Sol-gel immobilization of glutathione transferase: efficient tool for bioremediation

Glutathione transferases are multi-functional enzymes with an important role in xenobiotic detoxification. They catalyse the nucleophilic addition of the sulfur atom of glutathione (γ-L-Glu-L-Cys-Gly, GSH) to the electrophilic groups of a large variety of hydrophobic molecules including organic halides, epoxides, arene oxides, α- and β-unsaturated carbonyls, organic nitrate esters, and organic thiocyanates. The conjugation of GSH to such molecules increases their solubility and reduces their toxicity. GSTs represent a versatile tool with a variety of biotechnological applications, in the field of bioremediation to clean up environmentally contaminated sites. The purpose of this project was the study of GST immobilization for the biodegradation of toxic compounds

Inhibition of human glutathione transferases by pesticides: Development of a simple analytical assay for the quantification of pesticides in water

Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes that are involved in phase II cellular detoxification mechanism. Here, screening of the inhibition potency of a wide range of pesticides toward selected human GST isoenzymes (hGSTA1-1, hGSTP1-1, hGSTT2-2 and hGSTO1-1) was carried out. hGSTA1-1 was found more susceptible to inhibition by pesticides than other isoenzymes. The insecticides dieldrin and spiromesifen were identified as potent reversible inhibitors toward hGSTA1- 1 with IC50 values equal to 17.9 ± 1.7 M and 12.1 ± 3.4 M, respectively. Based on in silico docking analysis and kinetic inhibition studies it was concluded that dieldrin and spiromesifen bind specifically to the enzyme presumably at a distinct position that partially overlaps with both the G- and H-site. The ability of dieldrin and spiromesifen to inhibit hGSTA1-1 activity was exploited for the development of analytical quantification assays for these two pesticides. Linear calibration curves were obtained for dieldrin and spiromesifen, with useful concentration in the range of 0–10 M. The reproducibility of the assay response, expressed by relative standard deviation, was in the order of 4.1% (N = 28). The method was successfully applied to the determination of these pesticides in real water samples without sample preparation steps

Expanding the Plant GSTome Through Directed Evolution: DNA Shuffling for the Generation of New Synthetic Enzymes With Engineered Catalytic and Binding Properties

Glutathione transferases (GSTs, EC. 2.5.1.18) are inducible multifunctional enzymes that are essential in the detoxification and degradation of toxic compounds. GSTs have considerable biotechnological potential. In the present work, a new method for the generation of synthetic GSTs was developed. Abiotic stress treatment of Phaseolus vulgaris and Glycine max plants led to the induction of total GST activity and allowed the creation of a GST-enriched cDNA library using degenerated GST-specific primers and reverse transcription-PCR. This library was further diversified by employing directed evolution through DNA shuffling. Activity screening of the evolved library led to the identification of a novel tau class GST enzyme (PvGmGSTUG). The enzyme was purified by affinity chromatography, characterized by kinetic analysis, and its structure was determined by X-ray crystallography. Interestingly, PvGmGSTUG displayed enhanced glutathione hydroperoxidase activity, which was significantly greater than that reported so far for natural tau class GSTs. In addition, the enzyme displayed unusual cooperative kinetics toward 1-chloro-2,4-dinitrochlorobenzene (CDNB) but not toward glutathione. The present work provides an easy approach for the simultaneous shuffling of GST genes from different plants, thus allowing the directed evolution of plants GSTome. This may permit the generation of new synthetic enzymes with interesting properties that are valuable in biotechnology

Expanding the Plant GSTome Through Directed Evolution: DNA Shuffling for the Generation of New Synthetic Enzymes With Engineered Catalytic and Binding Properties

Glutathione transferases (GSTs, EC. 2.5.1.18) are inducible multifunctional enzymes that are essential in the detoxification and degradation of toxic compounds. GSTs have considerable biotechnological potential. In the present work, a new method for the generation of synthetic GSTs was developed. Abiotic stress treatment of Phaseolus vulgaris and Glycine max plants led to the induction of total GST activity and allowed the creation of a GST-enriched cDNA library using degenerated GST-specific primers and reverse transcription-PCR. This library was further diversified by employing directed evolution through DNA shuffling. Activity screening of the evolved library led to the identification of a novel tau class GST enzyme (PvGmGSTUG). The enzyme was purified by affinity chromatography, characterized by kinetic analysis, and its structure was determined by X-ray crystallography. Interestingly, PvGmGSTUG displayed enhanced glutathione hydroperoxidase activity, which was significantly greater than that reported so far for natural tau class GSTs. In addition, the enzyme displayed unusual cooperative kinetics toward 1-chloro-2,4-dinitrochlorobenzene (CDNB) but not toward glutathione. The present work provides an easy approach for the simultaneous shuffling of GST genes from different plants, thus allowing the directed evolution of plants GSTome. This may permit the generation of new synthetic enzymes with interesting properties that are valuable in biotechnology

Antioxidant capacity and immunomodulatory effects of a crysolaminarinenriched extract in Senegalese sole

The microalgae are an important source of bioactive molecules including &beta;-glucans that can be used as immunostimulants in aquaculture. In the present study, the antioxidant capacity, cytotoxicity and immunomodulatory activity of a chrysolaminarin-enriched extract obtained from the diatom Phaeodactylum tricornutum was evaluated. The extract showed a higher total antioxidant activity as determined by ORAC and FRAP assays and a lower DPPH scavenging activity than particulate yeast-&beta;-glucan. The cytotoxicity test indicated that extract concentrations higher than 0.01% w/v could impair cell viability of human dermal fibroblasts. To evaluate the immunomodulatory activity, juvenile soles were intraperitoneally injected with the chrysolaminarin-enriched extract suspended in coconut oil (1 mg/fish) followed by a reinjection at 7 days. A sham group injected with the carrier solution was maintained as a negative control. Cumulated mortality of fish injected with the chrysolaminarin-enriched extract was 29.4% after six days and no mortality was recorded after extract reinjection. Expression analyses of fifteen genes related to the innate immune system in kidney, spleen and intestine showed temporal and organ-specific responses. A rapid (2 days post-injection; dpi) and strong induction of the pro-inflammatory il1b and the antimicrobial peptide hamp1 in the three immunological organs, the hsp90aa in kidney and spleen, irf3 in intestine and c3 in spleen was observed indicating a potent inflammatory response. The recovery of steady-state levels for all activated genes at 5 dpi, and the down-regulation of c-lectin receptor as well as some interferon-related genes (ifn1, irf1, irf3, irf8, irf9 and mx) in kidney and cxc10 in spleen indicated that the soles were able to activate a homeostatic response against the &beta;-glucan insult. The reinjection of the chrysolaminarin-enriched extract did not activate a new inflammatory response but reduced the mRNA levels of hsp90aa and irf3 indicating that soles developed some resistance to &beta;-glucans. Overall, these results reveal this enriched extract as a novel and potent source of &beta;-glucans with antioxidant and immunomodulatory capacity suitable for immunostimulation in aquaculture. </p

A Key Role in Catalysis and Enzyme Thermostability of a Conserved Helix H5 Motif of Human Glutathione Transferase A1-1

Glutathione transferases (GSTs) are promiscuous enzymes whose main function is the detoxification of electrophilic compounds. These enzymes are characterized by structural modularity that underpins their exploitation as dynamic scaffolds for engineering enzyme variants, with customized catalytic and structural properties. In the present work, multiple sequence alignment of the alpha class GSTs allowed the identification of three conserved residues (E137, K141, and S142) at α-helix 5 (H5). A motif-directed redesign of the human glutathione transferase A1-1 (hGSTA1-1) was performed through site-directed mutagenesis at these sites, creating two single- and two double-point mutants (E137H, K141H, K141H/S142H, and E137H/K141H). The results showed that all the enzyme variants displayed enhanced catalytic activity compared to the wild-type enzyme hGSTA1-1, while the double mutant hGSTA1-K141H/S142H also showed improved thermal stability. X-ray crystallographic analysis revealed the molecular basis of the effects of double mutations on enzyme stability and catalysis. The biochemical and structural analysis presented here will contribute to a deeper understanding of the structure and function of alpha class GSTs

Overexpression of A Biotic Stress-Inducible <i>Pvgstu</i> Gene Activates Early Protective Responses in Tobacco under Combined Heat and Drought

Drought and heat stresses are major factors limiting crop growth and productivity, and their effect is more devastating when occurring concurrently. Plant glutathione transferases (GSTs) are differentially expressed in response to different stimuli, conferring tolerance to a wide range of abiotic stresses. GSTs from drought-tolerant Phaseolus vulgaris var. “Plake Megalosperma Prespon” is expected to play an important role in the response mechanisms to combined and single heat and drought stresses. Herein, we examined wild-type N. tabacum plants (cv. Basmas Xanthi) and T1 transgenic lines overexpressing the stress-induced Pvgstu3–3 and Pvgstu2–2 genes. The overexpression of Pvgstu3–3 contributed to potential thermotolerance and greater plant performance under combined stress. Significant alterations in the primary metabolism were observed in the transgenic plants between combined stress and stress-free conditions. Stress-responsive differentially expressed genes (DEGs) and transcription factors (TFs) related to photosynthesis, signal transduction, starch and sucrose metabolism, osmotic adjustment and thermotolerance, were identified under combined stress. In contrast, induction of certain DEGs and TF families under stress-free conditions indicated that transgenic plants were in a primed state. The overexpression of the Pvgstu3–3 is playing a leading role in the production of signaling molecules, induction of specific metabolites and activation of the protective mechanisms for enhanced protection against combined abiotic stresses in tobacco

Overexpression of A Biotic Stress-Inducible Pvgstu Gene Activates Early Protective Responses in Tobacco under Combined Heat and Drought

Drought and heat stresses are major factors limiting crop growth and productivity, and their effect is more devastating when occurring concurrently. Plant glutathione transferases (GSTs) are differentially expressed in response to different stimuli, conferring tolerance to a wide range of abiotic stresses. GSTs from drought-tolerant Phaseolus vulgaris var. “Plake Megalosperma Prespon” is expected to play an important role in the response mechanisms to combined and single heat and drought stresses. Herein, we examined wild-type N. tabacum plants (cv. Basmas Xanthi) and T1 transgenic lines overexpressing the stress-induced Pvgstu3–3 and Pvgstu2–2 genes. The overexpression of Pvgstu3–3 contributed to potential thermotolerance and greater plant performance under combined stress. Significant alterations in the primary metabolism were observed in the transgenic plants between combined stress and stress-free conditions. Stress-responsive differentially expressed genes (DEGs) and transcription factors (TFs) related to photosynthesis, signal transduction, starch and sucrose metabolism, osmotic adjustment and thermotolerance, were identified under combined stress. In contrast, induction of certain DEGs and TF families under stress-free conditions indicated that transgenic plants were in a primed state. The overexpression of the Pvgstu3–3 is playing a leading role in the production of signaling molecules, induction of specific metabolites and activation of the protective mechanisms for enhanced protection against combined abiotic stresses in tobacco

Genomic and phylogenetic analysis of choriolysins, and biological activity of hatching liquid in the flatfish Senegalese sole.

The hatching enzymes or choriolysins are key proteases in fish life cycle controlling the release of larvae to surrounding environment that have been suggested as target for novel biotechnological uses. Due to the large amounts of eggs released by the flatfish Solea senegalensis, during the spawning season, the hatching liquid properties and choriolysin-encoding genes were investigated in this species. A genomic analysis identified four putative genes referred to as SseHCEa, SseHCEb, SseLCE and SseHE. The phylogenetic analysis classified these paralogs into two clades, the clade I containing SseHCE paralogs and the clade II containing two well-supported subclades named as HE and LCE. The two SseHCE paralogs were intron-less and both genes were tandemly arrayed very close in the genome. The synteny and gene rearrangement identified in the flatfish lineage indicated that the duplication of these two paralogs occurred recently and they are under divergent evolution. The genes SseHE and SseLCE were structured in 8 exons and 7 introns and the synteny was conserved in teleosts. Expression studies confirmed that the four genes were expressed in the hatching gland cells and they migrate co-ordinately from the head to around the yolk sac close to the hatch with specific temporal and intensity expression profiles. Although the mRNA levels of the four genes peaked in the hours previous to larval hatching, the SseHCE and SseLCE paralogs kept a longer expression than SseHE after hatching. These expression patterns were consistent even when larvae were incubated at different temperatures that modified hatching times. The analysis of hatching-liquid using SDS-PAGE and zymography analyses of hatching liquid identified a major band of expected choriolysin size. The optimal pH for protease activity was 8.5 and inhibition assays using EDTA demonstrated that most of the activity in the hatching liquid was due to metalloproteases with Ca2+ ions acting as the most effective metal to restore the activity. All these data provide new clues about the choriolysin evolution and function in flatfish with impact in the aquaculture and the blue cosmetic industry

The Cosmeceutical Potential of the Yellow-Green Alga <i>Trachydiscus minutus</i> Aqueous Extract: Preparation of a Natural-Based Dermal Formula as a Proof of Concept

In the present study, selected cosmeceutical properties of aqueous extracts from the microalgae strain Trachydiscus minutus were assessed and compared with those obtained using three widely used Chlorella strains (C. vulgaris, C. sorokiniana, and C. minutissima). Among all extracts, T. minutus extracts showed the highest total antioxidant capacity (TAC) and inhibitory potency towards elastase, suggesting potential activity in controlling skin aging. Furthermore, the cytotoxicity, anti-inflammatory activity and UVA protection of T. minutus extract were evaluated employing normal human dermal fibroblasts (NHDF) and human keratinocyte HaCaT cells. The results showed that the T. minutus extract was able to significantly inhibit the transcription of selected marker genes involved in inflammation [interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor alpha (TNFα)]. In addition, treatment of NHDF and HaCaT cells with T. minutus extract ameliorate the UVA-induced cell damage by decreasing the accumulation of reactive oxygen species (ROS). Extracts from T. minutus were formulated into a skin care cream and an aqueous gel. Both formulas exhibited excellent compatibility and stability. Comprehensively, all these results suggest that T. minutus extract displays promising cosmeceutical properties by providing antioxidant, anti-aging, and anti-inflammatory activities, and therefore has potential for cosmeceutical use