Toxicological and Sedative Effects of Chipilin (Crotalaria longirostrata) Leaf Extracts Obtained by Maceration and Supercritical Fluid Extraction.

Chipilin (Crotalaria longirostrata) is consumed as a vegetable in the preparation of traditional dishes. As a folk medicine, Chipilin extracts are used as a hypnotic and sedative agent; however, there are few reports that support these uses. This study aimed to characterize the compounds present in Chipilin leaf extracts and to investigate their sedative effect using zebrafish as an in vivo model. Extracts were obtained by maceration with water (H2O), ethanol (EtOH), and EtOH-H2O, while oleoresin was obtained by supercritical fluid extraction (SFE). Total phenolic and flavonoid contents were quantified by colorimetric methods. Phytochemical constituents were identified by gas chromatography-mass spectrometry (GC-MS) analysis. The chronic and acute toxicities of Chipilin extracts were tested in zebrafish embryos and larvae, respectively. Chipilin sedative effect was tested by the larvae response to dark-light-dark transitions. EtOH-H2O extracts had the highest value of total phenolics (5345 ± 5.1 µg GAE/g), followed by water and oleoresin (1815 ± 5.1 and 394 ± 5.1 µg GAE/g, respectively). In water extracts were identified the alkaloid trachelanthamidine, 1,2ß-epoxy- and the alkyl ketone 7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, while oleamide, α-monostearin, and erucamide were detected in all samples except in water extracts. Oleoresin extract had the lowest embryotoxicity (LC50 = 4.99 µg/mL) and the highest sedative effects. SFE is a green alternative to obtain Chipilin extracts rich in erucamide, an endocannabinoid analogue, which plays an important role in the development of the central nervous system and in modulating neurotransmitter release.

Tracing of Di-Ethylhexyl Phthalate in the Tequila Production Process.

The purpose of this study was to determine the origin, presence, and fate of the endocrine disruptor di-ethylhexil phthalate (DEHP) during tequila production. For this, three tequila factories (small, medium, and large) were monitored. DEHP concentrations in water, agave, additives, lubricating greases, neoprene seals, and materials of each stage process were analyzed using gas chromatography/mass spectrometry. DEHP mass balances were performed to identify the processes with significant changes in the inputs/outputs. DEHP was detected in agave at up to 0.08 ± 0.03 mg kg-1, water 0.02 ± 0.01 mg kg-1, lubricant greases 131.05 ± 2.80 mg kg-1, and neoprene seals 369.11 ± 22.52 mg kg-1. Whereas, tequila produced in the large, medium, and small factories contained 0.05 ± 0.01, 0.24 ± 0.04, and 1.43 ± 0.48 mg kg-1 DEHP, respectively. Furthermore, in waste materials (vinasses and bagasse) released, 534.26 ± 349.02, 947.18 ± 65.84, and 5222.60 ± 2836.94 mg of DEHP was detected for every 1000 L of tequila produced. The most significant increase in DEHP occurred during the sugar extraction and distillation stages. Results demonstrate that main raw materials, such as agave and water, contain DEHP, but lubricant greases and neoprene seals are the major sources of DEHP contamination. Identification of the contamination sources can help the tequila industry to take actions to reduce it, protect consumer health and the environment, and prevent circular contamination.

Dietary Supplementation with Popped Amaranth Modulates the Gut Microbiota in Low Height-for-Age Children: A Nonrandomized Pilot Trial.

Amaranth has been recognized as a nutraceutical food because it contains high-quality proteins due to its adequate amino acid composition that covers the recommended requirements for children and adults. Since pre-Hispanic times, amaranth has been consumed as popped grain; the popping process improves its nutritive quality and improves its digestibility. Popped amaranth consumption has been associated with the recovery of malnourished children. However, there is no information on the impact that popped amaranth consumption has on gut microbiota composition. A non-randomized pilot trial was conducted to evaluate the changes in composition, structure, and function of the gut microbiota of stunted children who received four grams of popped amaranth daily for three months. Stool and serum were collected at the beginning and at the end of the trial. Short-chain fatty acids (SCFA) were quantified, and gut bacterial composition was analyzed by 16S rRNA gene sequencing. Biometry and hematology results showed that children had no pathology other than low height-for-age. A decrease in the relative abundance of Alistipes putredinis, Bacteroides coprocola, and Bacteroides stercoris bacteria related to inflammation and colitis, and an increase in the relative abundance of Akkermansia muciniphila and Streptococcus thermophiles bacteria associated with health and longevity, was observed. The results demonstrate that popped amaranth is a nutritious food that helps to combat childhood malnutrition through gut microbiota modulation.

Proteomic analysis of chemically transformed NIH-3T3 cells reveals novel mechanisms of action of amaranth lunasin-like peptide.

Food-derived biopeptides can interact with genes and proteins to preserve health and prevent the development of diseases. Lunasin is a soybean cancer-preventive peptide that has been well characterized; however, few studies have been carried out to characterize the function of amaranth lunasin-like peptide (AhLun). The aim of this work was to analyze the proteomic profile changes in NIH-3T3 cells when they are chemically transformed with the carcinogen 3-methylcholanthrene (3MC) in the absence or presence of AhLun. The addition of AhLun into the culture medium did not affect the cell morphology; however, as a chemopreventive agent, it significantly reduced anisokaryosis formation when cells were treated with 3MC. Changes in protein accumulation in NIH-3T3 cells were evaluated by gel-based proteomics analysis. Differentially accumulated protein spots that exhibited at least a twofold change in spot intensity (p < 0.05), when compared with control cells, were analyzed by LC-MS/MS. Successfully identified proteins were grouped into six main categories according to their localization and function (nuclear, ribosomal, mitochondrial, metabolism, cytoskeletal, and miscellaneous). The gel-based proteomic approach for the evaluation of the chemopreventive potential of AhLun reveals novel pathways of action and provides new clues about the possible mechanisms of action of this bioactive peptide present in amaranth seeds.

Lippia graveolens HBK oleoresins, extracted by supercritical fluids, showed bactericidal activity against multidrug resistance Enterococcus faecalis and Staphylococcus aureus strains.

OBJECTIVE: The aim of this work was to characterize Lippia graveolens oleoresins, obtained by Supercritical Fluid Extraction (SFE), from crops collected at different locations in Mexico. The antimicrobial effect of oleoresins was tested in reference strains and clinical isolates of susceptible and multidrug-resistant (MDR) strains of Enterococcus faecalis and Staphylococcus aureus. SIGNIFICANCE: The increasing of MDR strains is becoming a global public health problem that has led to the search for new treatments, and essential oils have resurged as a source of compounds with bactericidal functions. Oregano essential oil has attracted attention recently, however, this oil is mainly obtained by hydro-distillation (uses large amounts of water) or solvents extraction (potential contaminant). SFE has gained popularity as it represents an environmentally friendly technology. METHODS: L. graveolens oleoresins were obtained by SFE, total phenol contents were quantified by Folin-Ciocalteu method, the identification of compounds and thymol and carvacrol quantification was carried out by GC-MS. The antimicrobial activity was tested by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). RESULTS: SFE showed higher yields compared with the hydro-distillation process. L. graveolens grown in different Mexican locations showed differences in oleoresin composition and a slightly different antimicrobial capacity against clinical isolates. CONCLUSIONS: It was demonstrated that SFE is an efficient technology for extracting L. graveolens oleoresins. Additionally, the solvent-free extraction method and the observed antimicrobial effect increase the applications of these oleoresins in fields, such as cosmetics, food industry, medicine, amongst others.

Autodisplay of an endo-1,4-ß-xylanase from Clostridium cellulovorans in Escherichia coli for xylans degradation.

The goal of this work was the autodisplay of the endo ß-1,4-xylanase (XynA) from Clostridium cellulovorans in Escherichia coli using the AIDA system to carry out whole-cell biocatalysis and hydrolysate xylans. For this, pAIDA-xynA vector containing a synthetic xynA gene was fused to the signal peptide of the toxin subunit B Vibro cholere (ctxB) and the auto-transporter of the synthetic aida gene, which encodes for the connector peptide and ß-barrel of the auto-transporter (AT-AIDA). E. coli TOP10 cells were transformed and the biocatalyst was characterized using beechwood xylans as substrate. Optimal operational conditions were temperature of 55 °C and pH 6.5, and the Michaelis-Menten catalytic constants Vmax and Km were 149 U/gDCW and 6.01 mg/mL, respectively. Xylanase activity was inhibited by Cu2+, Zn2+ and Hg2+ as well as EDTA, detergents, and organic acids, and improved by Ca2+, Co2+ and Mn2+ ions. Ca2+ ion strongly enhanced the xylanolytic activity up to 2.4-fold when 5 mM CaCl2 were added. Also, Ca2+ improved enzyme stability at 60 and 70 °C. Results suggest that pAIDA-xynA vector has the ability to express functional xylanase to perform whole-cell biocatalysis in order to hydrolysate xylans from hemicellulose feedstock.

Biodegradation of diisononyl phthalate by a consortium of saline soil bacteria: optimisation and kinetic characterisation.

Diisononyl phthalate (DINP) is one of plasticisers most employed in the production of plastic materials and belongs to the most important environmental contaminants. In this work, a consortium of saline soil bacterial (SSB) capable of degrading DINP is presented. The genera of SSB-consortium were Serratia sp., Methylobacillus sp., Achromobacter sp., Pseudomonas sp., Stenotrophomonas sp., Methyloversatilis sp., Delftia sp. and Brevundimonas sp. Response surface methodology (RSM) study was employed to optimise and evaluate the culture conditions to improve the biodegradation of DINP. The optimal conditions were a pH 7.0, 31 °C and an initial DINP concentration of 500 mg L-1, resulting in almost complete biodegradation (99%) in 168 h. DINP degradation followed a first-order kinetic model, and the half-life was 12.76 h. During the biodegradation of DINP, 4-derived compounds were identified: monoisononyl phthalate, methyl nonyl phthalate, iso-nonanol and dimethyl phthalate. The metabolite profiling indicated that DINP was degraded through simultaneous pathways of de-esterification and ß-oxidation. Results suggest that the SSB-consortium could be useful for efficient biodegradation of the DINP-contaminated environments. KEY POINTS: • DINP degradation is mediated by de-esterification and ß-oxidation processes. • Temperature and the concentration of the substrate are key factors for DINP biodegradation • The SSB-consortium has the ability to biodegrade 99% of DINP (500 mg L-1).

Biodegradation of recalcitrant compounds and phthalates by culturable bacteria isolated from Liometopum apiculatum microbiota.

Liometopum apiculatum is a species of ants widely distributed in arid and semi-arid ecosystems where there is a relative food shortage compared with tropical ecosystems. L. apiculatum has established an ecological balance involving symbiotic interactions, which have allowed them to survive through mechanisms that are still unknown. Therefore, the aim of this study was to explore the metabolic potential of isolated bacteria from L. apiculatum using enzymatic activity assay and substrate assimilation. Results revealed a complex bacteria consortium belonging to Proteobacteria, Firmicutes, and Actinobacteria phylum. Most of the isolated bacteria showed activities associated with biopolymers degradation, from them Exiguobacterium and B. simplex showed the highest amylolytic activity (27 U/mg protein), while A. johnsonii and B. pumulis showed the highest cellulolytic and xylanolytic activities (1 and 2.9 U/mg protein, respectively). By other hand, some microorganisms such as S. ficaria, E. asburiae, P. agglomerans, A. johnsonii, S. rubidaea, S. marcescens, S. warneri, and M. hydrocarbonoxydans were able to grow up to 1000 mg/L of phthalates esters. These results not only revealed the important contribution of the symbionts in L apiculatum ants feeding habits, but also have shown a promising source of enzymes with potential biotechnological applications such as lignocellulosic biomass hydrolysis and bioremediation processes.

Autodisplay of alpha amylase from Bacillus megaterium in E. coli for the bioconversion of starch into hydrogen, ethanol and succinic acid.

In this work, the expression of an α-amylase from Bacillus megaterium on the cell surface of Escherichia coli strains WDHA (Δ hycA and Δ ldhA) and WDHFP (Δ hycA, Δ frdD and Δ pta) by the autodisplay adhesin involved in diffuse adherence (AIDA) system was carried out with the purpose to confer the ability to E. coli strains to degrade starch and thus produce hydrogen, ethanol and succinic acid. For the characterization of the biocatalyst, the effect of temperature (30-70 °C), pH (3-6) and CaCl2 concentration (0-25 mM), as well as the thermostability of the biocatalyst (55-80 °C) at several time intervals (15-60 min) were evaluated. The results showed that the biocatalyst had a maximum activity at 55 °C and pH 4.5. Calcium was required for the activity as well for the thermal stability of the biocatalyst. The calculated Vmax and Km values were 0.24 U/cm3 and 5.8 mg/cm3, respectively. Furthermore, a set of anaerobic batch fermentations was carried out using 10 g/dm3 of starch and 1 g/dm3 of glucose as carbon sources in 120 cm3 serological bottles, using WDHA and WDHFP strains harboring the pAIDA-amyA plasmid. The hydrogen production for WDHA was 1056.06 cm3/dm3 and the succinic acid yield was 0.68 g/gstarch, whereas WDHFP strain produced 1689.68 cm3/dm3 of hydrogen and an ethanol yield of 0.28 g/gstarch. This work represents a promising strategy to improve the exploitation of starchy biomass for the production of biofuels (hydrogen and ethanol) or succinate without the need of a pre-saccharification process.

Diminution of migration of phthalic acid esters in tequila beverage by the year of production.

The presence of diethyl-phthalate (DEP), dibutyl-phthalate (DBP), butylbenzyl-phthalate (BBP), diethylhexyl-phthalate (DEHP) and diisononyl-phthalate (DINP) was determined in 295 tequila samples. They were grouped by age of maturation (white, aged, extra aged or ultra aged) and year of production (between 2013 and 2018). Gas Chromatography coupled with Mass Spectrometry was used for identification and quantification. The results showed that 65 samples (22% of the total) were phthalate free. DEP (0.13-0.27 mg/kg), BBP (0.05-2.91 mg/kg) and DINP (1.64-3.43 mg/kg) were detected in 11 (3.73%), 37 (12.54%) and 5 (1.69%) samples, respectively. But, these concentrations did not exceed the maximum permitted limits (MPL) of phthalates for alcoholic beverages. DBP (0.01-2.20 mg/kg) and DEHP (0.03-4.64 mg/kg) were detected in 96 (32.54%) and 224 (75.93%) samples, from them only 10 (3.39%) and 15 (5.08%) samples, respectively, exceeded the MPL for alcoholic beverages and they were few tequilas produced in the year 2014 or before. DEHP was the most frequent phthalate found in tequila and observed DEHP concentrations were 2-times higher in ultra aged tequilas compared to those in white tequilas. We concluded that all tequilas produced in 2015 and after, satisfied the international standards for these compounds.

Engineering bioprintable alginate/gelatin composite hydrogels with tunable mechanical and cell adhesive properties to modulate tumor spheroid growth kinetics.

Tunable bioprinting materials are capable of creating a broad spectrum of physiological mimicking 3D models enabling in vitro studies that more accurately resemble in vivo conditions. Tailoring the material properties of the bioink such that it achieves both bioprintability and biomimicry remains a key challenge. Here we report the development of engineered composite hydrogels consisting of gelatin and alginate components. The composite gels are demonstrated as a cell-laden bioink to build 3D bioprinted in vitro breast tumor models. The initial mechanical characteristics of each composite hydrogel are correlated to cell proliferation rates and cell spheroid morphology spanning month long culture conditions. MDA-MB-231 breast cancer cells show gel formulation-dependency on the rates and frequency of self-assembly into multicellular tumor spheroids (MCTS). Hydrogel compositions comprised of decreasing alginate concentrations, and increasing gelatin concentrations, result in gels that are mechanically soft and contain a greater number of cell-adhesion moieties driving the development of large MCTS; conversely gels containing increasing alginate, and decreasing gelatin concentrations are mechanically stiffer, with fewer cell-adhesion moieties present in the composite gels yielding smaller and less viable MCTS. These composite hydrogels can be used in the biofabrication of tunable in vitro systems that mimic both the mechanical and biochemical properties of the native tumor stroma.

Diisononyl Phthalate Differentially Affects Sirtuin Expression in the HepG2 Cell Line.

Human exposure to phthalates has received special attention due to their possible adverse human health effects. Diisononyl phthalate (DINP) is a plasticizer still widely used in many products, despite being considered an endocrine disruptor. In this study, we evaluated DINP's cytotoxicity, its effect on the levels of reactive oxygen species (ROS), and its effect on sirtuin expression in HepG2 cells. Results showed that 1 µg/mL DINP significantly downregulated Sirt1, Sirt2, Sirt3, and Sirt5 gene expression (p < 0.05), while other sirtuins remained unaffected. Furthermore, protein levels of Sirt1 and Sirt3 were significantly downregulated by 1 µg/mL DINP. On the other hand, 100 µg/mL DINP doubled the levels of lysine acetylation proteins (increased 2-fold) as well as reactive oxygen species (ROS) compared with the controls. In conclusion, our study suggests, for the first time, that DINP regulates the potential epigenetic disruptor sirtuin family and leads to induction of ROS via sirtuins.

Phthalates affect the in vitro expansion of human hematopoietic stem cell.

Phthalates are esters of phthalic acid used industrially as plastic additives, however, these are not covalently bound to the polymer matrix and therefore can be released to the environment. The aim of this study was to evaluate the effect of four phthalates: dibutyl phthalate (DBP), benzyl butyl phthalate (BBP), diethyl phthalate (DEP) and diethylhexyl phthalate (DEHP) on the in vitro expansion of human hematopoietic cells from umbilical cord blood. For this, 0.5 × 106 cells/mL were exposure to concentrations ranging from 0.1 to 100 µg/mL and the total cell expansion was determined after 14 days of culture in IMDM-cytokines medium. The control cultures attained 1.31 ± 0.21 × 106 cell/mL, whereas the cultures exposed to DBP, BBP and DEHP showed a reduction from 23 to 81%, 17 to 69% and 15 to 93.5%, respectively. DEP did not affect the total cell expansion. The most significant decrease on total cell expansion was observed at 0.1 µg/mL DBP, 100 µg/mL BBP and 10 µg/mL DEHP (p < 0.05). Additionally, the effect of these compounds on the expansion of hematopoietic progenitors was analyzed by clonogenic assays as colony forming units (CFU). The CFU decreased considerably compared with respect to the control cultures. The reduction was 74.6 and 99.1% at 10 and 100 µg/mL DBP respectively, whereas 100 µg/mL BBP and 100 µg/mL DEHP reduced the CFU expansion in 97.1% and 81%, respectively. Cultures exposed to DEP did not show significant differences. The results demonstrate the toxicity of DBP, BBP and DEHP on the human hematopoietic stem cells.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (> 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Microbiota of edible Liometopum apiculatum ant larvae reveals potential functions related to their nutritional value.

Edible insects, due to their high nutritive value, are currently considered as a potential renewable source for food and feed production. Liometopum apiculatum ants are widely distributed in arid and semi-arid ecosystems and their larvae (escamoles) are considered as a delicacy, however the microbial importance in L. apiculatum nutritional ecology is unknown. The aim of this research was to characterize the microorganisms associated with both L. apiculatum larvae and the reproductive adult ants using the 16S rRNA gene sequencing and culturomics approaches. The obligate endosymbionts were also investigated through microscopic analysis. The most abundant Phylum identified by sequencing in the larvae was Firmicutes while in adult ants was Proteobacteria. Interestingly, the culturomics results showed 15 genera corresponding to the bacteria identified by sequencing analysis. Particularly, it was observed a large population of nitrogen-fixing bacteria, which could be linked with the high protein content in escamoles. Endosymbionts were detected in bacteoriocytes, these bacteria are related with vitamins and essential amino acids biosynthesis, and both compounds contributing to the high nutritional value of escamoles. This is the first report of the microorganisms present in the escamolera ant ensuring their safety as food and opening new areas of nutritional ecological and food processing.

Constitutive expression of the active fragment of human vasostatin Vs30 in Pichia pastoris SMD1168H.

Vasostatin 30 (Vs30) is an active fragment derived from the N-terminal region (135-164 aa) of human calreticulin and has the ability to inhibit angiogenesis. In this work, the expression of Vs30 was performed using a protease-deficient strain of the methylotrophic yeast Pichia pastoris. The vs30 gene was optimized for P. pastoris preferential codon usage and inserted into constitutive expression vector pGAPZαA. In addition, a plasmid with four copies of the expression cassette was obtained and transformed into P. pastoris. The flask fermentation conditions were: culture volume of 25 mL in 250 mL baffled flasks at 28 °C, pH 6 and harvest time of 48 h. Up to 21.07 mg/L Vs30 were attained and purified by ultrafiltration with a 30-kDa cut-off membrane and the recovery was 49.7%. Bioactivity of Vs30 was confirmed by the inhibition of cell proliferation, as well as the inhibition of the capillary-like structures formation of EA.hy926 cells in vitro. This work constitutes the first report on the expression of Vs30 in Pichia pastoris using a constitutive promoter and multi-copy approach such as strategies to improve the recombinant Vs30 expression.

2D-DIGE as a strategy to identify serum biomarkers in Mexican patients with Type-2 diabetes with different body mass index.

Obesity and type 2 diabetes(T2D) are the most prevalent and serious metabolic diseases affecting people worldwide. However racial and ethnic disparities seems to be a risk factor for their development. Mexico has been named as one of the largest populations with the highest prevalence of diabetes and obesity. The aim of this study was to identify novel T2D-associated proteins in Mexican patients. Blood samples were collected from 62 Mexican patients with T2D and they were grouped according to their body mass index(BMI). A panel of 10 diabetes and obesity serum markers was determined using MAGPIX. A comparative proteomics study was performed using two-dimensional difference in-gel electrophoresis(2D-DIGE) followed by mass spectrometry(LC-MS/MS). We detected 113 spots differentially accumulated, in which 64 unique proteins were identified, proteins that were involved in metabolism pathways, molecular transport, and cellular signalling. Four proteins(14-3-3, ApoH, ZAG, and OTO3) showing diabetes-related variation and also changes in relation to obesity were selected for further validation by western blotting. Our results reveal new diabetes related proteins present in the Mexican population. These could provide additional insight into the understanding of diabetes development in Mexican population and may also be useful candidate biomarkers.

Indole modifies the central carbon flux in the anaerobic metabolism of Escherichia coli: application to the production of hydrogen and other metabolites.

Indole is a bicyclic signaling molecule with effects on both eukaryotic and prokaryotic cells. The majority of studies of indole action have been performed with bacteria cultured under aerobic conditions and little information is available about its effects under anaerobic conditions. Here the effect of the indole on anaerobic metabolism of Escherichia coli WDHL was studied. Indole in the range 0.5-8mM was added to the culture medium and cell growth, hydrogen and metabolite production were compared to cultures lacking indole. Results showed that while 8mM indole abolished growth completely, 4mM indole had a partial bacteriostatic effect and the maximum optical density of the culture decreased by 44% compared to the control cultures. In addition, 4mM indole had an important effect on anaerobic metabolism. Hydrogen production increased from 650±115 to 1137±343mL H2/L, and hydrogen yield increased from 0.45±0.1 to 0.94±0.34mol H2/mol glucose, compared to the control culture. Carbon flux was also affected and the composition of the final by-products changed. Lactate (41mM) was the main metabolite in the control cultures, whereas ethanol (56.2mM) and acetate (41.2mM) were the main metabolites in the cultures with 2mM indole. We conclude that the supplementation of E. coli cultures with exogenous indole is a simple and novel strategy to improve the production of hydrogen as well as other metabolites such as ethanol used as biofuels.

Biochemical and Molecular Characterization of a Novel Cu/Zn Superoxide Dismutase from Amaranthus hypochondriacus L.: an Intrinsically Disordered Protein.

A novel Cu/ZnSOD from Amaranthus hypochondriacus was cloned, expressed, and characterized. Nucleotide sequence analysis showed an open reading frame (ORF) of 456 bp, which was predicted to encode a 15.6-kDa molecular weight protein with a pI of 5.4. Structural analysis showed highly conserved amino acid residues involved in Cu/Zn binding. Recombinant amaranth superoxide dismutase (rAhSOD) displayed more than 50 % of catalytic activity after incubation at 100 °C for 30 min. In silico analysis of Amaranthus hypochondriacus SOD (AhSOD) amino acid sequence for globularity and disorder suggested that this protein is mainly disordered; this was confirmed by circular dichroism, which showed the lack of secondary structure. Intrinsic fluorescence studies showed that rAhSOD undergoes conformational changes in two steps by the presence of Cu/Zn, which indicates the presence of two binding sites displaying different affinities for metals ions. Our results show that AhSOD could be classified as an intrinsically disordered protein (IDP) that is folded when metals are bound and with high thermal stability.

Expression and characterization of a recombinant psychrophilic Cu/Zn superoxide dismutase from Deschampsia antarctica E. Desv. [Poaceae].

We present here the structural modeling and biochemical characterization of a recombinant superoxide dismutase (SOD) from Deschampsia antarctica E. Desv. [Poaceae] produced in Escherichia coli. The recombinant protein was purified by affinity chromatography nickel-nitrilotriacetic acid (Ni-NTA), and its identity was demonstrated by immunoblotting and inhibition by H2O2 and KCN. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis confirmed the presence of Cu and Zn. Modeling of the D. antarctica Cu/Zn-SOD (DaSOD) amino acid sequence using the SWISS-MODEL and 2Q2L_B monomer of the psychrophilic Cu/Zu-SOD from Potentilla atrosanguinea (PaSOD) as template produced a structure similar to that of the typical eukaryotic Cu/Zn-SODs. Activity assays using the p-nitro blue tetrazolium chloride (NBT) solution method showed that the purified DaSOD had a specific activity of 5818 U/mg at 25 °C and pH 7.2 and that it was active in a pH interval of 5-8 and a temperature interval of 0-40 °C. Furthermore, DaSOD was still active at -20 °C as observed by a zymogram assay. We found 100 % activity when it was heated at 80 °C for 60 min, indicating a high thermostability. DaSOD properties suggest that this enzyme could be useful for preventing the oxidation of refrigerated or frozen foods, as well as in the preparation of cosmetic and pharmaceutical products.