Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity.

Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 105 M-1 s-1 against paraoxon hydrolysis at 70°C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.

Biodegradation of mycotoxin fumonisin B1 by a novel bacterial consortium SAAS79.

Fumonisin B1 (FB1) contamination in cereals and cereal products remains an important aspect of food safety because of its wide distribution and the potential health hazard. However, only a few microorganisms have been reported to effectively degrade FB1. In this present study, a bacterial consortium SAAS79 with highly FB1-degrading activity was isolated from the spent mushroom compost. The combination of antibiotic-driven selection and 16S rDNA sequencing identified the Pseudomonas genus as the key FB1-degrading member. The microbial consortium could degrade more than 90% of 10 µg/mL FB1 after incubation for 24 h at pH of 5-7 and temperature of 28-35 °C. The enzymes from the intracellular space were proved to be responsible for FB1 degradation, which eliminated about 90% of 10 µg/mL FB1 in 3 h. Besides, liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) analysis identified two degradation products of FB1, and their toxicity on the monkey kidney cells (MARC-145) was significantly lower (p < 0.05) compared with the parent FB1. Overall, the consortium SAAS79 and its crude enzymes may be a potential choice for the decontamination of FB1 in the feed and food industry. Also, the bacterial consortium provides a new source of genes for the development of enzymatic detoxification agent.

Evidence for a complex formation between CYP2J5 and mEH in living cells by FRET analysis of membrane protein interaction in the endoplasmic reticulum (FAMPIR).

The potential complex formation between microsomal epoxide hydrolase (mEH) and cytochrome P450-dependent monooxygenase (CYP) has been a subject of research for many decades. Such an association would enable efficient substrate channeling between CYP and mEH and as such represent an attractive strategy to prevent deleterious accumulation of harmful metabolic by-products such as CYP-generated epoxide intermediates. However, such complex formation is experimentally difficult to prove, because CYP and mEH are membrane-bound proteins that are prone to unspecific aggregation after solubilization. Here, we report the development of a FRET-based procedure to analyze the mEH-CYP interaction in living cells by fluorescence-activated cell sorting. With this non-invasive procedure, we demonstrate that CYP2J5 and mEH associate in the endoplasmic reticulum of recombinant HEK293 cells to the same extent as do CYP2J5 and its indispensible redox partner cytochrome P450 reductase. This presents final proof for a very close proximity of CYP and mEH in the endoplasmic reticulum, compatible with and indicative of their physical interaction. In addition, we provide with FAMPIR a robust and easy-to-implement general method for analyzing the interaction of ER membrane-resident proteins that share a type I topology.

Removal of phenolic inhibitors from lignocellulose hydrolysates using laccases for the production of fuels and chemicals.

Lignocellulose is the most abundant biopolymer in the biosphere. It is inexpensive and therefore considered an attractive feedstock to produce biofuels and other biochemicals. Thermochemical and/or enzymatic pretreatment is used to release fermentable monomeric sugars. However, a variety of inhibitory by-products such as weak acids, furans, and phenolics that inhibit cell growth and fermentation are also released. Phenolic compounds are among the most toxic components in lignocellulosic hydrolysates and slurries derived from lignin decomposition, affecting overall fermentation processes and production yields and productivity. Ligninolytic enzymes have been shown to lower inhibitor concentrations in these hydrolysates, thereby enhancing their fermentability into valuable products. Among them, laccases, which are capable of oxidizing lignin and a variety of phenolic compounds in an environmentally benign manner, have been used for biomass delignification and detoxification of lignocellulose hydrolysates with promising results. This review discusses the state of the art of different enzymatic approaches to hydrolysate detoxification. In particular, laccases are used in separate or in situ detoxification steps, namely in free enzyme processes or immobilized by cell surface display technology to improve the efficiency of the fermentative process and consequently the production of second-generation biofuels and bio-based chemicals.

Improvement of catalytic activity of sorbose dehydrogenase for deoxynivalenol degradation by rational design.

Deoxynivalenol (DON) is the most frequently contaminated mycotoxin in food and feed worldwide, causing significant economic losses and health risks. Physical and chemical detoxification methods are widely used, but they cannot efficiently and specifically remove DON. In the study, the combination of bioinformatics screening and experimental verification confirmed that sorbose dehydrogenase (SDH) can effectively convert DON to 3-keto-DON and a substance that removes four hydrogen atoms for DON. Through rational design, the Vmax of the mutants F103L and F103A were increased by 5 and 23 times, respectively. Furthermore, we identified catalytic sites W218 and D281. SDH and its mutants have broad application conditions, including temperature ranges of 10-45 °C and pH levels of 4-9. Additionally, the half-lives of F103A at 90 °C (processing temperature) and 30 °C (storage temperature) were 60.1 min and 100.5 d, respectively. These results suggest that F103A has significant potential in the detoxification application of DON.

Biodegradation of Aflatoxin B1 in Peanut Oil by an Amphipathic Laccase-Inorganic Hybrid Nanoflower.

Aflatoxin B1 (AFB1) contamination is an important issue for the safety of edible oils. Enzymatic degradation is a promising approach for removing mycotoxins in a specific, efficient, and green manner. However, enzymatic degradation of mycotoxins in edible oil is challenging as a result of the low activity and stability of the enzyme. Herein, a novel strategy was proposed to degrade AFB1 in peanut oil using an amphipathic laccase-inorganic hybrid nanoflower (Lac NF-P) as a biocatalyst. Owing to the improved microenvironment of the enzymatic reaction and the enhanced stability of the enzyme structure, the proposed amphipathic Lac NF-P showed 134- and 3.2-fold increases in the degradation efficiency of AFB1 in comparison to laccase and Lac NF, respectively. AFB1 was removed to less than 0.96 µg/kg within 3 h when using Lac NF-P as a catalyst in the peanut oil, with the AFB1 concentration ranging from 50 to 150 µg/kg. Moreover, the quality of the peanut oil had no obvious change, and no leakage of catalyst was observed after the treatment of Lac NF-P. In other words, our study may open an avenue for the development of a novel biocatalyst for the detoxification of mycotoxins in edible oils.

Transcriptome analysis reveals differential effects of beta-cypermethrin and fipronil insecticides on detoxification mechanisms in Solenopsis invicta.

Insecticide resistance poses many challenges in insect pest control, particularly in the control of destructive pests such as red imported fire ants (Solenopsis invicta). In recent years, beta-cypermethrin and fipronil have been extensively used to manage invasive ants, but their effects on resistance development in S. invicta are still unknown. To investigate resistance development, S. invicta was collected from populations in five different cities in Guangdong, China. The results showed 105.71- and 2.98-fold higher resistance against fipronil and beta-cypermethrin, respectively, in the Guangzhou population. The enzymatic activities of acetylcholinesterase, carboxylases, and glutathione S-transferases significantly increased with increasing beta-cypermethrin and fipronil concentrations. Transcriptomic analysis revealed 117 differentially expressed genes (DEGs) in the BC-ck vs. BC-30 treatments (39 upregulated and 78 downregulated), 109 DEGs in F-ck vs. F-30 (33 upregulated and 76 downregulated), and 499 DEGs in BC-30 vs. F-30 (312 upregulated and 187 downregulated). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEGs associated with insecticide resistance were significantly enriched in metabolic pathways, the AMPK signaling pathway, the insulin signaling pathway, carbon metabolism, peroxisomes, fatty acid metabolism, drug metabolism enzymes and the metabolism of xenobiotics by cytochrome P450. Furthermore, we found that DEGs important for insecticide detoxification pathways were differentially regulated under both insecticide treatments in S. invicta. Comprehensive transcriptomic data confirmed that detoxification enzymes play a significant role in insecticide detoxification and resistance development in S. invicta in Guangdong Province. Numerous identified insecticide-related genes, GO terms, and KEGG pathways indicated the resistance of S. invicta workers to both insecticides. Importantly, this transcriptome profile variability serves as a starting point for future research on insecticide risk evaluation and the molecular mechanism of insecticide detoxification in invasive red imported fire ants.

Using Cholinesterases and Immobilized Luminescent Photobacteria for the Express-Analysis of Mycotoxins and Estimating the Efficiency of Their Enzymatic Hydrolysis.

Novel sensitive analytical agents that can be used for simple, affordable, and rapid analysis of mycotoxins are urgently needed in scientific practice, especially for the screening of perspective bio-destructors of the toxic contaminants. We compared the characteristics of a rapid quantitative analysis of different mycotoxins (deoxynivalenol, ochratoxin A, patulin, sterigmatocystin, and zearalenone) using acetyl-, butyrylcholinesterases and photobacterial strains of luminescent cells in the current study. The best bioindicators in terms of sensitivity and working range (µg/mL) were determined as follows: Photobacterium sp. 17 cells for analysis of deoxynivalenol (0.8-89) and patulin (0.2-32); Photobacterium sp. 9.2 cells for analysis of ochratoxin A (0.4-72) and zearalenone (0.2-32); acetylcholinesterase for analysis of sterigmatocystin (0.12-219). The cells were found to be more sensitive than enzymes. The assayed strains of photobacterial cells ensured 44%-83% lower limit of detection for deoxynivalenol and sterigmatocystin as compared to the previously known data for immobilized luminescent cells, and the range of working concentrations was extended by a factor of 1.5-3.5. Calibration curves for the quantitative determination of patulin using immobilized photobacteria were presented in this work for the first time. This calibration was applied to estimate the enzyme efficiency for hydrolyzing mycotoxins using zearalenone and His6-tagged organophosphorus hydrolase as examples.

Designing a cocktail containing redox enzymes to improve hemicellulosic hydrolysate fermentability by microorganisms.

Bioproducts production using monomeric sugars derived from lignocellulosic biomass presents several challenges, such as to require a physicochemical pretreatment to improve its conversion yields. Hydrothermal lignocellulose pretreatment has several advantages and results in solid and liquid streams. The former is called hemicellulosic hydrolysate (HH), which contains inhibitory phenolic compounds and sugar degradation products that hinder microbial fermentation products from pentose sugars. Here, we developed and applied a novel enzyme process to detoxify HH. Initially, the design of experiments with different redox activities enzymes was carried out. The enzyme mixture containing the peroxidase (from Armoracia rusticana) together with superoxide dismutase (from Coptotermes gestroi) are the most effective to detoxify HH derived from sugarcane bagasse. Butanol fermentation by the bacteria Clostridium saccharoperbutylacetonicum and ethanol production by the yeast Scheffersomyces stipitis increased by 24.0× and 2.4×, respectively, relative to the untreated hemicellulosic hydrolysates. Detoxified HH was analyzed by chromatographic and spectrometric methods elucidating the mechanisms of phenolic compound modifications by enzymatic treatment. The enzyme mixture degraded and reduced the hydroxyphenyl- and feruloyl-derived units and polymerized the lignin fragments. This strategy uses biocatalysts under environmentally friendly conditions and could be applied in the fuel, food, and chemical industries.

An In Silico Target Fishing Approach to Identify Novel Ochratoxin A Hydrolyzing Enzyme.

Ochratoxin A (OTA), a mycotoxin that is of utmost concern in food and feed safety, is produced by fungal species that mainly belong to the Aspergillus and Penicillium genera. The development of mitigation strategies to reduce OTA content along the supply chains is key to ensuring safer production of food and feed. Enzyme-based strategies are among the most promising methods due to their specificity, efficacy, and multi-situ applicability. In particular, some enzymes are already known for hydrolyzing OTA into ochratoxin alpha (OTα) and phenylalanine (Phe), eventually resulting in detoxification action. Therefore, the discovery of novel OTA hydrolyzing enzymes, along with the advancement of an innovative approach for their identification, could provide a broader basis to develop more effective mitigating strategies in the future. In the present study, a hybrid in silico/in vitro workflow coupling virtual screening with enzymatic assays was applied in order to identify novel OTA hydrolyzing enzymes. Among the various hits, porcine carboxypeptidase B was identified for the first time as an effective OTA hydrolyzing enzyme. The successful experimental endorsement of findings of the workflow confirms that the presented strategy is suitable for identifying novel OTA hydrolyzing enzymes, and it might be relevant for the discovery of other mycotoxin- mitigating enzymes.

A quinone-dependent dehydrogenase and two NADPH-dependent aldo/keto reductases detoxify deoxynivalenol in wheat via epimerization in a Devosia strain.

The Fusarium mycotoxin deoxynivalenol (DON) is typically controlled by fungicides. Here, we report DON detoxification using enzymes from the highly active Devosia strain D6-9 which degraded DON at 2.5 µg/min/108 cells. Strain D6-9 catabolized DON to 3-keto-DON and 3-epi-DON, completely removing DON in wheat. Genome analysis of three Devosia strains (D6-9, D17, and D13584), with strain D6-9 transcriptomes, identified three genes responsible for DON epimerization. One gene encodes a quinone-dependent DON dehydrogenase QDDH which oxidized DON into 3-keto-DON. Two genes encode the NADPH-dependent aldo/keto reductases AKR13B2 and AKR6D1 that convert 3-keto-DON into 3-epi-DON. Recombinant proteins expressed in Escherichia coli efficiently degraded DON in wheat grains. Molecular docking and site-directed mutagenesis revealed that residues S497, E499, and E535 function in QDDH's DON-oxidizing activity. These results advance potential microbial and enzymatic elimination of DON in agricultural samples and lend insight into the underlying mechanisms and molecular evolution of DON detoxification.

Detoxification of the Fumonisin Mycotoxins in Maize: An Enzymatic Approach.

Enzymatic detoxification has become a promising approach for control of mycotoxins postharvest in grains through modification of chemical structures determining their toxicity. In the present study fumonisin esterase FumD (EC 3.1.1.87) (FUMzyme®; BIOMIN, Tulln, Austria), hydrolysing fumonisin (FB) mycotoxins by de-esterification, was utilised to develop an enzymatic reduction method in a maize kernel enzyme incubation mixture. Efficacy of the FumD FB reduction method in "low" and "high" FB contaminated home-grown maize was compared by monitoring FB1 hydrolysis to the hydrolysed FB1 (HFB1) product utilising a validated LC-MS/MS analytical method. The method was further evaluated in terms of enzyme activity and treatment duration by assessing enzyme kinetic parameters and the relative distribution of HFB1 between maize kernels and the residual aqueous environment. FumD treatments resulted in significant reduction (≥80%) in "low" (≥1000 U/L, p < 0.05) and "high" (100 U/L, p < 0.05; ≥1000 U/L, p < 0.0001) FB contaminated maize after 1 h respectively, with an approximate 1:1 µmol conversion ratio of FB1 into the formation of HFB1. Enzyme kinetic parameters indicated that, depending on the activity of FumD utilised, a significantly (p < 0.05) higher FB1 conversion rate was noticed in "high" FB contaminated maize. The FumD FB reduction method in maize could find application in commercial maize-based practices as well as in communities utilising home-grown maize as a main dietary staple and known to be exposed above the tolerable daily intake levels.

Enzymatic detoxification of organophosphorus pesticides and related toxicants.

Millions of cases of pesticide intoxication occur yearly and represent a public health problem. In addition, pesticide poisoning is the preferred suicidal method in rural areas. The use of enzymes for the treatment of intoxication due to organophosphorus pesticides was proposed decades ago. Several enzymes are able to transform organophosphorus compounds such as pesticides and nerve agents. Some specific enzymatic treatments have been proposed, including direct enzyme injection, liposome and erythrocytes carriers, PEGylated preparations and extracorporeal enzymatic treatments. Nevertheless, no enzymatic treatments are currently available. In this work, the use of enzymes for treating of organophosphorus pesticide intoxication is critically reviewed and the remaining challenges are discussed.

Degradation of Aflatoxins by Means of Laccases from Trametes versicolor: An In Silico Insight.

Mycotoxins are secondary metabolites of fungi that contaminate food and feed, and are involved in a series of foodborne illnesses and disorders in humans and animals. The mitigation of mycotoxin content via enzymatic degradation is a strategy to ensure safer food and feed, and to address the forthcoming issues in view of the global trade and sustainability. Nevertheless, the search for active enzymes is still challenging and time-consuming. The in silico analysis may strongly support the research by providing the evidence-based hierarchization of enzymes for a rational design of more effective experimental trials. The present work dealt with the degradation of aflatoxin B1 and M1 by laccase enzymes from Trametes versicolor. The enzymes-substrate interaction for various enzyme isoforms was investigated through 3D molecular modeling techniques. Structural differences among the isoforms have been pinpointed, which may cause different patterns of interaction between aflatoxin B1 and M1. The possible formation of different products of degradation can be argued accordingly. Moreover, the laccase gamma isoform was identified as the most suitable for protein engineering aimed at ameliorating the substrate specificity. Overall, 3D modeling proved to be an effective analytical tool to assess the enzyme-substrate interaction and provided a solid foothold for supporting the search of degrading enzyme at the early stage.

Antioxidant defence systems in the protozoan pathogen Giardia intestinalis.

The microaerophilic protist Giardia intestinalis is the causative agent of giardiasis, one of the most common intestinal infectious diseases worldwide. The pathogen lacks not only respiratory terminal oxidases (being amitochondriate), but also several conventional antioxidant enzymes, including catalase, superoxide dismutase and glutathione peroxidase. In spite of this, since living attached to the mucosa of the proximal small intestine, the parasite should rely on an efficient antioxidant system to survive the oxidative and nitrosative stress conditions found in this tract of the human gut. Here, we review current knowledge on the antioxidant defence systems in G. intestinalis, focusing on the progress made over the last decade in the field. The relevance of this research and future perspectives are discussed.

The furofuran-ring selectivity, hydrogen peroxide-production and low Km value are the three elements for highly effective detoxification of aflatoxin oxidase.

AFO (aflatoxin oxidase), an enzyme from Armillariella tabescens previously named aflatoxin detoxifizyme, exhibits oxidative detoxification activity toward aflatoxin B1 and sterigmatocystin. Bioinformatics reveals that AFO is a newly discovered oxidase because AFO does not share any significant similarities with any known oxidase. It is critically important to understand how AFO acts on aflatoxin B1. In this study, in addition to aflatoxin B1 (AFB1) and sterigmatocystin (ST), five other chemicals that have furan or pyran structures were investigated. The results indicated that in addition to AFB1 and ST, AFO is also able to act on versicolorin A, 3,4-dihydro-2H-pyran and furan. These results suggested that 8,9-unsaturated carboncarbon bond of aflatoxin B1 is the potential reactive site for AFO. Further findings indicated that the action of AFO is oxygen-dependent and hydrogen peroxide-producing. The simultaneously produced-hydrogen peroxide possibly plays the essential role in detoxification of AFO. In addition, the extremely low Km value of 0.33 µmol/l for AFO-AFB1 and 0.11 µmol/l for AFO-ST signifies that AFO is highly selective for AFB1 as well as ST.