Oxidase Enzyme Research Articles

Nonylphenol displays immunotoxicity by triggering hemocyte extracellular traps in Manila clam via ROS burst, ERK pathway and glycolysis

Nonylphenol (NP), an endocrine disruptor, has been demonstrated to be a harmful environmental contaminant and toxic to organisms. In this study, to address concerns regarding the immunotoxicity of NP, we treated clam Ruditapes philippinarum hemocytes with NP in vitro and explored the underlying mechanisms of NP-induced extracellular traps (ETs). NP could induce the formation of hemocytes ETs in a dose-dependent manner. Transcriptomics analysis revealed changes of signaling pathway involved in immunity and energy metabolism in hemocytes after NP stimulation. In this process, both reactive oxygen species (ROS) and myeloperoxidase (MPO) were up-regulated. Moreover, mitogen-activated protein kinase (MAPK) signaling pathway was proved to be activated in the formation of NP-induced ETs, manifested as enhanced phosphorylation of extracellular signal-regulated kinase (ERK) but not p38 or c-Jun N-terminal kinase (JNK). In the presence of U0126, an ERK phosphorylation inhibitor, the NP-induced expression of NADPH oxidase enzyme (NOX) was significantly decreased, which further alleviated the ROS production and ultimately limited the release of ETs. NP exposure increased glucose uptake, along with enhanced activities of glycolysis-related enzymes such as hexokinase (HK) and pyruvate kinase (PK). After inhibiting glycolysis by the inhibitor 2-DG, the formation of NP-induced ETs was significantly suppressed. ERK could regulate mTOR signaling and the PI3K/AKT pathway, potentially directing ETs formation by orchestrating the glycolysis through the activation of key transcription factors c-Myc and HIF-1α. Collectively, the results preliminary confirm that the ERK-NOX-ROS axis and glycolysis are involved in NP-induced ETs formation, contributing to the cellular immunotoxicity in clam.

Extraction, Purification and Characterization of Poly Phenol Oxidase from Potato and Apple and Inhibition of Enzymatic Browning

The purpose of this study was to determine the optimum conditions for the extraction of polyphenol oxidase (PPO) from potato and apple. PPO is found in a variety of fruits and vegetables and is the enzyme that causes browning reactions when handling damaged cells. At 405 nm, the enzyme activity was determined using spectrophotometry. PPO was researched to stop potato and apple from browning, which reduces their marketability. The outcomes showed that using an extraction buffer containing phosphate buffer (0.05 M, pH 6.6) was the optimum condition for the extraction of polyphenol oxidase from potato and apple. In addition, the results showed that as the temperature increased, the (PPO) activity increased, and the activity reached its peak at a temperature of 35 ͦC. The optimum pH value of (PPO) activity was 6.5 and we observed during the research that (PPO) decreased in strong acid condition and alkaline environments. Moreover, the results showed that the highest activity of polyphenol oxidase enzyme extracted from apple and potato was by using (O-dihydroxybenzene) as a substrate. As the concentration of the substrate increased, the (PPO) activity increased, reaching the optimum substrate concentration of 0.8%, and then its activity decreased. Also, we found that when using ascorbic acid, citric acid, and Na2SO3 as inhibitors, the activity of the polyphenol oxidase enzyme decreased with an increase in the concentration of ascorbic acid, citric acid, and Na2SO3. Sodium thiosulfate, 0.01 M ascorbic acid and 0.5% polyethylene glycol were extracted with an extraction ratio of 1:4 (w/v) for three minutes by using a blender. The purpose of study was to determine the optimum conditions for polyphenol oxidase extraction from potato and apple.

Colorimetric approach based on iron oxide magnetic molecularly imprinted nanozyme for sensitive determination of antipyrine and benzocaine: Application to environmental water samples and pharmaceutical dosage form

Nanozymes provide the required enzymatic activity however, they lack the selectivity to target a certain drug during analysis. Herein, a novel polydopamine molecularly imprinted polymer (MIP) was fabricated onto Fe3O4 nanozymes surface for sensitive and selective determination of antipyrine (ANT) and benzocaine HCl (BEN). Synthesized Fe3O4 nanozyme with peroxidase like activity catalyzed the oxidation of ortho phenylenediamine by the aid of H2O2 to produce intense yellow color that can be measured via colorimetric assay. The MIPs were prepared and characterized using field-emission scanning electronic microscopy, Fourier-transform IR spectroscopy, surface area analysis, and X-ray diffraction spectrum. Two Fe3O4 @ MIP NPs nanozyme with peroxidase like activity of two drugs were prepared. Upon binding of the target drug with its respective MIP, an inhibition in the Fe3O4 enzymatic activity occurred and thus a decrease in the yellow color produced. This quantitative decrease in the color intensity represents the drug concentration. Factors affecting the enzymatic colorimetric reaction have been investigated and optimized. The formed MIPs increased the sensitivity of the proposed method with LOD 1.405, 0.658 ng mL−1, LOQ 4.259, 1.993 and recovery percentage of 100.07, 100.57 for ANT and BEN, respectively. The reusability of the Fe3O4 @ MIP NPs was assessed for six cycles without significant loss in enzymatic oxidase activity. Furthermore, the method was validated following ICH guidelines. Then, it was successfully applied for determination of the two drugs in spiked environmental water samples and pharmaceutical formulation.

Electrodeposited ZnO Nanostructures on ITO Surfaces: Exploring Their Efficacy for Cholesterol Biosensing Applications

In this study, ZnO nanostructures were prepared by electrochemical anodization of electrodeposited Zn on ITO/glass substrates for cholesterol detection. The efficiency of the developed ZnO nanostructures in the detection of the Cholesterol oxidase (ChOx) enzyme was determined by the cyclic voltammetry method. The XRD and SEM results confirmed the synthesis of ZnO nanostructures prepared by the anodization method with various parameters. The effect of electrodeposition and anodization time on the morphology was observed. Cyclic voltammetry of ZnO/Zn/ITO/glass and Pt/ZnO/Zn/ITO/glass electrodes in electrolytes with various cholesterol concentrations was performed. The detection limit of the obtained Pt/ZnO/Zn/ITO/glass structured electrode was calculated as 0.965x10-3M. The resulting material with a layered structure may have potential applications in electrochemical sensors and biosensors in biomedical applications. In addition to biosensing performance, this study proposes a new approach for the development of ZnO-based biosensors that does not require expensive infrastructure and raw material costs, making it possible to develop high-sensitivity biosensor electrodes with lower detection limits with improvements to be made in future studies.

Effect of konjac glucomannan on gut microbiota from hyperuricemia subjects in vitro: fermentation characteristics and inhibitory xanthine oxidase activity.

The disorder of uric acid metabolism is closely associated with gut microbiota and short-chain fatty acids (SCFAs) dysregulation, but the biological mechanism is unclear, limiting the development of uric acid-lowering active polysaccharides. Konjac glucomannan (KGM) could attenuate metabolic disturbance of uric acid and modulate the gut microbiota. However, the relationship between uric acid metabolism and gut microbiota is still unknown. In this study, The fecal samples were provided by healthy volunteers and hyperuricemia (HUA) patients. Fecal samples from healthy volunteers was regarded as the NOR group. Similarly, 10% HUA fecal suspension was named as the HUA group. Then, fecal supernatant was inoculated into a growth basal medium containing glucose or KGM, and healthy fecal samples were designated as the NOR-GLU and NOR-KGM groups, while HUA fecal samples were designated as the HUA-GLU and HUA-KGM groups. All samples were cultured in an anaerobic bag system. After fermentation for 24 h, the samples were collected for further analysis of composition of intestinal microbiota, SCFAs concentration and XOD enzyme activity. The results showed that KGM could be utilized and degraded by the gut microbiota from HUA subjects, and it could modulate the composition and structure of their HUA gut microbiota to more closely resemble that of a healthy group. In addition, KGM showed a superior modulated effect on HUA gut microbiota by increasing Megasphaera, Faecalibacterium, Lachnoclostridium, Lachnospiraceae, Anaerostipes, and Ruminococcus levels and decreasing Butyricicoccus, Eisenbergiella, and Enterococcus levels. Furthermore, the fermentation solution of KGM showed an inhibitory effect on xanthine oxidase (XOD) enzyme activity, which might be due to metabolites such as SCFAs. In conclusion, the effect of KGM on hyperuricemia subjects was investigated based on the gut microbiota in vitro. In the present study. It was found that KGM could be metabolized into SCFAs by HUA gut microbiota. Furthermore, KGM could modulate the structure of HUA gut microbiota. At the genus level, KGM could decrease the relative abundances of Butyricicoccus, Eisenbergiella, and Enterococcus, while Lachnoclostridium and Lachnospiraceae in HUA gut microbiota were significantly increased by the addition of KGM. The metabolites of gut microbiota, such as SCFAs, might be responsible for the inhibition of XOD activity. Thus, KGM exhibited a superior probiotic function on the HUA gut microbiota, which is expected as a promising candidate for remodeling the HUA gut microbiota.

In silico study of the xanthine oxidase enzyme inhibition by the essential oil compounds of Myrtus communis plant in the treatment of gout disease

In silico study of the xanthine oxidase enzyme inhibition by the essential oil compounds of Myrtus communis plant in the treatment of gout disease

Changes in polyphenol oxidase and guaiacol peroxidase enzymes and the expression of pathogenesis-related genes in benzothiadiazole, mycorrhiza-induced or genetic resistance of sunflower plants affected by Sclerotinia sclerotiorum

Changes in polyphenol oxidase and guaiacol peroxidase enzymes and the expression of pathogenesis-related genes in benzothiadiazole, mycorrhiza-induced or genetic resistance of sunflower plants affected by Sclerotinia sclerotiorum

A single diiron enzyme catalyses the oxidative rearrangement of tryptophan to indole nitrile.

Nitriles are uncommon in nature and are typically constructed from oximes through the oxidative decarboxylation of amino acid substrates or from the derivatization of carboxylic acids. Here we report a third nitrile biosynthesis strategy featuring the cyanobacterial nitrile synthase AetD. During the biosynthesis of the eagle-killing neurotoxin, aetokthonotoxin, AetD transforms the 2-aminopropionate portion of 5,7-dibromo-L-tryptophan to a nitrile. Employing a combination of structural, biochemical and biophysical techniques, we characterized AetD as a non-haem diiron enzyme that belongs to the emerging haem-oxygenase-like dimetal oxidase superfamily. High-resolution crystal structures of AetD together with the identification of catalytically relevant products provide mechanistic insights into how AetD affords this unique transformation, which we propose proceeds via an aziridine intermediate. Our work presents a unique template for nitrile biogenesis and portrays a substrate binding and metallocofactor assembly mechanism that may be shared among other haem-oxygenase-like dimetal oxidase enzymes.

Subcellular distribution of the β-N-methylamino-L-alanine-containing proteins in marine diatoms

Neurotoxin β-N-methylamino-L-alanine (BMAA) has been implicated as a major inducer of human neurodegenerative diseases. In recent years, marine diatoms were verified to produce BMAA-containing proteins. It will be an important cue to elucidate the subcellular distribution of BMAA in marine diatoms for disclosing its biosynthesis pathway. In this study, three species of Thalassiosira (T. andamanica, T. allenii and T. minima) were used to investigate the subcellular distribution of BMAA in organelles. Results showed that the crushing efficiency of diatoms was species-specific and increased with the rise of ultrasonic intensity of 22, 50 and 100 W (pulse = 0.2 s/s, 4 min), of which T. andamanica and T. allenii obtained the lowest and highest crushing efficiency, respectively. Interestingly, although T. allenii and T. minima were more efficiently crushed at 50 W and 100 W power (pulse = 0.2 s/s), their organelles were largely fragmented, which was verified by cytochrome c oxidase (CCO) enzyme analysis and transmission electron microscopy (TEM) observation. Their organelles were not fragmented only at 22 W. However, the crushing efficiency of T. andamanica was more reliable, and its organelles were essentially intact and only damaged at 100 W. Analysis of the BMAA-containing proteins showed that these proteins exclusively distribute in the Golgi and endoplasmic reticulum (ER) organelles. The nearly intact membranes of nucleus, mitochondria, Golgi and ER organelles testified that the absence of BMAA in other organelles was not caused by damage of nucleus or mitochondria. Results demonstrated that the BMAA-containing proteins were produced and accumulated in the ER and Golgi of diatoms.

One-Pot Synthesis of a pH-Sensitive MOF Integrated with Glucose Oxidase for Amplified Tumor Photodynamic/Photothermal Therapy.

Photothermal therapy (PTT) and photodynamic therapy (PDT) provide targeted approaches to cancer treatment, but each therapy has inherent limitations such as insufficient tissue penetration, uneven heat distribution, extreme hypoxia, and overexpressed HSP90 in tumor cells. To address these issues, herein, by encapsulating the IR780 dye and glucose oxidase (GOx) enzyme within ZIF-8 nanoparticles, we created a versatile system capable of combining photodynamic and enhanced photothermal therapy. The integration of the IR780 dye facilitated the generation of reactive oxygen species and hyperthermia upon light activation, enabling dual-mode cancer cell ablation. Moreover, GOx catalyzes the decomposition of glucose into gluconic acid and hydrogen peroxide, leading to the inhibition of ATP production and downregulation of heat shock protein 90 (HSP90) expression, sensitizing cancer cells to heat-induced cytotoxicity. This synergistic combination resulted in significantly improved therapeutic outcomes. Both in vitro and in vivo results validated that the nanoplatform demonstrated superior specificity and favorable therapeutic responses. Our innovative approach represents a promising strategy for overcoming current limitations in cancer treatments and offers the potential for clinical translation in the future.

Purification of Glycolate Oxidase Enzymes from Peasm (Pisum sativum l.) and Sorghum (Sorghum sudanense J.) Using Ion-Exchange Chromatography and their Physicochemical Characteristics

Purification of Glycolate Oxidase Enzymes from Peasm (Pisum sativum l.) and Sorghum (Sorghum sudanense J.) Using Ion-Exchange Chromatography and their Physicochemical Characteristics

Bio-functionalized conductive poly(acrylic acid):poly(3,4-Ethylenedioxythiophene)-Prussian blue hybrid transducer for biosensors and bioelectronics interfaces

This study presents an innovative organic-inorganic hybrid transducer of poly(acrylic acid) (PAA), poly(3,4-ethylenedioxythiophene) (PEDOT) and Prussian blue (PB) nanocatalysts. The transducer demonstrated functionality conducive to enzyme conjugation and exhibited favorable electrochemical properties for biosensor signal transduction. Fabricated by a one-tube chemical redox method, the PAA:PEDOT-PB transducer showed long-term electrocatalytic and structural stability. The performance of the transducer was characterized by high transduction activity and low charge transfer resistance, particularly for H2O2 reduction. It achieves a linear detection range from 1.0 μM to 4.0 mM, with a sensitivity of 494 ± 10 μA mM−1 cm−1 and a LOD of 0.34 μM. The PAA:PEDOT-PB transducer featured a high density of carboxyl groups (DCOOH = 14.64 ± 0.05 μmol cm−2) that promoted the immobilization of the H2O2-dependent oxidase enzyme lactate oxidase (LOx) with an EDC/S–NHS coupling agent. The LOx-PAA:PEDOT-PB transducer was developed for lactate biosensing. The transducer provided high LOx-enzyme affinity (KMapp = 1.47 ± 0.05 mM), and a rapid response time (10 s) for lactate detection across a concentration range of 5.0 μM to 4.0 mM, showing a sensitivity of 223 ± 3 μA mM−1 cm−1 and an LOD of 1.45 μM. The LOx-PAA:PEDOT-PB transducer was integrated with a flexible screen-printed electrode, incorporating a wireless, battery-free near-field communication potentiostat module to measure lactate in artificial sweat on a skin model via smartphone. The PAA:PEDOT-PB transducer could enable connections with multiple bio-recognition molecules through polycarboxylic acid groups, providing potential avenues for the development of advanced biosensors.

Methanol extract of black garlic protects against hyperuricemia by xanthine oxidase inhibitory activity in-vitro

Garlic (Allium sativum L.) has been used as a medicative food for a long time. However, some individuals are averse to raw garlic owing to its pungent taste and aroma. As a result, a variety of garlic formulations have been created to minimize these characteristics without reducing their functions. Extensive in vitro and in vivo research has shown that black garlic has several benefits, including anti-oxidant, anti-inflammatory, anticancer, anti-obesity, anti-diabetic, anti-allergic, cardioprotective, and hepatoprotective characteristics. However, it remains unclear which compounds are active ingredients in the extract and the contributions of the main identified compounds to the extract's XO inhibitory activity are unknown. This study aims to look into the potent XO inhibitors found in black garlic methanol extract. The research methodology is experimental, involving the inhibition of xanthine oxidase activity, specifically measuring the amount of uric acid formed during the reaction catalyzed by xanthine oxidase. This is accomplished in vitro via enzymatic reactions and spectrophotometric measurements. The Maillard reaction is responsible for the production of black color in garlic, and it is influenced by heating temperature, humidity, and fermentation time. Starting on the 17th day of the fermentation process, the brownish intensity of the garlic samples heated at 70 ° C and 90% humidity changed to brown. The optimum concentration is 0.3 units/mL at an optimum temperature of 30oC and an optimum pH of 8.0 at a substratum concentration of 0.30 mM at a wavelength of 284 nm. The activity of the xanthine oxidase enzyme was inhibited by allopurinol at various concentrations, with allopurinol having the highest percentage inhibition value of 78.74 percent at a concentration of 10 ppm. The present study showed that meanwhile, the highest inhibition occurred on the 29th day of fermentation, with a percentage value of 68.01 percent, and decreased the next day

Synthesis and Evaluation of 1,4-Dihydropyridine-Based Urea Derivatives as Polyphenol Oxidase Inhibitors

This study investigated the potential inhibitory effects of nine novel synthesized urea-substituted 1,4-dihydropyridine derivatives (DT-DEN-1-9) on polyphenol oxidase (PPO) activity. The compounds were synthesized via the Hantzsch reaction, providing a series of structurally diverse urea and thiourea-modified 1,4-dihydropyridines. Polyphenol oxidase enzyme was extracted from banana (Musa cavendishii) and purified using affinity chromatography with a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity gel. The purified enzyme's activity was measured spectrophotometrically using catechol as the substrate, monitoring the increase in absorbance at 420 nm. The inhibitory effects of the synthesized compounds on PPO activity were evaluated through in vitro assays. Various concentrations of each compound were incorporated into the enzyme reaction mixture, and the residual PPO activity was determined. The percentage of PPO activity was calculated relative to a control reaction without inhibitors. IC50 values, representing the concentration of inhibitor required to reduce enzyme activity by 50%, were determined using Lineweaver-Burk plots. Among the tested compounds, DT-DEN-6, featuring a phenyl thiourea substituent, exhibited the most potent inhibition with an IC50 value of 100.14 μM. DT-DEN-8, containing a 2,5-dichlorophenyl thiourea moiety, also showed strong inhibitory activity with an IC50 below 150 μM. Structure-activity relationships were observed, with electron-withdrawing substituents generally enhancing inhibitory potency. Conversely, DT-DEN-5, bearing a 4-(trifluoromethyl)phenyl thiourea substituent, exhibited the weakest inhibition profile (IC50: 233.33 μM). Our findings provide valuable insights for the design of next-generation PPO inhibitors, potentially leading to the development of novel anti-browning agents for applications in food preservation and other industries where control of enzymatic browning is crucial.

Effect of halogens on 3-[4-(dimethylamino) phenyl]-1-phenylprop-2-en-1-ones: development of a new class of monoamine oxidase-B inhibitors

Five dimethylamino-based chalcone derivatives (AC) were synthesized and evaluated for their inhibition degree against monoamine oxidase (MAO) enzymes. All AC compounds showed better inhibitory activity against MAO-B than that against MAO-A. AC4 showed the highest inhibitory ability with an IC50 value of 0.020 µM, similar to that of a reference drug safinamide (IC50 = 0.019 µM) against MAO-B, followed by AC1 (IC50 = 0.068 µM) and AC3 (IC50 = 0.083 µM). Substituent -F in ring A (AC4) increased the MAO-B inhibition, followed by -H (AC1), -Br (AC3), and -Cl (AC2). The selectivity index (SI) value of AC4 was high (SI = 82.00) as well as other compounds (44.41 to 98.15). AC4 was found to be a reversible inhibitor as confirmed through analysis using the dialysis method. Interestingly, AC4 was observed to be a noncompetitive MAO-B inhibitor with a rare case and with Ki values of 0.011 ± 0.0036 µM. These experiments confirmed that AC4 is a reversible and potent selective inhibitor of MAO-B. Molecular docking experiments revealed that AC4 showed the highest inhibitory activity with a docking score (-9.510 kcal/mol). A study using molecular dynamics modeling revealed that the protein–ligand complex was more stable. It was observed that AC4 was non-cytotoxic in the study using L929 cell line. In conclusion, compound AC4 shows promise as a MAO-B inhibitor.

UPLC-MS/MS-based metabolomics and chemometrics unveil metabolic patterns and their regulatory pathways over the course of pumpkin seeds (Cucurbita maxima) germination

While pumpkin seeds (Cucurbita maxima) are regarded as agro-industrial waste, they serve as powerhouses packed with a plethora of valuable compounds with potential nutraceutical significance. The present work offers an integrated view of metabolites dynamics and their regulatory pathways over the course of pumpkin seeds germination and their potential entanglements on Alzheimerʼs disease (AD) mitigation through Ultra Performance Liquid Chromatography-mass spectrometry (UPLC-MS/MS) analysis and chemometrics. A total of 76 compounds spanning amino acids, phenolic acid, flavonoids, lignans, vitamins, fatty acids, phospholipids and phytosterols were annotated revealing changing trends during germination process. Coincidentally, orthogonal projection to latent structures-discriminant analysis (OPLS-DA) demonstrated a clear separation trend among the five pumpkin samples assuring their chemical heterogeneity. Complementarily, OPLS-DA models were constructed in different timepoint comparisons to pick out the significant metabolic features which were subsequently imported into MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/) software for metabolic pathway enrichment analysis. Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed biosynthesis of unsaturated fatty acids, phenylpropanoid biosynthesis, glycerophospholipid metabolism and phenylalanine, tyrosine and tryptophan biosynthesis as the top predictive metabolic pathways associated with significant phytochemical shifts during germination process. Importantly, our efficacy experimental results dissected that 5 d and 7 d sprouted samples exerted noteworthy inhibitory potentials against acetylcholine esterase (AChE) and monoamine oxidase (MAO-B) enzymes. OPLS-derived coefficients plots portrayed that docosahexaenoic acid, oleic acid, lirioresinol A and tocotrienols dramatically augmented during germination were probably ascribed to promising AChE and MAO-B inhibitory activities. These findings offer a turning point for using pumpkin sprouts with an appreciable biochemical content to pursue the concept of nutritional therapy and accelerate perpetual influx of novel bioproducts to rectify AD.

Cryo-EM Structure of the Mnx Protein Complex Reveals a Tunnel Framework for the Mechanism of Manganese Biomineralization.

The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O2 directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from Bacillus sp. strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The β-sheet-rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxE3F3 cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.

Recombinant C-Terminal Catalytic Domain of Rat L-Gulono Lactone Oxidase Produced in Bacterial Cells Is Enzymatically Active.

The L-gulonolactone oxidase enzyme (GULO) catalyzes the last step of L-ascorbic acid (vitamin C) biosynthesis. This enzymatic activity is lost in primates. The full-length rat GULO has been previously produced in plants and demonstrated to be active. In this study, we compared the activity of two variants of GULO produced in Escheriachia coli cells, full-length rat GULO (fGULO) and its C-terminal catalytic domain (cGULO). The expression and purification of the recombinant proteins were optimized, and their biological activity was confirmed by two methods, the GULO activity assay in the protein extracts and the 'in-gel' staining for GULO activity. Both variants of recombinant GULO were biologically active in both assays. However, cGULO is more promising than fGULO for ascorbic acid production because it is more efficiently produced by bacteria. Furthermore, the optimal activities of the fGULO and cGULO recombinant proteins were observed at pH 7 and 6.5, and at temperatures of 40 and 30 °C, respectively. Kinetic studies revealed that at low substrate concentrations, Km values for fGULO and cGULO were 53.5 ± 5 and 42 ± 6.3 µM, respectively.

Composite PEDOT-PSS based highly sensitive electrochemical sensors for sensing glucose from human saliva

Here we report two composites, especially two different 2D materials and Au nanoparticle composite with conducting polymer PEDOT-PSS as electrodes for highly sensitive, cost effective and reliable biosensors. Conducting polymer composite electrodes of AuNP/WS2/PEDOT-PSS and AuNP/GO/PEDOT-PSS were prepared by electrochemical deposition and were studied using various structural and electrochemical techniques. XRD, FTIR and SEM have been used for structural characterization which confirmed the elemental compositions of these composite electrodes. Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV) were used to examine the charge transfer kinetics and electroactive properties of the modified electrodes. Impedance spectra analysis had shown the reduced charge transfer resistance and diffusive property of AuNP/WS2/PEDOT-PSS electrodes compared to AuNP/GO/PEDOT-PSS. To demonstrate biosensing advantages of these electrodes, interaction between covalently immobilized glucose oxidase (GlOx) enzyme and D-glucose was monitored via chronoamperometry and CV. GlOx/AuNP/WS2/PEDOT-PSS/ITO yielded wider linear range from 0.74 to 440.67 µM towards the oxidation of glucose with high selectivity and lower limit of detection (LOD) of 1 µM. AuNP/GO/PEDOT-PSS/ITO composite electrode exhibited linearity in the range 3.84–373.33 µM and LOD 2.33 µM. Better sensitivity and wider linear range of GlOx/AuNP/WS2/PEDOT-PSS/ITO has been assigned to special layered structure as sheets formed by incorporation of WS2 to PEDOT-PSS. Validation of the glucose sensors has shown satisfactory results using real saliva samples. Study suggests use of conducting polymer, 2D layered material and metal nanoparticle composite electrodes based sensors offer wider linearity range and higher sensitivity for stable, reliable, cost- effective sensor electrodes which can be used easily in non-clinical diagnostics.

Cytochrome c Oxidase Detection in Human Fibroblasts Using Scanning Electrochemical Microscopy

Cytochrome C Oxidase Deficiency (COXD) is a disorder caused by the mutation in genes encoding for the cytochrome c oxidase enzyme (COX),1 a mitochondrial multimeric enzyme which acts as the terminal electron acceptor in the electron transport chain.2 COXD causes serious health issues in affected individuals, including severe muscle weakness, brain damage and disorders in organs such as heart, liver and kidneys.1 Diagnosis is mostly performed by muscle biopsy,3,4 which is invasive and time-consuming. Finding new effective and early diagnostic strategies is therefore crucial to minimize symptoms and long-term disabilities. This research proposes the use of scanning electrochemical microscopy (SECM) as a promising method to quantify COX activity in normal and COXD fibroblasts as cell models. We explore the interaction of the redox probe N,N,N’,N’-tetramethyl-p-phenylene diamine (TMPD) with COX as an indicator of enzymatic activity.5,6 A platinum ultramicroelectrode was used to monitor electrochemical currents at different scan speeds, which were later used to determine an apparent heterogeneous rate constant (k 0) for TMPD regeneration by each cell line using numerical modelling. A significant difference in k 0 values (p < 0.1) was observed when comparing normal and COXD fibroblasts. The further treatment of normal fibroblasts with a COX inhibitor, NaN3,7 induced significant decreases in k 0 (p < 0.01). The COX activity results determined using SECM were in good agreement with a standard spectrophotometric assay. Taken together, our findings confirm SECM as an effective and rapid method to detect COXD in living tissues and provides a foundation for the development of new assays to diagnose COXD in infants. References M. Brischigliaro and M. Zeviani, Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1862, 148335 (2021).A. Timón-Gómez et al., Semin Cell Dev Biol, 76, 163–178 (2018).M. L. Simard, A. Mourier, L. C. Greaves, R. W. Taylor, and J. B. Stewart, Journal of Pathology, 245, 311–323 (2018).S. B. Wortmann, J. A. Mayr, J. M. Nuoffer, H. Prokisch, and W. Sperl, Neuropediatrics, 48, 309–314 (2017).S. Kuss, E. E. L. Tanner, M. Ordovas-Montanes, and R. G. Compton, Chem Sci, 8, 7682–7688 (2017).S. Thind et al., Proc Natl Acad Sci U S A (2023). In press.M. J. Fei et al., Acta Crystallogr D Biol Crystallogr, 56, 529–535 (2000).