Harmful H2O2 Research Articles

A neoteric antibacterial ceria-silver nanozyme for abiotic surfaces

Community-associated and hospital-acquired infections caused by bacteria continue to yield major global challenges to human health. Bacterial contamination on abiotic surfaces is largely spread via high-touch surfaces and contemporary standard disinfection practices show limited efficacy, resulting in unsatisfactory therapeutic outcomes. New strategies that offer non-specific and broad protection are urgently needed. Herein, we report our novel ceria-silver nanozyme engineered at a molar ratio of 5:1 and with a higher trivalent (Ce3+) surface fraction. Our results reveal potent levels of surface catalytic activity on both wet and dry surfaces, with rapid, and complete eradication of Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin resistant S. aureus, in both planktonic and biofilm form. Preferential electrostatic adherence of anionic bacteria to the cationic nanozyme surface leads to a catastrophic loss in both aerobic and anaerobic respiration, DNA damage, osmodysregulation, and finally, programmed bacterial lysis. Our data reveal several unique mechanistic avenues of synergistic ceria-Ag efficacy. Ag potentially increases the presence of Ce3+ sites at the ceria-Ag interface, thereby facilitating the formation of harmful H2O2, followed by likely permeation across the cell wall. Further, a weakened Ag-induced Ce–O bond may drive electron transfer from the Ec band to O2, thereby further facilitating the selective reduction of O2 toward H2O2 formation. Ag destabilizes the surface adsorption of molecular H2O2, potentially leading to higher concentrations of free H2O2 adjacent to bacteria. To this end, our results show that H2O2 and/or NO/NO2−/NO3− are the key liberators of antibacterial activity, with a limited immediate role being offered by nanozyme-induced ROS including O2•- and OH•, and likely other light-activated radicals. A mini-pilot proof-of-concept study performed in a pediatric dental clinic setting confirms residual, and continual nanozyme antibacterial efficacy over a 28-day period. These findings open a new approach to alleviate infections caused by bacteria for use on high-touch hard surfaces.

Functionalization of Glucose Oxidase in Organic Solvent: Towards Direct Electrical Communication across Enzyme-Electrode Interface.

Enzymatic biosensors based on glucose oxidase has been proven to be one of the effective strategies for the detection of glucose and contributed to health improvements. Therefore, research and debates to date are ongoing in an attempt to find the most effective way to detect this analyte using this enzyme as the recognition center. The 3rd generation biosensors using direct electron transfer (DET) type enzymes are a great way towards practical devices. In this work, we developed a simple method for the functionalization of glucose oxidase with redoxable ferrocene groups in chloroform. The enzyme retained its activity after storage in this organic solvent and after the functionalization procedures. This enzyme functionalization strategy was employed to develop the biosensing monolayer-based platforms for the detection of glucose utilizing the quasi-DET mechanism. As a result of an electrochemical regeneration of the catalytic center, the formation of harmful H2O2 is minimized during enzymatic electrocatalysis.

On the Mediated Electron Transfer of Immobilized Galactose Oxidase for Biotechnological Applications.

The use of enzymes as catalysts in chemical synthesis offers advantages in terms of clean and highly selective transformations. Galactose oxidase (GalOx) is a remarkable enzyme with several applications in industrial conversions as it catalyzes the oxidation of primary alcohols. We have investigated the wiring of GalOx with a redox polymer; this enables mediated electron transfer with the electrode surface for its potential application in biotechnological conversions. As a result of electrochemical regeneration of the catalytic center, the formation of harmful H2O2 is minimized during enzymatic catalysis. The introduced bioelectrode was applied to the conversion of bio‐renewable platform materials, with glycerol as model substrate. The biocatalytic transformations of glycerol and 5‐hydroxymethylfurfural (HMF) were investigated in a circular flow‐through setup to assess the possibility of substrate over‐oxidation, which is observed for glycerol oxidation but not during HMF conversion.

Improving bioconversion of eugenol to coniferyl alcohol by constitutive promoters in Escherichia coli

Coniferyl alcohol is a common monolignol, which is a precursor of lignin. Coniferyl alcohol is rarely found as free molecules in plants based on its conversion to lignins by peroxidases or laccases. It can be used for biosynthesis of silybin and a lot of pinoresinol derivatives. Although increased output of coniferyl alcohol has been attempted, few methods are effective. In this study, we evaluated 20 promoters for their ability to express genes in Penicillium simplicissimum encoding vanillyl alcohol oxidase (PsVAO) and catalase (ScCTA1) for producing coniferyl alcohol, while eliminating harmful H2O2. We constructed 100 combinations of constitutive promoters in E. coli to enhance gene expression. Then orthogonal experiments, fed-batch kinetic model and CRISPR/Cas9 system were followed to improve the yield of coniferyl alcohol, while eliminate the pinoresinol. Finally, the coniferyl alcohol titers, bioconversion rates, and productivity levels reaching 53.90 g/L, 86.72 %, and 2.45 g/(L × h), respectively.

In silico methods predict new blood-brain barrier permeable structure for the inhibition of kynurenine 3-monooxygenase

Kynurenine 3-monooxygenase (KMO) regulates the levels of bioactive substances in the kynurenine pathway of tryptophan catabolism and its activity is tied to so many diseases that finding an appropriate inhibitor for KMO has become an urgent task. This especially proved to be difficult for the central nervous system related diseases due to the requirement that the supposed inhibitor should be both blood brain barrier permeable and should not cause hydrogen peroxide as a harmful side product. In this in silico study, we present our step-wise approach, whose starting point is based on the important experimental observations. To tackle the problem, a library of 7561938 structures was obtained from Zinc15 database utilizing the tranche browser. From this library, a subset of 501777 structures was determined with the considerations of their functional groups that constrain their applicability. Then, the binding affinity ranking of this set of structures was determined via virtual screening. Starting from the structures whose affinities are the highest among this subset, the ADMET properties were checked through in silico methods and the binding properties of the selected inhibitor candidates were further investigated via molecular dynamics simulations and MM/GBSA calculations. According to the computational results of this study, ZINC_71915355 has passed all the evaluations and is a potentially BBB permeable structure that can inhibit KMO. Additionally, ZINC_19827377 was identified as a new potential KMO inhibitor which may be more suitable for peripheral administration. From the in silico study presented herein, ZINC_71915355 and ZINC_19827377 structures, which showed high binding affinity without harmful H2O2 production, along with the tailored properties can now serve as powerful candidates for KMO inhibition and these hits are worth of further experimental validation.

Ionic silicon improves endothelial cells' survival under toxic oxidative stress by overexpressing angiogenic markers and antioxidant enzymes.

Oxidative stress, induced by harmful levels of reactive oxygen species, is a common occurrence that impairs proper bone defect vascular healing through the impairment of endothelial cell function. Ionic silicon released from silica-based biomaterials, can upregulate hypoxia-inducible factor-1α (HIF-1α). Yet it is unclear whether ionic Si can restore endothelial cell function under oxidative stress conditions. Therefore, we hypothesized that ionic silicon can help improve human umbilical vein endothelial cells' (HUVECs') survival under toxic oxidative stress. In this study, we evaluated the ionic jsilicon effect on HUVECs viability, proliferation, migration, gene expression, and capillary tube formation under normal conditions and under harmful hydrogen peroxide levels. We demonstrated that 0.5-mM Si4+ significantly enhanced angiogenesis in HUVECs under normal condition (p<0.05). HUVECs exposed to 0.5-mM Si4+ presented a morphological change, even without the bed of Matrigel, and formed significantly more tube-like structures than the control (p<0.001). In addition, 0.5-mM Si4+ enhanced cell viability in HUVECs under harmful H2 O2 levels. HIF-1α, vascular endothelial growth factor-A, and vascular endothelial growth factor receptor-2 were overexpressed more than twofold in silicon-treated HUVECs, under normal and toxic H2 O2 conditions. Moreover, the HUVECs were treated with 0.5-mM Si4+ overexpressed superoxide dismutase-1 (SOD-1), catalase-1 (Cat-1), and nitric oxide synthase-3 (NOS3) under normal and oxidative stress environment (p<0.01). A computational model was used for explaining the antioxidant effect of Si4+ in endothelial cells and human periosteum cells by SOD-1 enhancement. In conclusion, we demonstrated that 0.5-mM Si4+ can recover the HUVECs' viability under oxidative stress conditions by reducing cell death and upregulating expression of angiogenic and antioxidant factors.

Improving bioconversion of eugenol to coniferyl alcohol by in situ eliminating harmful H2O2

Coniferyl alcohol is a valuable chemical. However, the current approaches to obtain coniferyl alcohol are either unefficient or expensive. Penicillium simplicissimum vanillyl alcohol oxidase (PsVAO) can be used to produce coniferyl alcohol. However, PsVAO intrinsically produces harmful byproduct H2O2. Utilizing catalase to decompose H2O2 is a potential straightforward approach; however, catalase can also exhibit peroxidase activity to facilitate coniferyl alcohol over-oxidation. In this study, catalases exhibiting both high catalase activity and low peroxidase activity were found out, and introduced into the bioconversion systems. Our results showed that eliminating H2O2in situ released H2O2 inhibition of PsVAO, improved coniferyl alcohol production and eliminated coniferyl alcohol over-oxidation. Finally, coniferyl alcohol titer, molar yield, and productivity reached 22.9 g/L, 78.7%, and 0.5 g/(L × h) respectively. An efficient coniferyl alcohol production method was developed by overcoming the intrinsic disadvantages of PsVAO.

Sparks fly between ascorbic acid and iron-based nanozymes: A study on Prussian blue nanoparticles.

Herein we reported Prussian blue nanoparticles (PBNPs) possess ascorbic acid oxidase (AAO)- and ascorbic acid peroxidase (APOD)-like activities, which suppressed the formation of harmful H2O2 and finally inhibited the anti-cancer efficiency of ascorbic acid (AA). This newly revealed correlation between iron and AA could provide new insight for the studies of nanozymes and free radical biology.

Co-immobilization of glucose oxidase and catalase for enhancing the performance of a membraneless glucose biofuel cell operated under physiological conditions.

Glucose oxidase (GOx)-catalase co-immobilized catalyst (CNT/PEI/(GOx-Cat)) was synthesized, and its catalytic activity and electrical performance were investigated and compared, whereas the amount of immobilized catalase was optochemically inspected by chemiluminescence (CL) assay. With the characterizations, it was confirmed that the catalase was well immobilized on the CNT/PEI surface, whereas both the GOx and catalase play their roles well in the catalyst. According to the measurements of the current density peak of the flavin adenine dinucleotide (FAD) redox reaction, electron transfer rate, Michaelis-Menten constants and sensitivity, CNT/PEI/(GOx-Cat) shows the best values, and this is attributed to the excellent catalytic activity of GOx and the H2O2 decomposition capability of the catalase. To evaluate the electrical performance, a membraneless glucose biofuel cell (GBFC) adopting the catalyst was operated under physiological conditions and produced a maximum power density (MPD) of 180.8 ± 22.3 μW cm-2, which is the highest value compared to MPDs obtained by adoption of other catalysts. With such results, it was clarified that the CNT/PEI/(GOx-Cat) manufactured by co-immobilization of GOx and catalase leads to enhancements in the catalytic activity and GBFC performance due to the synergetic effects of (i) effective removal of harmful H2O2 moiety by catalase and (ii) superior activation of desirable reactions by GOx.

Glutathione Peroxidase 7 Utilizes Hydrogen Peroxide Generated by Ero1α to Promote Oxidative Protein Folding

Ero1 flavoproteins catalyze oxidative folding in the endoplasmic reticulum (ER), consuming oxygen and generating hydrogen peroxide (H2O2). The ER-localized glutathione peroxidase 7 (GPx7) shows protein disulfide isomerase (PDI)-dependent peroxidase activity in vitro. Our work aims at identifying the physiological role of GPx7 in the Ero1α/PDI oxidative folding pathway and at dissecting the reaction mechanisms of GPx7. Our data show that GPx7 can utilize Ero1α-produced H2O2 to accelerate oxidative folding of substrates both in vitro and in vivo. H2O2 oxidizes Cys57 of GPx7 to sulfenic acid, which can be resolved by Cys86 to form an intramolecular disulfide bond. Both the disulfide form and sulfenic acid form of GPx7 can oxidize PDI for catalyzing oxidative folding. GPx7 prefers to interact with the a domain of PDI, and intramolecular cooperation between the two redox-active sites of PDI increases the activity of the Ero1α/GPx7/PDI triad. Our in vitro and in vivo evidence provides mechanistic insights into how cells consume potentially harmful H2O2 while optimizing oxidative protein folding via the Ero1α/GPx7/PDI triad. Cys57 can promote PDI oxidation in two ways, and Cys86 emerges as a novel noncanonical resolving cysteine. GPx7 promotes oxidative protein folding, directly utilizing Ero1α-generated H2O2 in the early secretory compartment. Thus, the Ero1α/GPx7/PDI triad generates two disulfide bonds and two H2O molecules at the expense of a single O2 molecule.

Actin Stimulates Reduction of the MICAL-2 Monooxygenase Domain

MICALs are large, multidomain flavin-dependent monooxygenases that use redox chemistry to cause actin to depolymerize. Little enzymology has been reported for MICALs, and none has been reported for MICAL-2, an enzyme vital for the proliferation of prostate cancer. The monooxygenase domains of MICALs resemble aromatic hydroxylases, but their substrate is the sulfur of a methionine of actin. In order to determine how closely MICAL-2 conforms to the aromatic hydroxylase paradigm, we studied its reaction with NAD(P)H. The enzyme has a strong preference for NADPH over NADH caused by a large difference in binding NADPH. A comparison of the reduction kinetics using protio-NADPH and [4R-(2)H]-NADPH showed that MICAL-2 is specific for the proR hydride of NADPH, as evidenced by a 4.8-fold kinetic isotope effect. The reductive half-reaction of the MICAL-2 hydroxylase domain is stimulated by f-actin. In the absence of actin, NADPH reduces the flavin relatively slowly; actin speeds that reaction significantly. The separate monooxygenase domain of MICAL-2 has the classic regulatory behavior of flavin-dependent aromatic hydroxylases (Class A monooxygenases): slow reduction of the flavin when the substrate to be oxygenated is absent. This prevents the wasteful consumption of reduced pyridine nucleotide and the production of harmful H2O2. Our results show that this strategy is used by MICAL-2. Thus, our data suggest that MICAL-2 could regulate catalysis through the monooxygenase domain alone; control by interactions with other domains of MICAL in the full-length enzyme may not be needed.

Dynamics of nonphotochemical superoxide production in the Great Barrier Reef lagoon

Superoxide (O2–) and hydrogen peroxide (H2O2) concentrations ranging from 87 to 1120 pmol L−1 and 5 to 107 nmol L−1, respectively, were measured in samples of surface water from the Great Barrier Reef (GBR) lagoon in the absence of photochemistry. Nonphotochemical, particle‐associated net production rates of O2‐ ranging from 1 to 16 pmol L‐1 s‐1 were also determined and calculated to be similar in magnitude to the likely abiotic photochemical O2‐ production rates in GBR surface waters. Manipulative experiments using 0.22‐µm filtration and addition of biological inhibitors demonstrated that the majority of this particle‐associated production was biological and likely driven by photosynthetic organisms. Pseudo‐first‐order O2‐ decay rate constants were very low at O2‐ concentrations &lt; 1 nmol L‐1 (values in filtered samples ranged from 0.7 to 4.3 x 10−2 s−1) but increased with increasing O2‐ concentration toward a value of ~ 0.2 s−1 at O2‐ concentrations &gt; 10 nmol L‐1. This was thought to occur because reduced forms of metals such as iron and copper, or redox‐active organic moieties, preferentially react with O2 rather than O2‐ at low O2‐ concentrations, thereby inhibiting catalyzed O2‐ disproportionation. This notion was supported by the observation that addition of superoxide dismutase dramatically increased rates of H2O2 production in samples. We suggest that, under these conditions, O2‐ can maintain a biologically useful reducing microenvironment around cells without resulting in significant accumulation of potentially harmful H2O2.

Horseradish Peroxidase Combined With Oxidase Enzymes a Valuable Bioanalytical Tool: Lactate Oxidase – A Case Study

AbstractEnzymatic biosensors have been extensively investigated for real‐time bioprocess monitoring and other online analysis. However, implementation of biosensors has been strongly hindered by their limited stability. This work reports a significant improvement of the stability of the immobilized oxidases by in situ reduction of the harmful H2O2. Thus, stabilized oxidases can serve as the basis for ethanol, glucose, and lactate sensors, with the ability to operate for long periods of time with virtually no change in activity. As an example, a lactate sensor, containing lactate oxidase aimed for bioprocess monitoring, has been described and characterized. Operational stabilities that allow up to 8 h continuous lactate conversion with virtually no activity loss have been achieved. The described system based on the in situ stabilization strategy is a promising new tool for the development of online analyses.

Mineralization in Ferritin: An Efficient Means of Iron Storage

Ferritins are a class of iron storage and mineralization proteins found throughout the animal, plant, and microbial kingdoms. Iron is stored within the protein shell of ferritin as a hydrous ferric oxide nanoparticle with a structure similar to that of the mineral “ferrihydrite.” The eight hydrophilic channels that traverse the protein shell are thought to be the primary avenues by which iron gains entry to the interior of eukaryotic ferritins. Twenty-four subunits constitute the protein shell and, in mammalian ferritins, are of two types, H and L, which have complementary functions in iron uptake. The H chain contains a dinuclear ferroxidase site that is located within the four-helix bundle of the subunit; it catalyzes the oxidation of ferrous iron by O2, producing H2O2. The L subunit lacks this site but contains additional glutamate residues on the interior surface of the protein shell which produce a microenvironment that facilitates mineralization and the turnover of iron(III) at the H subunit ferroxidase site. Recent spectroscopic studies have shown that a di-Fe(III) peroxo intermediate is produced at the ferroxidase site followed by formation of a μ-oxobridged dimer, which then fragments and migrates to the nucleation sites to form incipient mineral core species. Once sufficient core has developed, iron oxidation and mineralization occur primarily on the surface of the growing crystallite, thus minimizing the production of potentially harmful H2O2.

Heterologous Expression and Characterization of Soybean Cytosolic Ascorbate Peroxidase

Ascorbate peroxidase is a widespread plant enzyme that catalyzes the removal of potentially harmful H2O2. This enzyme is particularly important in legume root nodules due to their high potential for generating activated forms of oxygen. A cDNA clone of soybean nodule ascorbate peroxidase was used to construct an expression system inEscherichia coli.The recombinant protein had an N-terminal tag of six consecutive histidine residues to allow for purification by Ni2+-agarose affinity chromatography. Large amounts of recombinant peroxidase (about 27% of total soluble protein) were produced but most of the peroxidase was present in the apo-form (without heme). Addition of δ-aminolevulinic acid to the growth media resulted in an increase in production of holoprotein. Apoprotein was easily converted to the holo-form byin vitroreconstitution with hemin. The reconstituted protein was catalytically, spectrally, and immunologically indistinguishable from native ascorbate peroxidase.

Mitochondrial short-chain acyl-CoA dehydrogenase of human liver and kidney can function as an oxidase.

During an attempt to purify the peroxisomal acyl-CoA oxidases from human liver and kidney, we discovered a novel short-chain acyl-CoA oxidase, which was well separated from the known peroxisomal oxidases on various chromatographic columns. However, further experiments demonstrated that the novel oxidase is identical with the mitochondrial short-chain acyl-CoA dehydrogenase. (1) Subcellular fractionation revealed that the short-chain acyl-CoA oxidase is present in mitochondria and absent from peroxisomes. (2) The molecular mass (43 kDa) of the subunit of the purified oxidase was similar to that reported for the dehydrogenase. (3) The substrate spectrum of the oxidase was comparable with that described for the dehydrogenase. (4) On column chromatography, the oxidase and dehydrogenase activities co-eluted. Our results indicate that, in the absence of suitable electron acceptors, the short-chain acyl-CoA dehydrogenase is capable of transferring electrons directly to molecular oxygen, yielding potentially harmful H2O2. This raises the question as to whether the dehydrogenase might function as an oxidase in conditions in which the activity of the electron-transport chain is decreased, such as reperfusion after ischaemia.

Correlations between the ascorbate‐glutathione pathway and effectiveness in legume root nodules

Legume root nodules use the ascorbate‐glutathione pathway to remove harmful H2O2. In the present study. effective and ineffective nodules from soybean and alfalfa were compared with regard to this pathway. Effective nodules had higher activity of all 4 enzymes (ascorbate peroxidase, EC 1. 11. 1. 11: monodehydroascorbate reductase, EC 1. 6. 5. 4: dehydroascorbate reductase, EC 1. 8. 5. 1: and glutathione reductase, EC 1. 6. 4. 2). The concentration of thiol tripeptides (primarily homoglutathione) was about 1 mM in effective nodules – a level 3–4‐fold higher than in ineffective nodules. Effective nodules contained higher levels of NAD+. NADP+ and NADPH. but not of NADH or ascorbate. The increased capacity for peroxide scavenging in effective nodules as compared to ineffective nodules emphasizes the important protective role that this pathway may play in processes related to nitrogen fixation.