudioside A, with Selected Drugs.

Organic anion transporter 3 (OAT3) plays a critical role in the renal excretion of many xenobiotics. Because steviol acyl glucuronide (SVAG), an OAT3 substrate, is the major circulating metabolite after oral ingestion of steviol glycosides and is excreted into the urine, inhibition of OAT3 activity may alter pharmacokinetic profiles of SVAG. The present study showed that drugs such as probenecid and glimepiride displayed potent inhibition toward the OAT3-mediated SVAG transport, with IC50 values of 4.9 and 0.8 µM, respectively. No species differences were observed. Probenecid and glimepiride could significantly elevate plasma concentrations of SVAG after oral administration of rebaudioside A, with significant increases in plasma maximum (Cmax) and area under the plasma time-concentration curve values. The inhibitory effect on the OAT3-mediated SVAG transport exemplified a unique case between drugs and the metabolite of a food additive. Our data suggest that caution should be exercised when giving steviol glycoside products to human subjects with compromised renal function.

Carboxylesterase, a de-esterification enzyme, catalyzes the degradation of chlorimuron-ethyl in Rhodococcus erythropolis D310-1.

Microbial degradation is considered to be the most acceptable method for degradation of chlorimuron-ethyl, a typical long-term residual sulfonylurea herbicide, but the underlying mechanism at the genetic and biochemical levels is unclear. In this work, the genome sequence of the chlorimuron-ethyl-degrading bacterium Rhodococcus erythropolis D310-1 was completed, and the gene clusters responsible for the degradation of chlorimuron-ethyl in D310-1 were predicted. A carboxylesterase gene, carE, suggested to be responsible for carboxylesterase de-esterification, was cloned from D310-1. CarE was expressed in Escherichia coli BL21 and purified to homogeneity. The active site of the chlorimuron-ethyl-degrading enzyme CarE and the biochemical activities of CarE were elucidated. The results demonstrated that CarE is involved in catalyzing the de-esterification of chlorimuron-ethyl. A carE deletion mutant strain, D310-1ΔcarE, was constructed, and the chlorimuron-ethyl degradation rate in the presence of 100 mg L-1 chlorimuron-ethyl within 120 h decreased from 86.5 % (wild-type strain D310-1) to 58.2 % (mutant strain D310-1ΔcarE). Introduction of the plasmid pNit-carE restored the ability of the mutant strain to utilize chlorimuron-ethyl. This study is the first to demonstrate that carboxylesterase can catalyze the de-esterification reaction of chlorimuron-ethyl and provides new insights into the mechanism underlying the degradation of sulfonylurea herbicides and a theoretical basis for the utilization of enzyme resources.