NADH Oxidase Research Articles

Photothermally Reinforced Nanozyme Remodeling Tumor Microenvironment of Redox and Metabolic Homeostasis to Enhance Ferroptosis in Tumor Therapy.

The acidity and high GSH level in the tumor microenvironment (TME) greatly limit the antitumor activity of nanozymes. Thus, enhancing nanozymes' activity is fundamentally challenging in tumor therapy. Although the combination of photothermal therapy (PTT) and nanozymes can enhance the catalytic activity, cancer cells will overexpress heat shock proteins (HSPs) at high temperature, aggravating the heat resistance of tumor cells, which in turn compromises the outcome of chemodynamic therapy. Herein, we propose an iron-doped metal-organic framework nanozyme (IB@Fe-ZIF8@PDFA) that can be activated under the weak acidity and high level of GSH, demonstrating the activities of GSH oxidation (GSH-OXD), peroxidase (POD), and NADH oxidase (NADH-OXD). Under laser irradiation, it displays photothermal-enhanced multienzyme activities to simultaneously eliminate tumors and inhibit tumor metastasis. While consuming endogenous GSH, IB@Fe-ZIF8@PDFA promotes the decomposition of H2O2 into ·OH, enhancing ferroptosis in tumor cells. Surprisingly, IB@Fe-ZIF8@PDFA nanozyme can oxide NADH and subsequently limit the ATP supply, reducing the expression of HSPs and significantly weakening the heat resistance of tumor cells during PTT. Meanwhile, H2O2 is generated during this procedure, which can endogenously replenish the consumed H2O2. Thus, this IB@Fe-ZIF8@PDFA nanozyme constitutes a self-cascading platform to consume GSH and NADH, endogenously replenish the H2O2 and continuously generate ·OH to facilitate ferroptosis by disrupting the redox and metabolic homeostasis in tumor cells, achieving tumor elimination and tumor metastasis inhibition.

Advancements in NADH Oxidase Nanozymes: Bridging Nanotechnology and Biomedical Applications.

Nicotinamide adenine dinucleotide (NADH) oxidase (NOX) is key in converting NADH to NAD+, crucial for various biochemical pathways. However, natural NOXs are costly and unstable. NOX nanozymes offer a promising alternative with potential applications in bio-sensing, antibacterial treatments, anti-aging, and anticancer therapies. This review provides a comprehensive overview of the types, functional mechanisms, biomedical applications, and future research perspectives of NOX nanozymes. It also addresses the primary challenges and future directions in the research and development of NOX nanozymes, underscoring the critical need for continued investigation in this promising area. These challenges include optimizing the catalytic efficiency, ensuring biocompatibility, and achieving targeted delivery and controlled activity within biological systems. Additionally, the exploration of novel materials and hybrid structures holds great potential for enhancing the functional capabilities of NOX nanozymes. Future research directions can involve integrating advanced computational modeling with experimental techniques to better understand the underlying mechanisms and to design more effective nanozyme candidates. Collaborative efforts across disciplines such as nanotechnology, biochemistry, and medicine will be essential to unlock the full potential of NOX nanozymes in future biomedical applications.

The Role of Propionate-Induced Rearrangement of Membrane Proteins in the Formation of the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Adhesive-invasive E. coli has been suggested to be associated with the development of Crohn's disease (CD). It is assumed that they can provoke the onset of the inflammatory process as a result of the invasion of intestinal epithelial cells and then, due to survival inside macrophages and dendritic cells, stimulate chronic inflammation. In previous reports, we have shown that passage of the CD isolate ZvL2 on minimal medium M9 supplemented with sodium propionate (PA) as a carbon source stimulates and inhibits the adherent-invasive properties and the ability to survive in macrophages. This effect was reversible and not observed for the laboratory strain K12 MG1655. We were able to compare the isogenic strain AIEC in two phenotypes-virulent (ZvL2-PA) and non-virulent (ZvL2-GLU). Unlike ZvL2-GLU, ZvL2-PA activates the production of ROS and cytokines when interacting with neutrophils. The laboratory strain does not cause a similar effect. To activate neutrophils, bacterial opsonization is necessary. Differences in neutrophil NADH oxidase activation and ζ-potential for ZvL2-GLU and ZvL2-PA are associated with changes in membrane protein abundance, as demonstrated by differential 2D electrophoresis and LC-MS. The increase in ROS and cytokine production during the interaction of ZvL2-PA with neutrophils is associated with a rearrangement of the abundance of membrane proteins, which leads to the activation of Rcs and PhoP/Q signaling pathways and changes in the composition and/or modification of LPS. Certain isoforms of OmpA may play a role in the formation of the virulent phenotype of ZvL2-PA and participate in the activation of NADPH oxidase in neutrophils.

Catalytic Potential-Guided Design of Multi-Enzymatic System for DHA Production from Glycerol

The growing demand for sustainable chemical production has spurred significant interest in biocatalysis. This study is framed within the biocatalytic production of 1,3-dihydroxyacetone (DHA) from glycerol, a byproduct of biodiesel manufacturing. The main goal of this study is to address the challenge of identifying the optimal operating conditions. To achieve this, catalytic potential, a lumped parameter that considers both the activity and stability of immobilized biocatalysts, was used to guide the design of a multi-enzymatic system. The multi-enzymatic system comprises glycerol dehydrogenase (GlyDH) and NADH oxidase (NOX). The enzymatic oxidation of glycerol to DHA catalyzed by GlyDH requires the cofactor NAD+. The integration of NOX into a one-pot reactor allows for the in situ regeneration of NAD+, enhancing the overall efficiency of the process. Furthermore, immobilization on Ni+2 agarose chelated supports, combined with post-immobilization modifications (glutaraldehyde crosslinking for GlyDH), significantly improved the stability and activity of both enzymes. The catalytic potential enabled the identification of the optimal operating conditions, which were 30 °C and pH 7.5, favoring NOX stability. This work establishes a framework for the rational design and optimization of multi-enzymatic systems. It highlights the crucial interplay between individual enzyme properties and process conditions to achieve efficient and sustainable biocatalytic transformations.

The compartment-specific manipulation of the NAD+/NADH ratio affects the metabolome and the function of glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive type of cancer in the brain and has an inferior prognosis because of the lack of suitable medicine, largely due to its tremendous invasion. GBM has selfish metabolic pathways to promote migration, invasion, and proliferation compared to normal cells. Among various metabolic pathways, NAD (nicotinamide adenine dinucleotide) is essential in generating ATP and is used as a resource for cancer cells. LbNOX (Lactobacillus brevis NADH oxidase) is an enzyme that can directly manipulate the NAD+/NADH ratio. In this study, we found that an increased NAD+/NADH ratio by LbNOX or mitoLbNOX reduced intracellular glutamate and calcium responses and reduced invasion capacity in GBM. However, the invasion was not affected in GBM by rotenone, an ETC (Electron Transport Chain) complex I inhibitor, or nicotinamide riboside, a NAD+ precursor, suggesting that the crucial factor is the NAD+/NADH ratio rather than the absolute quantity of ATP or NAD+ for the invasion of GBM. To develop a more accurate and effective GBM treatment, our findings highlight the importance of developing a new medicine that targets the regulation of the NAD+/NADH ratio, given the current lack of effective treatment options for this brain cancer.

Engineering Oxygen-Independent NADH Oxidase Integrated with Electrocatalytic FAD Cofactor Regeneration.

An electrochemically mediated enzyme process for nicotinamide adenine dinucleotide (NADH) oxidation and biosensing has been developed in which the oxygen-dependent activities of wild-type NADH oxidase are replaced by electrochemical regeneration of the flavin adenine dinucleotide (FAD) cofactor in the active site. Consequently, the present bioelectrocatalysis does not rely on a continuous oxygen supply through bubbling air or pure oxygen in biosynthetic applications, which reduces enzyme stability. The coupled electrochemical and enzymatic catalysis is achieved through a combination of enzyme immobilization on the electrode and electrochemical oxidation of FADH2 in the active site mediated by the electron transfer mediator ferrocene carboxylic acid (FcCA). Furthermore, to minimize the effect of dissolved oxygen when the electrocatalytic process is exposed to air, we successfully designed mutations at the Leu40 and Cys42 sites of Leuconostoc mesenteroides (LmNOx) to block the oxygen passage into the active site and to eliminate the native FAD cofactor regeneration half-reaction. The engineered enzymes, whose activities are significantly reduced or inactive in solution, are electrocatalytically active toward conversion of NADH to NAD+, demonstrating successful FAD cofactor regeneration in the active site via electrochemistry. Finally, we developed two highly responsive electrochemical biosensors for NADH detection which has a superior substrate specific to standard detectors using metal electrodes, and comparable detection range and detection limit (1-3 μM).

Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem.

2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.

Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of porcine epidemic diarrhea virus, Brachyspira hyodysenteriae, and Lawsonia intracellularis.

PEDV, Brachyspira hyodysenteriae, and Lawsonia intracellularis, are highly contagious diarrheal pathogens that have caused significant harm to the global swine industry. Co-infections with multiple pathogens are common, making it challenging to identify the actual causative agents depending only on clinical information. It is crucial to develop a reliable method to simultaneously detect and differentiate these pathogens. Based on the conserved regions of the M gene of PEDV, NADH oxidase gene of B. hyodysenteriae, and the 16S rDNA gene of L. intracellularis, specific probes and primers for the multiplex real-time PCR assay were designed. The concentrations of primers and probes were optimized using a matrix method. The approach demonstrated high specificity and no cross-reactivity with major pathogens related to diarrheal diseases. It showed high sensitivity with a detection limit of 10 copies/μL for B. hyodysenteriae and L. intracellularis, and 100 copies/μL for PEDV, respectively. It also demonstrated high reproducibility and stability with low coefficients of variation. Results from the multiplex real-time PCR method were in complete agreement with the commercial singleplex real-time PCR kit for detecting PEDV, B. hyodysenteriae and L. intracellularis. Clinical data revealed single infection rates of 31.46% for PEDV, 58.43% for B. hyodysenteriae, and 98.6% for L. intracellularis. The co-infection rates were 16.85% for PEDV + B. hyodysenteriae, 31.46% for PEDV + L. intracellularis, 57.86% for B. hyodysenteriae + L. intracellularis, and 16.85% for PEDV + B. hyodysenteriae + L. intracellularis, respectively. The new multiplex real-time PCR method can simultaneously differentiate PEDV, B. hyodysenteriae and L. intracellularis, making it a valuable diagnostic tool for preventing and controlling infectious diseases, as well as aiding in epidemiological investigations.

Hypoxia decreases mitochondrial ROS production in cells

We re-examined the reported increase in mitochondrial ROS production during acute hypoxia in cells. Using the Amplex Ultrared/horseradish peroxidase assay we found a decrease, not increase, in hydrogen peroxide release from HEK293 cells under acute hypoxia, at times ranging from 1 min to 3 h. The rates of superoxide/hydrogen peroxide production from each of the three major sites (site IQ in complex I and site IIIQo in complex III in mitochondria, and NADH oxidases (NOX) in the cytosol) were decreased to the same extent by acute hypoxia, with no change in the cells’ ability to degrade added hydrogen peroxide. A similar decrease in ROS production under acute hypoxia was found using the diacetyldichlorofluorescein assay. Using a HIF1α reporter cell line we confirmed earlier observations that suppression of superoxide production by site IIIQo decreases HIF1α expression, and found similar effects of suppressing site IQ or NOX. We conclude that increased mitochondrial ROS do not drive the response of HIF1α to acute hypoxia, but suggest that cytosolic H2O2 derived from site IQ, site IIIQo and NOX in cells is necessary to permit HIF1α stabilization by other signals.

Innovative Colorimetric NQO1 Detection Strategy via Substrate Competitive and Biomimetic Cascade Reactions with a Highly Active NADH Oxidase Mimic.

NAD(P)H: quinone oxidoreductase-1 (NQO1) plays critical roles in antioxidation and abnormally overexpresses in tumors. Developing a fast and sensitive method of monitoring NQO1 will greatly promote cancer diagnosis in clinical practice. This study introduces a transformative colorimetric detection strategy for NQO1, harnessing an innovative competitive substrate mechanism between NQO1 and a new NADH oxidase (NOX) mimic, cobalt-nitrogen-doped carbon nanozyme (CoNC). This method ingeniously exploits the differential consumption of NADH in the presence of NQO1 to modulate the generation of H2O2 from CoNC catalysis, which is then quantified through a secondary, peroxidase-mimetic cascade reaction involving Prussian blue (PB) nanoparticles. This dual-stage reaction framework not only enhances the sensitivity of NQO1 detection, achieving a limit of detection as low as 0.67 μg mL-1, but also enables the differentiation between cancerous and noncancerous cells by their enzymatic activity profiles. Moreover, CoNC exhibits exceptional catalytic efficiency, with a specific activity reaching 5.2 U mg-1, significantly outperforming existing NOX mimics. Beyond mere detection, CoNC serves a dual role, acting as both a robust mimic of cytochrome c reductase (Cyt c) and a cornerstone for enzymatic regeneration, thereby broadening the scope of its biological applications. This study not only marks a significant step forward in the bioanalytical application of nanozymes but also sets the stage for their expanded use in clinical diagnostics and therapeutic monitoring.

Novel cofactor regeneration-based magnetic metal–organic framework for cascade enzymatic conversion of biomass-derived bioethanol to acetoin

Upgrading biomass-derived bioethanol to higher-order alcohols using conventional biotechnological approaches is challenging. Herein, a novel, magnetic metal–organic-framework-based cofactor regeneration system was developed using ethanol dehydrogenase (EtDH:D46G), NADH oxidase (NOX), formolase (FLS:L482S), and nicotinamide adenine dinucleotide (NAD+) for converting rice straw-derived bioethanol to acetoin. A magnetic zeolitic imidazolate framework-8@Fe3O4/NAD+ (ZIF-8@Fe3O4/NAD+) regeneration system for cell-free cascade reactions was introduced and used to encapsulate EtDH:D46G, NOX, and FLS:L482S (ENF). ZIF-8@Fe3O4/NAD+ENF created an efficient microenvironment for three-step enzyme cascades. Under the optimized conditions, the yield of acetoin from 100 mM bioethanol using ZIF-8@Fe3O4/NAD+ENF was 90.4 %. The regeneration system showed 97.1 % thermostability at 50 °C. The free enzymes retained only 16.3 % residual conversion, compared with 91.2 % for ZIF-8@Fe3O4/NAD+ENF after ten cycles. The magnetic metal–organic-framework-based cofactor regeneration system is suitable for enzymatic cascade biotransformations and can be extended to other cascade systems for potential biotechnological applications.

Water-accommodated fractions of crude oil and its mixture with chemical dispersant impairs oxidase stress and energy metabolism disorders in Oryzias melastigma embryos

In this study, marine medaka (Oryzias melastigma) embryos were exposed to different concentrations of water-accommodated fractions (WAFs) and chemically enhanced water-accommodated fractions (CEWAFs) of Oman crude oil for 14 d by semi-static exposure methods. The effects on growth and development and energy metabolism process were evaluated. Results showed that embryo survival and hatchability were decreased in a dose-dependent manner with an increase in the concentration of petroleum hydrocarbon compounds, whereas the malformation exhibited a dose-dependent increase. Compared to the control, the adenosine triphosphate (ATP) content and Na+-K+–ATPase (NKA) activities of embryos exposed to both WAFs and CEWAFs were reduced, while intracellular reactive oxygen species (ROS) levels and NADH oxidase (NOX) activities were increased. Our study demonstrated that exposure to crude oil dispersed by chemical dispersant affected the growth and development of marine medaka embryos, caused oxidative stress while produced a series of malformations in the body and dysregulation in energy metabolism. In comparison, the toxic effects of chemically dispersed crude oil might be more severe than the oil itself in the equivalent diluted concentration treatment solution. These would provide more valuable and reliable reference data for the use of chemical dispersants in oil spills.

Pt-Cluster-Embedded Metal-Organic Frameworks-Derived Fe@C as Dual-Enzyme Mimics for NADH Detection in Serum.

Dihydro-nicotinamide adenine dinucleotide (NADH) detection is crucial since it is a vital coenzyme in organism metabolism. Compared to the traditional method based on natural NADH oxidase (NOX), nanozymes with multienzyme-like activity can catalyze multistage reactions in a singular setup, simplifying detection processes and enhancing sensitivity. In this study, an innovative NADH detection method was developed using iron-doped carbon (Fe@C) nanozyme synthesized from metal-organic frameworks with in situ reduced Pt clusters. This nanozyme composite (Pt/Fe@C) demonstrated dual NOX and peroxidase-like characteristics, significantly enhancing the catalytic efficiency and enabling NADH conversion to NAD+ and H2O2 with subsequent detection. The collaborative research involving both experimental and theoretical simulations has uncovered the catalytic process and the cooperative effect of Fe and Pt atoms, leading to enhanced oxygen adsorption and activation, as well as a decrease in the energy barrier of the key step in the H2O2 decomposition process. These findings indicate that the catalytic performance of Pt/Fe@C in NOX-like and POD-like reactions can be significantly improved. The colorimetric sensor detects NADH with a limit of detection as low as 0.4 nM, signifying a breakthrough in enzyme-mimicking nanozyme technology for precise NADH measurement.

Regulatory effects of antioxidants on indoxyl sulfate-enhanced intracellular oxidation and impaired phagocytic activity in differentiated U937 human macrophage cells.

Indoxyl sulfate (IS), a uremic toxin, is a physiologically active sulfated metabolite, specifically in kidney failure patients. Our previous studies have shown that IS downregulates phagocytic immune function in a differentiated HL-60 human macrophage cell model. However, it remains unclear whether IS exerts similar effects on macrophage function in other cell types or in lipopolysaccharide (LPS)-sensitive immune cell models. Therefore, this study aimed to investigate the effects of IS on intracellular oxidation levels and phagocytic activity in a differentiated U937 human macrophage cell model, both in the absence and presence of LPS. Our results demonstrated that IS significantly increases intracellular oxidation levels and decreases phagocytic activity, particularly in cells activated by LPS. Furthermore, we found that 2-acetylphenothiazine, an NADH oxidase inhibitor, attenuates the effects of IS in LPS-activated macrophage cells. Representative antioxidants, trolox, α-tocopherol, and ascorbic acid, significantly mitigated the effects of IS on the macrophages responding to LPS.

Catalytic Differences between Flavohemoglobins of Giardia intestinalis and E. coli.

The sole known heme enzyme of the parasitic protist Giardia intestinalis is a flavohemoglobin (gFlHb) that acts as a nitric oxide dioxygenase (NOD) and protects the organism from the free radical nitric oxide. To learn more about the properties of this enzyme, we measured its nitric oxide dioxygenase, NADH oxidase, and cytochrome c reductase activities and compared these to the activities of the E. coli flavohemoglobin (Hmp). The turnover number for the NOD activity of gFlHb (23 s-1) is about two-thirds of that of Hmp (34 s-1) at pH 6.5 and 37 °C. The two enzymes differ in their sensitivity towards molecules that act as heme ligands. For both gFlHb and Hmp, inhibition with miconazole, a large imidazole ligand, is adequately described by simple competitive inhibition, with KI = 10 μM and 0.27 μM for gFlHb and Hmp, respectively. Inhibition plots with the small ligand imidazole were biphasic, which is consistent with previous experiments with carbon monoxide as a probe that show that the active site of flavohemoglobins exists in two conformations. Interestingly, the largest difference is observed with nitrite, which, like imidazole, also shows a biphasic inhibition plot; however, nitrite inhibits gFlHb at sub-millimolar concentrations while Hmp is not significantly affected. NADH oxidase activity measured under aerobic conditions in the absence of nitric oxide for Hmp was more than twice the activity of gFlHb. The addition of 1 mM hydrogen peroxide in these assays stimulated the NADH oxidase activity of gFlHb but not Hmp. Both enzymes had nearly identical cytochrome c reductase activities but the extent of the contribution of indirect reduction by flavohemoglobin-generated superoxide was much lower with gFlHb (4% SOD-inhibited) than with Hmp (17% SOD-inhibited). Although the active sites of the two enzymes share the same highly conserved residues that are important for catalysis, differences in the distal ligand binding site may account for these differences in activity and sensitivity towards NOD inhibitors. The differences observed in the NADH oxidase and cytochrome c reductase assays suggest that gFlHb may have evolved to protect the protist, which lacks both superoxide dismutase and catalase, from the damaging effects of superoxide by minimizing its production and from peroxide by actively reducing it.

Antioxidant, anti-amylase, anti-lipase, and efficiency of Satureja fatty acid on the anti-inflammatory parameters in lipopolysaccharide-stimulated macrophage through Nrf2/NF-kB/NADH oxidase pathway

Satureja is an aromatic plant that is used for flavoring, perfume, and food manufacturing due to its pleasant essential oil. Modern medicine research revealed several biological activities of Satureja essential oil, including antifungal, antibacterial, antiviral, antioxidant, anticancer, and anti-inflammatory. However, the functional properties of Satureja fatty acid have not been explored. This study examined the fatty acid profile, lipid nutritional quality, antioxidant, anti-amylase, and anti-lipase capacities of Satureja. The efficiency of Satureja fatty acid on the anti-oxidative and anti-inflammatory parameters in LPS-induced macrophage through the Nrf2/NF-kB/NADH oxidase pathway was examined. The whole lipid extract was prepared with chloroform/methanol/water solution. Fatty acids methyl ester from whole lipid extract were prepared with methanol/sulfuric acid reagent. The fatty acid profile was analyzed using gas chromatography-mass spectrometry. Total antioxidant was determined by ABTS decolorization. Lipase and amylase activities were determined by monitoring the decomposition of p-nitrophenyl butyrate and starch. The macrophage cell line was grown in DMEM media in the presence of fatty acid. The hydrogen peroxide production in treated cells was monitored using the FOX reagent. NADH oxidase activity was measured by monitoring NADH breakdown. The expression of NOX, NF-kB, and NRF2, were tested in the treated cells by real-time PCR. The main components of the Satureja fatty acid were linolenic acid (24.67–37.32%), palmitic acid (10.65–20.29%), linoleic acid (8.31–13.39%), oleic acid (4.42–14.35%), stearic acid (2.76–8.77%) and palmitoleic acid (1.77–4.95%). Given the nutritional quality, omega-3 PUFA (23.58–37.32%), SFA (21.53–26.70%), omega-6 PUFA (10.86–16.14%), omega-9 MUFA (4.42–14.35%), and omega-7 MUFA (1.77–4.95%) comprise the majority of fatty acids. Satureja fatty acid has a promising unsaturation index (120.77–164.27), PUFA/MUFA (2.07–6.41), hypocholesterolemic index (2.44–3.47), health-promoting index (2.03–2.42), PUFA/SFA (1.37–1.94), nutritive value index (0.53–1.71), MUFA/SFA (0.30–0.80) omega-6/omega-3 (0.34–0.65), atherogenicity index (0.41–0.49), and thrombogenicity index (0.17–0.27). Satureja fatty acid displayed strong antioxidant capacity (with IC50 ranging from 354 to 428 µg/mL), anti-lipase capacity (with IC50 ranging from 354 to 428 µg/mL), and anti-amylase capacity (with IC50 ranging from 370 to 390 µg/mL). LPS induced the expression of NOX, NRF2, and NF-kB and the synthesis of hydrogen peroxide in macrophage cells. In LPS-stimulated macrophages, Satureja fatty acid reduced NOX expression, hydrogen peroxide, and NF-kB expression and increased NRF2 at 0.04 mg/mL. In conclusion, Satureja fatty acids have potent antioxidant, anti-amylase, anti-lipase, and anti-inflammatory activities. The mechanisms in lowering oxidative stress markers depended on down-regulating superoxide-producing enzymes at gene and protein levels. Satureja polyunsaturated omega-3 fatty acids could be recommended for healthy products combined with dietary therapy to treat obesity, diabetes, and oxidative stress.

Antioxidant and immunomodulatory potential of cell wall polysaccharides from Harungana madagascariensis Lam

Numerous plant polysaccharides have demonstrated immunomodulatory and antioxidant potentials, and can be investigated as novel molecules with biological properties that may enhance immune function. The aim of this study was to assess the antioxidant and immunomodulatory properties of cell wall polysaccharides fractions isolated from Harungana madagascariensis (HM). Pectin fraction (PF) and hemicellulose fraction (HCF) were extracted and tested for their antioxidant properties using reducing power, 1,1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2′-azino-bis-3-éthylbenzylthiazoline6-sulphonic acid (ABTS) radical scavenging assays, chelating activity and inhibition of lipid peroxidation. Their immunomodulatory activities were carried out on peritoneal macrophages targeting NADH oxidase and lysosomal enzyme activities as well as the production of nitric oxide (NO) and hydroperoxides (H2O2) during phagocytosis of Saccharomyces cereviceae. The polysaccharides fractions showed significant antioxidant and immunostimulatory activities via stimulation of NADH oxidase and lysosomal enzyme of peritoneal macrophages and production of NO and H2O2. Taken together, the results of this study indicate the potential of polysaccharides from H. madagascariensis as antioxidant and immunomodulator agents.

Engineering Styrene Oxide Isomerase Towards Enhanced Chemoenzymatic Synthesis of (S)‐Flurbiprofen

AbstractThe conventional synthesis of (S)‐flurbiprofen, a valuable non‐steroidal anti‐inflammatory drug (NSAID), involves inefficient chiral resolution with low‐yielding multiple reaction steps, or asymmetric synthesis with low enantioselectivity and/or regioselectivity. Here, we developed high‐yielding, enantioselective, and sustainable synthesis of (S)‐flurbiprofen via two chemoenzymatic approaches from a simple alkene: (A) enzymatic epoxidation‐isomerization‐oxidation cascade and Pd‐catalyzed arylation; and (B) chemical epoxidation, enzymatic isomerization‐oxidation cascade, and Pd‐catalyzed arylation. Styrene oxide isomerase (SOI)‐catalyzed enantio‐retentive Meinwald rearrangement was the key step in the two pathways, but SOI suffers from low activity. SOI was engineered via two rounds of iterative saturation mutagenesis and screening with a three‐enzyme cascade assay, generating a double‐mutant SOIDM (N99A/D95A) with 5.5‐fold improvement in catalytic efficiency (kcat/KM). In Pathway A, cascade biotransformation of 3‐fluoro‐4‐bromo‐α‐methylstyrene with engineered E. coli strain expressing SOIDM, styrene monooxygenase (SMO) and aldehyde dehydrogenase (ALDH), and subsequent Pd‐catalyzed arylation afforded (S)‐flurbiprofen in 98% yield and 91% ee. In Pathway B, chemical epoxidation (Sharpless dihydroxylation and ring‐closing), cascade conversion using an engineered E. coli strain expressing SOIDM, ALDH and NADH oxidase (NOX), and Pd‐catalyzed arylation produced (S)‐flurbiprofen in 83% yield and 98% ee. Compared to conventional synthesis, the developed methods reduced reaction steps, minimized waste generation and improved enantioselectivity or overall yield.

Towards a mechanistic model of oxidase deactivation in a bubble column

Despite the many advantages of biocatalysis, enzyme stability remains an issue. This work investigates the long-term kinetic stability of a water-forming NAD(P)H-oxidase (NOX2), an enzyme of potential industrial interest for NAD(P)H regeneration, in a bubble column sparged with air, which enables reasonable oxygen transfer but without drastic enzyme deactivation. Experiments have been performed with the particular goal of explaining the observed two-stage deactivation trend. We show evidence supporting the hypothesis that the first stage is related to the adsorption of enzymes to the interface, followed by a subsequent deactivation stage at the interface.The characterization of NOX deactivation in the bubble column setup, complemented by additional’quiescent’ experiments, has enabled the development of a first principles model as the first step towards a complete mechanistic model of oxidase deactivation at gas–liquid interfaces.

Whole-cell one-pot biosynthesis of dodecanedioic acid from renewable linoleic acid

BackgroundDodecanedioic acid (DDA), a typical medium-chain dicarboxylic fatty acid with widespread applications, has a great synthetic value and a huge industrial market demand. Currently, a sustainable, eco-friendly and efficient process is desired for dodecanedioic acid production.ResultsHerein, a multi-enzymatic cascade was designed and constructed for the production of DDA from linoleic acid based on the lipoxygenase pathway in plants. The cascade is composed of lipoxygenase, hydroperoxide lyase, aldehyde dehydrogenase, and unidentified double-bond reductase in E. coli for the main cascade reactions, as well as NADH oxidase for cofactor recycling. The four component enzymes involved in the cascade were co-expressed in E. coli, together with the endogenous double-bond reductase of E. coli. After optimizing the reaction conditions of the rate-limiting step, 43.8 g L− 1 d− 1 of DDA was obtained by a whole-cell one-pot process starting from renewable linoleic acid.ConclusionsThrough engineering of the reaction system and co-expressing the component enzymes, a sustainable and eco-friendly DDA biosynthesis route was set up in E. coli, which afforded the highest space time yield for DDA production among the current artificial multi-enzymatic routes derived from the LOX-pathway, and the productivity achieved here ranks the second highest among the current research progress in DDA biosynthesis.Graphical