Iron porphyrin-based porous organic polymer with high peroxidase-like activity as colorimetric sensor for glutathione and ascorbic acid assay.

A new iron porphyrin-based organic polymer (Fe-POP) was synthesized through the William ether reaction. The as-prepared Fe-POP presented high chemical stability, wide pore distribution, high iron content, and strong affinity with 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2), which contributed to its excellent peroxidase-mimicking performance. In the presence of H2O2, Fe-POP could catalyze the transparent TMB into blue ox-TMB, which could be easily distinguished by the naked eyes. Moreover, glutathione (GSH) and ascorbic acid (AA) could convert blue ox-TMB into colorless TMB due to the inhibitory effect of GSH/AA to the catalytic oxidation of TMB. Based on this phenomenon, a rapid and sensitive colorimetric method for the assay of H2O2, GSH, and AA was developed using Fe-POP as sensor. The detection limits of H2O2, GSH, and AA  were 1.37, 0.44, and 0.33 µM, respectively. Finally, the colorimetric method based on Fe-POP was used to evaluate the GSH and AA content in real samples, which provided the guidance for GSH and AA supplements in our daily diet, suggesting the significant potential of Fe-POP in practical applications.

A G-quadruplex/hemin structure-undamaged method to inhibit peroxidase-mimic DNAzyme activity for biosensing development.

Damaging the structure of the G-quadruplex (G4) to prevent the formation of the G4/hemin complex is presently the only available method to inhibit the activity of the peroxidase-mimic DNAzyme. In this study, a unique intramolecular inhibitory effect of the adjacent base-pair (InE(N:N)), by installing a rationally adjacent base-pair of the G4 core sequence, is proposed for the inhibition of the DNAzyme activity, which eliminates the need to damage the entire G4 structure. Various base pairs show different abilities to inhibit DNAzyme activity. The adjacent adenine: thymine pair possesses the best inhibitory efficiency (17 times). Through detailed investigations of the InE(N:N), it was revealed that the adjacent adenine: thymine pair downregulated the formation of compound I in the catalytic process, thus inhibiting the G4 DNAzyme activity. The mechanism of inhibition indicated that the carbonyl group on the hexatomic ring of the complementary base played an important role. To further reflect the advantages of the proposed strategy, two InE(N:N)-based biosensors were developed for DNA analysis and Uracil-DNA glycosylase (UDG) detection. Compared with existing DNAzyme-based methods, the application of InE(N: N) facilitates the real-time assay and simplifies the design difficulty. Therefore, InE(N:N) provides new insights into the regulation of the DNAzyme activity and offers an efficient approach for the future application of DNAzyme.

Deployment of response surface methodology to optimize microencapsulation of peroxidases from turnip roots (Brassica rapa L.) by double emulsion in PLA polymer.

In order to improve the preservation conditions and stability of peroxidase catalytic properties, a number of immobilization techniques have been widely developed. In this context, we set as objective, the optimization of synthesis and stability of microcapsules of peroxidases (POD) from turnip using polylactic acid (PLA) polymer with the double emulsion technique. The surfactant, polymer, and peroxidase concentrations were the optimized parameters. According to the results obtained using the Box-Behnken design, the optimal parameters found were 1.55% of PVA, 55 mg/mL of peroxidases, and 30 mg/mL of PLA polymer with an encapsulation efficiency of 57.29%. The scanning electron microscopy morphological characterization of the optimized microcapsules showed a regular spherical structure. Fourier transform infrared spectroscopy identified the specific functional groups and chemical bonds before and after microencapsulation. The elaborated microcapsules were characterized by an average size of 200 µm (mainly from 150 to 500 µm) with a low residual moisture content (2.26%) and the encapsulated peroxidases showed better thermal stability. The in vitro release of peroxidases confirmed that the microcapsules have an excellent sustained release in simulated gastric digestion. Encapsulated peroxidases' storage under 25 and 4 °C displays a good residual POD activity with about 60% of initial activities during 80 days of storage, whereas free POD losses its initial activity within 15 and 30 days, respectively. The obtained results are promising for the development of effective therapeutic treatment of some intestinal troubles due to oxidative stress. PRACTICAL APPLICATION: Brassica rapa L. root is well known for its richness on peroxidases and thus presents an interesting potential for developing high added value products. In order to preserve the activity of extracted peroxidases (POD) from turnip roots, microencapsulation was optimized using a polylactic acid polymer. The encapsulated POD showed the maintenance of its activity under the effect of different storage conditions (time and temperature) and demonstrated resistance to gastric acidity. According to the obtained results, the encapsulation of peroxidases opens up medicine and pharmaceutical applications such as intestinal and colic protection against inflammations.

High peroxidase-mimicking activity of gold@platinum bimetallic nanoparticle-supported molybdenum disulfide nanohybrids for the selective colorimetric analysis of cysteine.

Herein, gold@platinum (Au@Pt) bimetallic nanoparticles with high catalytic ability were in situ decorated onto a molybdenum disulfide (MoS2) surface to obtain nanocomposites (MoS2-Au@Pt) with high peroxidase-mimicking activity, which were used to construct a colorimetric sensor for cysteine (Cys) detection. Interestingly, this sensor can efficiently distinguish Cys from homocysteine (Hcy), glutathione (GSH) and 19 other amino acids with high sensitivity. As expected, the colorimetric sensor can determine the Cys content in Cys supplement tablets due to its high stability and repeatability. Finally, the detection mechanism was studied.

A colorimetric aptamer-based method for detection of cadmium using the enhanced peroxidase-like activity of Au-MoS2 nanocomposites.

In this work, a colorimetric aptamer-based method for detection of cadmium using gold nanoparticles modified MoS2 nanocomposites as enzyme mimic is established. In short, biotinylated Cd2+ aptamers are immobilized by biotin-avidin binding on the bottoms of the microplate, the complementary strands of Cd2+ aptamers are connected to the Au-MoS2 nanocomposites which have the function of enhanced peroxidase-like activity. The csDNA-Au-MoS2 signal probe and target Cd2+ compete for binding Cd2+ aptamer, the color change can be observed by addition of chromogenic substrate, thereby realizing visual detection of Cd2+. The absorbance of the solution at 450 nm has a clear linear relationship with the Cd2+ concentration. The linear range is 1-500 ng/mL, and the limit of detection is 0.7 ng/mL. The assay was used to test white wine samples, the results are consistent with those of atomic absorption spectrometry; which prove that this method can be used for detection of Cd2+ in real samples.

Enhanced biodegradation of sediment-bound heavily weathered crude oil with ligninolytic enzymes encapsulated in calcium-alginate beads.

The degradation of crude and weathered crude oil following the application of crude and calcium-alginate encapsulated ligninolytic enzymes was studied using in situ microcosms. Changes in the chemical composition of the oil were monitored in crude enzyme extracts, as well as a sediment matrix, for as long as 70 days. Compound-specific effects of ligninolytic enzymes applied to the sediments were observed over time through changes in concentration of total petroleum hydrocarbons (TPH), polycyclic aromatic hydrocarbons (PAHs) and fractions of saturates, aromatics, resins and asphaltenes (SARA). As the oil weathered, most TPH and PAH fractions showed a rapid decrease in concentration. As sediment oil concentrations decreased following treatment with ligninolytic enzymes, the microbial population was enriched with hydrocarbon-degrading species. This trend demonstrates that the oil fractions initially not bioavailable for microbial degradation, were subsequently released to the sediment via catalytic conversion with laccase and manganese peroxidase, and the oil continues to be biodegraded by microbial populations.

Biosynthesis of gold and selenium nanoparticles by purified protein from Acinetobacter sp. SW 30.

Synthesis of nanoparticles is an enzymatic reduction process in microorganisms. In the present study, a protein, lignin peroxidase has been purified by DEAE-Cellulose anion exchange chromatography and Biogel P-150 gel filtration chromatography from the cell suspension of Acinetobacter sp. SW30 responsible for the synthesis of gold nanoparticles (AuNP) and selenium nanoparticles (SeNP). The purified fraction has a specific activity of 29.4U/mg/min with 959 fold purification. Native and SDS PAGE confirmed that purified lignin peroxidase is monomeric enzyme with 97.4KDa molecular weight. The enzyme synthesized spherical crystalline AuNP (10±2nm) and amorphous SeNP (100±10nm). It has maximum activity at pH 2 and temperature 40°C, with 1.0mMKm value, when n-propanol was used as a substrate. Activity was completely inhibited by sodium thiosulphate and zinc sulphate. This is the first report on association of lignin peroxidase in the synthesis of AuNP and SeNP from Acinetobacter sp. SW30.

Quantitative urine test strip reading for leukocyte esterase and hemoglobin peroxidase.

BACKGROUND: Recently, urine test strip readers have become available for automated test strip analysis. We explored the possibilities of the Sysmex UC-3500 automated urine chemistry analyzer based on complementary metal oxide semiconductor (CMOS) sensor technology with regard to accuracy of leukocyte esterase and hemoglobin peroxidase results. We studied the influence of possible confounders on these measurements. METHODS: Reflectance data of leukocyte esterase and hemoglobin peroxidase were measured using CMOS technology on the Sysmex UC-3500 automated urine chemistry analyzer. Analytical performance (imprecision, LOQ) as well as the correlation with white blood cell (WBC) and red blood cell (RBC) counts (Sysmex UF-5000) were studied. Furthermore, the influence of urinary dilution, haptoglobin, pH and ascorbic acid as confounders was determined. RESULTS: Within- and between-run imprecision (reflectance signal) ranged from 1.1% to 3.6% and 0.9% to 4.2% for peroxidase and 0.4% to 2.5% and 0.4% to 3.3% for leukocyte esterase. Good agreement was obtained between the UF-5000 for RBCs and peroxidase reflectance (r=0.843) and for WBCs and leukocyte esterase (r=0.821). Specific esterase activity decreased for WBC counts exceeding 100 cells/µL. Haptoglobin influenced the peroxidase activity, whereas leukocyte esterase and peroxidase activities showed a pH optimum between 5.0 and 6.5. A sigmoidal correlation was observed between urinary osmolality and peroxidase activity. CONCLUSIONS: CMOS technology allows to obtain high quality test strip results for assessing WBC and RBC in urine. Quantitative peroxidase and leukocyte esterase are complementary with flow cytometry and have an added value in urinalysis, which may form a basis for expert system development.

Defense-related Proteins from Chelidonium majus L. as Important Components of its Latex.

The aim of this review is to cover most recent research on plant pathogenesis- and defenserelated proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesisrelated (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores.

Recovery of lignin peroxidase from submerged liquid fermentation of Amauroderma rugosum (Blume & T. Nees) Torrend using polyethylene glycol/salt aqueous two-phase system.

Amauroderma rugosum is a wild mushroom species widely distributed in tropics and is classified under the class of Basidiomycetes. Basidiomycetes are well-known for their abilities of producing lignocellulolytic enzymes such as lignin peroxidase (LiP), laccase (Lac) and manganese peroxidase (MnP). Different factors such as nutrient sources, incubation period and agitation affect the production of lignocellulolytic enzymes. The A. rugosum produced LiP in the medium supplemented with potato dextrose broth (PDB), 0.5% yeast and 1.0% saw dust at 26.70±3.31 U/mL. However, the LiP activity was increased to 106.32±5.32 U/mL when supplemented with 150 µm of copper (CuSO4). The aqueous two-phase system (ATPS) is a simple, rapid and low cost method for primary extraction and recovery of LiP. A total of 25 systems made from five different molecular weights of polyethylene glycol (PEG)/dipotassium hydrogen phosphate (K2HPO4) were tested. PEG 600 produced the highest top phase purification factor (PFT) of 1.33±0.62 with yield of 72.18±8.50%. The optimization of the ATPS parameters, such as volume ratio VR, pH and crude enzyme loading are the factors controlling the phase partition. Our results showed that significant improvement (PFT of 6.26±2.87 with yield of 87.31±3.14%) of LiP recovery can be achieved by optimized the parameters.

Expression of a fungal manganese peroxidase in Escherichia coli: a comparison between the soluble and refolded enzymes.

BACKGROUND: Manganese peroxidase (MnP) from Irpex lacteus F17 has been shown to have a strong ability to degrade recalcitrant aromatic pollutants. In this study, a recombinant MnP from I. lacteus F17 was expressed in Escherichia coli Rosetta (DE3) in the form of inclusion bodies, which were refolded to achieve an active enzyme. Further, we optimized the in vitro refolding conditions to increase the recovery yield of the recombinant protein production. Additionally, we attempted to express recombinant MnP in soluble form in E. coli, and compared its activity with that of refolded MnP. RESULTS: Refolded MnP was obtained by optimizing the in vitro refolding conditions, and soluble MnP was produced in the presence of four additives, TritonX-100, Tween-80, ethanol, and glycerol, through incubation at 16 °C. Hemin and Ca2+ supplementation was crucial for the activity of the recombinant protein. Compared with refolded MnP, soluble MnP showed low catalytic efficiencies for Mn2+ and H2O2 substrates, but the two enzymes had an identical, broad range substrate specificity, and the ability to decolorize azo dyes. Furthermore, their enzymatic spectral characteristics were analysed by circular dichroism (CD), electronic absorption spectrum (UV-VIS), fluorescence and Raman spectra, indicating the differences in protein conformation between soluble and refolded MnP. Subsequently, size exclusion chromatography (SEC) and dynamic light scattering (DLS) analyses demonstrated that refolded MnP was a good monomer in solution, while soluble MnP predominantly existed in the oligomeric status. CONCLUSIONS: Our results showed that two forms of recombinant MnP could be expressed in E. coli by varying the culture conditions during protein expression.

Removal of pigments from molasses wastewater by combining micro-electrolysis with biological treatment method.

Pigments in molasses wastewater (MWW) effluent, such as melanoidins, were considered as kinds of the most recalcitrant and hazardous colorant contaminants to the environment. In this study, de-coloring the MWW by a synergistic combination of micro-electrolysis with bio-treatment was performed. Aiming to a high de-colorization yield, levels of nutrition source supplies, MWW dilution ratio, and micro-electrolysis reaction time were optimized accordingly. For a diluted (50 %, v/v) MWW, an maximum overall de-colorization yield (97.1 ± 0.5 %, for absorbance at 475 nm) was achieved through the bio-electrolysis treatment. In electrolysis bio-treatment, the positive effect of micro-electrolysis was also revealed by a promoted growth of fungal biomass as well as activities of ligninolytic enzymes. Activities of lignin peroxidase, manganese peroxidase, and laccase were promoted by 111.2, 103.9, and 7.7 %, respectively. This study also implied that the bio-treatment and the micro-electrolysis had different efficiencies on removal of pigments with distinct polarities.

Chitosan beads immobilized manganese peroxidase catalytic potential for detoxification and decolorization of textile effluent.

Textile industry has led to severe environmental pollution and is posing a serious threat to the ecosystems. Immobilized biocatalysts have gained importance as potential bio-remediating agent. Manganese peroxidase (MnP) was immobilized onto glutaraldehyde activated chitosan beads by crosslinking and employed for the degradation and detoxification of dyes in textile effluents. The efficiency of chitosan-immobilized MnP (CI-MnP) was evaluated on the basis of decolorization, water quality improvement and toxicity reduction. Maximum color removal of 97.31% was recorded and up to 82.40%, 78.30% and 91.7% reductions in COD, TOC, and BOD were achieved, respectively. The cytotoxicity of bio-treated effluents reduced significantly and 38.46%, 43.47% and 41.83% Allium cepa root length, root count and mitotic index were increased, respectively, whereas brine shrimp nauplii death reduced up to 63.64%. Mutagenicity (Ames test) reduced up to 73.44% and 75.43% for TA98 and TA100 strains, respectively. The CI-MnP retained 60% activity after 10 repeated decolorization batches. The CI-MnP showed excellent efficiency for the bioremediation of textile effluents and can be used for the remediation of toxic agents in wastewater. The monitoring of processed wastewater using bioassays is suggested to evaluate bio-efficiency of treatment method for safe disposal of effluents into water bodies.

[Physiological responses of sugar beet (Beta vulgaris) to drought stress during vegetative development period under drip irrigation].

Sugar beet (Beta vulgaris cv. Beta 356) was subjected to drought stress during vegetative development by maintaining the soil water content in the 0-40 cm soil depth at 70%, 50% or 30% of field capacity to study the physiological traits of the leaves. Results showed that the compensation index was the highest in the 50% field capacity treatment. Malonaldehyde (MDA) content, relative conductivity, catalase (CAT) activity, and soluble sugar content began to increase 24 h after rehydration. Proline content began to increase 48 h after rehydration. In contrast, no compensation effect was observed in peroxidase (POD) activity after rehydration. Among the active oxygen scavenging enzymes, CAT was most sensitive to drought stress. Supplemental irrigation should be carried out promptly when the soil water content dropped to 50% of field capacity during vegetative development. Rehydration could promote self-repair functions in leaves, thus reducing the effects of drought on sugar beet yield and sugar content.

Comparative Cold Shock Expression and Characterization of Fungal Dye-Decolorizing Peroxidases.

Dye-decolorizing peroxidases (DyPs) from Auricularia auricula-judae, Bjerkandera adusta, Pleurotus ostreatus and Marasmius scorodonius (Basidiomycota) were expressed in Escherichia coli using the cold shock-inducible expression system pCOLD I DNA. Functional expression was achieved without the addition of hemin or the co-expression of any chaperones. The presence or absence of the native signal sequence had a strong impact on the success of the expression, but the effect was not consistent for the different DyPs. While BaDyP and AajDyP were stable at 50 °C, the more thermolabile MsP2 and PoDyp, upon catalytic intervention, lend themselves to more rapid thermal inactivation. The bleaching of norbixin (E 160b) using MsP2 was most efficient at pH 4.0, while BaDyP and AajDypP worked best in the weakly acidic to neutral range, indicating a choice of DyPs for a broad field of applications in different food matrices.

Immobilized soybean hull peroxidase for the oxidation of phenolic compounds in coffee processing wastewater.

Chitosan beads were prepared, using glutaraldehyde as a crosslinking agent for the immobilization of soybean hull peroxidase (SBP). The activity of free and immobilized SBP was studied. The optimum pH was 6.0 for both the free and immobilized enzyme; however, enzyme activity became more dependent on the temperature after immobilization. This study evaluated the potential use of immobilized and free enzyme in the oxidation of caffeic acid, of synthetic phenolic solution (SPS) and of total phenolic compounds in coffee processing wastewater (CPW). Some factors, such as reaction time, amount of H2O2 and caffeic acid were evaluated, in order to determine the optimum conditions for enzyme performance. Both enzymes showed a potential in the removal of caffeic acid, SPS and CPW, and immobilized SBP had the highest oxidation performance. The immobilized enzyme showed a potential of 50% in the oxidation of caffeic acid after 4 consecutive cycles.

PINUS cembra L: histo-anatomical features, antioxidant enzyme activities and heavy metal contents of leaves and long shoots.

AIM: This study aimed to investigate the histo-anatomical features of the long shoots and leaves (young and mature) of Pinus cembra L.. The activity of antioxidant enzymatic systems and the content of heavy metals were also evaluated. MATERIAL AND METHODS: For the histo-anatomical study, the cross-sections were performed by usual techniques. The activity of antioxidant enzymatic systems (superoxide dismutase, catalase and peroxidase) was evaluated by spectrophotometric methods. The content of heavy metals was determined by atomic absorption spectroscopy. RESULTS: The cross-section through the long shoots shows many resiniferous canals and a periderm of variable thickness. The leaf has a triangular shape and only two vascular bundles in the inferior and upper levels. The highest level of superoxide dismutase activity (344.90 U/mg protein) was determined in the long shoots collected from a cembran pine in Vatra Dornei, while the highest level of peroxidase activity (7611.11 U/mg protein) was found in the leaves collected in Calimani Mountains. Cd level in all samples was under the quantification limit. Higher levels of Pb were determined in the long shoots (3 µg/g dry weight for the vegetal material collected in Vatra Dornei and 2.86 µg/g dry weight for the vegetal material collected in Calimani Mountains). CONCLUSIONS: Pinus cembra L. leaves show specific elements of subgenus Strobus (a triangular shape of the cross section, one single vascular bundle and two resiniferous canals). The results obtained for the superoxide dismutase and peroxidase activities corroborated with those obtained for the heavy metal contents indicate that antioxidant enzymes play an important role in the protection of Pinus cembra L. against exogenous stress factors.

Manganese peroxidases from Ganoderma applanatum degrade ß-carotene under alkaline conditions.

A ß-carotene-degrading enzyme activity was observed in liquid cultures of the basidiomycete Ganoderma applanatum. Supplementing the cultures with ß-carotene induced the bleaching activity. Purification via hydrophobic interaction, ion exchange and size exclusion chromatography followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resulted in a single protein band. LC-ion-trap-MS analyses and gene amplification identified two manganese peroxidase isoenzymes with 97.8 % identity on the amino acid level. These showed an estimated molecular mass of 48 kDa and an isoelectric point of 2.6. Properties not yet described for other manganese peroxidases were hydrogen-peroxide-independent catalysis and two maxima of the bleaching activity, a distinct one at pH 5 and a lower one at pH 8. During simulated washing studies, the applicability of the isoenzymes for the brightening of carotenoids under alkaline conditions was proven. The new enzymes may replace common bleaching agents to produce environmentally more compatible detergent formulations.

cDNA cloning and characterization of vanadium-dependent bromoperoxidases from the red alga Laurencia nipponica.

The marine red alga genus Laurencia is one of the richest producers of unique brominated compounds in the marine environment. The cDNAs for two Laurencia nipponica vanadium-dependent bromoperoxidases (LnVBPO1 and LnVBPO2) were cloned and expressed in Escherichia coli. Enzyme assays of recombinant LnVBPO1 and LnVBPO2 using monochlorodimedone revealed that they were thermolabile but their Km values for Br(-) were significantly lower than other red algal VBPOs. The bromination reaction was also assessed using laurediol, the predicted natural precursor of the brominated ether laurencin. Laurediol, protected by trimethylsilyl at the enyne, was converted to deacetyllaurencin by the LnVBPOs, which was confirmed by tandem mass spectrometry. Native LnVBPO partially purified from algal bodies was active, suggesting that LnVBPO is functional in vivo. These results contributed to our knowledge of the biosynthesis of Laurencia brominated metabolites.

Purification and characterization of novel cationic peroxidases from Asparagus acutifolius L. with biotechnological applications.

Four novel basic peroxidases, named AaP-1, AaP-2, AaP-3, and AaP-4, were purified from Asparagus acutifolius L. seeds by cation-exchange and gel filtration chromatographies. The four proteins showed a similar electrophoretic mobility of 46 kDa while, by MALDI-TOF MS, different Mr values of 42758.3, 41586.9, 42796.3, and 41595.5 were determined for AaP-1, AaP-2, AaP-3, and AaP-4, respectively. N-terminal sequences of AaPs 1-4 up to residue 20 showed a high percentage of identity with the peroxidase from Glycine max. In addition, AaP-1, AaP-2, AaP-3, and AaP-4 were found to be glycoproteins, containing 21.75, 22.27, 25.62, and 18.31 % of carbohydrates, respectively. Peptide mapping and MALDI-TOF MS analysis of AaPs 1-4 showed that the structural differences between AaP-1 and AaP-2 and AaP-3 and AaPs-4 were mainly due to their glycan content. We also demonstrate that AaPs were able to remove phenolic compounds from olive oil mill wastewaters with a higher catalytic efficiency with respect to horseradish peroxidase, thus representing candidate enzymes for potential biotechnological applications in the environmental field.