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Background: Chronic alcohol intake is associated with alterations of choline metabolism in various tissues. Here, we assessed if an oral choline supplementation attenuated the development of alcohol-related liver disease (ALD) in mice. Methods: Female C57BL/6 J mice (n = 8/group) were either pair-fed a liquid control diet, or a Lieber DeCarli liquid diet (5% ethanol) ± 2.7 g choline/kg diet for 29 days. Liver damage, markers of intestinal permeability and intestinal microbiota composition were determined. Moreover, the effects of choline on ethanol-induced intestinal permeability were assessed in an ex vivo model. Results: ALD development as determined by liver histology and assessing markers of inflammation (e.g., nitric oxide, interleukin 6 and 4-hydroxynonenal protein adducts) was attenuated by the supplementation of choline. Intestinal permeability in small intestine being significantly higher in ethanol-fed mice was at the level of controls in ethanol-fed mice receiving choline. In contrast, no effects of the choline supplementation were found on intestinal microbiota composition. Choline also significantly attenuated the ethanol-induced intestinal barrier dysfunction in small intestinal tissue ex vivo, an effect almost entirely abolished by the choline oxidase inhibitor dimbunol. Conclusion: Our results suggest that an oral choline supplementation attenuates the development of ALD in mice and is related to a protection from intestinal barrier dysfunction. [ABSTRACT FROM AUTHOR]

Abstract Background Chronic alcohol intake is associated with alterations of choline metabolism in various tissues. Here, we assessed if an oral choline supplementation attenuated the development of alcohol-related liver disease (ALD) in mice. Methods Female C57BL/6 J mice (n = 8/group) were either pair-fed a liquid control diet, or a Lieber DeCarli liquid diet (5% ethanol) ± 2.7 g choline/kg diet for 29 days. Liver damage, markers of intestinal permeability and intestinal microbiota composition were determined. Moreover, the effects of choline on ethanol-induced intestinal permeability were assessed in an ex vivo model. Results ALD development as determined by liver histology and assessing markers of inflammation (e.g., nitric oxide, interleukin 6 and 4-hydroxynonenal protein adducts) was attenuated by the supplementation of choline. Intestinal permeability in small intestine being significantly higher in ethanol-fed mice was at the level of controls in ethanol-fed mice receiving choline. In contrast, no effects of the choline supplementation were found on intestinal microbiota composition. Choline also significantly attenuated the ethanol-induced intestinal barrier dysfunction in small intestinal tissue ex vivo, an effect almost entirely abolished by the choline oxidase inhibitor dimbunol. Conclusion Our results suggest that an oral choline supplementation attenuates the development of ALD in mice and is related to a protection from intestinal barrier dysfunction.

Background: Choline oxidase, a flavoprotein, is an enzyme that catalyzes the reaction which converts choline into glycine betaine. Choline oxidase started its journey way back in 1933. However, the impact of the high temperature on its structure has not been explored despite the long history and availability of its crystal structure. Both choline oxidase and its product, glycine betaine, have enormous applications spanning across multiple industries. Understanding how the 3D structure of the enzyme will change with the temperature change can open new ways to make it more stable and useful for industry. Process: This research paper presents the in-silico study and analysis of the structural changes of A. globiformis choline oxidase at temperatures from 25 °C to 60 °C. A step-wise process is depicted in Fig. 1. Results: Multiple sequence alignment (MSA) of 11 choline oxidase sequences from different bacteria vs Arthrobacter globiformis choline oxidase showed that active site residues are highly conserved.The available crystal structure of A. globiformis choline oxidase with cofactor Flavin Adenine Dinucleotide (FAD) in the dimeric state (PDB ID: 4MJW)1 was considered for molecular dynamics simulations. A simulated annealing option was used to gradually increase the temperature of the system from 25 °C to 60 °C. Analysis of the conserved residues, as well as residues involved in Flavin Adenine Dinucleotide (FAD) binding, substrate binding, substate gating, and dimer formationwas done. At high temperatures, the formation of the inter-chain salt bridge between Arg50 and Glu63 was a significant observation near the active site of choline oxidase. Conclusion: Molecular dynamics studies suggest that an increase in temperature has a significant impact on the extended Flavin Adenine Dinucleotide (FAD) binding region. These changes interfere with the entry of substrate to the active site of the enzyme and make the enzyme inactive.

Transgenic tobacco plants overexpressing the choline oxidase gene from A. globiformis showed an increase in resistance at the level of primary and secondary biosynthesis of metabolites, removing the damage characteristic of salinity and stabilizing the condition of plants. We used 200 mM NaCl, which inhibits the growth of tobacco plants at all stages of development. Leaves of transgenic and wild-type (WT) plants Nicotiána tabácum were used for biochemical, cytological and molecular biological analysis. However, for transgenic lines cultivated under normal conditions (without salinity), we noted juvenile characteristics, delay in flowering, and slowing down of development, including the photosynthetic apparatus. This caused changes in the amount of chlorophyll, a delay in the plastid grana development with the preservation of prolamellar bodies. It also caused changes in the amount of sugars and indirectly downstream processes. A significant change in the activity of antioxidant enzymes and a change in metabolism is probably compensated by the regulation of a number of genes, the expression level of which was also changed. Thus, the tolerance of transgenic tobacco plants to salinity, which manifested itself as a result of the constitutive expression of codA, demonstrates an advantage over WT plants, but in the absence of salinity, transgenic plants did not have such advantages due to juvenilization. [ABSTRACT FROM AUTHOR]

As choline levels can be associated with several diseases such as Alzheimer's, Parkinson's, and so on, monitoring the choline levels has great importance. In this study, a metal–organic framework (Cu-MOF) with peroxidase-like activity was synthesized through the employment of an organic amine as a template and used as an efficient immobilization matrix for choline oxidase (ChOx). The Km values of Cu-MOF toward TMB and H2O2 are 1.4 mM and 0.084 mM, respectively. Results showed that the optimal pH for the ChOx@Cu-MOF complex in the presence of choline was obtained 5.5 in which it showed the high activity from 40 to 55 °C. Reusability studies showed that the residual activity of the ChOx@Cu-MOF complex toward choline had no apparent reduction up to five cycles of reusing. In comparison, it can maintain about 80% of the initial activity after ten cycles of reusing. Furthermore, this system displayed a robust linear relationship from 0.2 to 250 µM of choline with a detection limit of 0.2 μM. The obtained results showed that the recovery of these samples ranged between 95.07 and 102.77%. This strategy provides a rapid and facile approach to construct stable and functional biocatalysts, which have great promise for applications in bio-sensing and industrial catalysis. [ABSTRACT FROM AUTHOR]

Transgenic tobacco plants overexpressing the choline oxidase gene from A. globiformis showed an increase in resistance at the level of primary and secondary biosynthesis of metabolites, removing the damage characteristic of salinity and stabilizing the condition of plants. We used 200 mM NaCl, which inhibits the growth of tobacco plants at all stages of development. Leaves of transgenic and wild-type (WT) plants Nicotiána tabácum were used for biochemical, cytological and molecular biological analysis. However, for transgenic lines cultivated under normal conditions (without salinity), we noted juvenile characteristics, delay in flowering, and slowing down of development, including the photosynthetic apparatus. This caused changes in the amount of chlorophyll, a delay in the plastid grana development with the preservation of prolamellar bodies. It also caused changes in the amount of sugars and indirectly downstream processes. A significant change in the activity of antioxidant enzymes and a change in metabolism is probably compensated by the regulation of a number of genes, the expression level of which was also changed. Thus, the tolerance of transgenic tobacco plants to salinity, which manifested itself as a result of the constitutive expression of codA, demonstrates an advantage over WT plants, but in the absence of salinity, transgenic plants did not have such advantages due to juvenilization.

Proteins immobilized on biosilica which have superior reactivity and specificity and are innocuous to natural environments could be useful biological materials in industrial processes. One recently developed technique, living diatom silica immobilization (LiDSI), has made it possible to immobilize proteins, including multimeric and redox enzymes, via a cellular excretion system onto the silica frustule of the marine diatom Thalassiosira pseudonana. However, the number of application examples so far is limited, and the type of proteins appropriate for the technique is still enigmatic. Here, we applied LiDSI to six industrially relevant polypeptides, including protamine, metallothionein, phosphotriesterase, choline oxidase, laccase, and polyamine synthase. Protamine and metallothionein were successfully immobilized on the frustule as protein fusions with green fluorescent protein (GFP) at the N terminus, indicating that LiDSI can be used for polypeptides which are rich in arginine and cysteine. In contrast, we obtained mutants for the latter four enzymes in forms without green fluorescent protein. Immobilized phosphotriesterase, choline oxidase, and laccase showed enzyme activities even after the purification of frustule in the presence of 1% (wt/vol) octylphenoxy poly(ethyleneoxy)ethanol. An immobilized branched-chain polyamine synthase changed the intracellular polyamine composition and silica nanomorphology. These results illustrate the possibility of LiDSI for industrial applications. [ABSTRACT FROM AUTHOR]

Today, quantitative and qualitative diagnosis of choline is clinically essential. One way to deal with the limitations of traditional methods is using the bimetallic metal–organic framework (MOF)/enzyme-based biosensors. Unlike single metal, the bimetallic MOFs have important properties such as increasing MOF peroxidation like activity, increasing the stability, and maintenance of the enzyme in the MOF structure, etc. In this research, a biosensor system based on MOF‏(Fe–Cu)–ChOx is designed for colorimetric detection of choline. Our results showed that the optimum pH and temperature for the peroxidase-like activity of MOF-enzyme complex were 5.5 and 25–40‏ °C, respectively. Also, the linear area and the detection limit of this biosensor were 20–200 and 0.006 micromoles, respectively. The obtained recovery in the human serum choline assay was in the range of 95.15–103.1%, which, compared to other similar biosensors, shows the sensitivity, specificity, and reliability of the biosensor in biological samples. These results indicated that the synthesized peroxidase-mimicking MOF‏(Fe–Cu) could stabilize the choline oxidase and sense choline in the biological fluids. [ABSTRACT FROM AUTHOR]

Environment-friendly nanobiosensors can improve the detection performance of traditional biomolecular sensors and are of importance in practical applications. In this study, a kind of room temperature phosphorescence (RTP) quantum dots (QDs) (ChOx RTP QDs) nanobiosensor was prepared by mineralization at room temperature (25 ℃), using choline oxidase (ChOx) as the template. The specific enzyme substrate reaction between ChOx and choline oxidase and the photoinduced electron transfer (PIET) were used to detect the RTP of choline oxidase. The linear range of chox detection is 0. 05~20 mmol/L, and the detection limit is 0. 02 μmol/L. This method is based on the RTP property of QDs, which can effectively avoid the interference of background fluorescence of biological samples and does not need complex sample pretreatment process. Therefore, this method is more suitable for the quantitative detection of choline oxidase in biological samples. [ABSTRACT FROM AUTHOR]

Nanosized polymer-enzyme films were fabricated on graphite surfaces via a sequential two-stage adsorption of the pH- and thermoresponsive polymer, viz., linear poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA), and the enzyme, viz., choline oxidase (ChO). The influence of the conditions (pH and temperature), which are applied for deposition of the polymer onto graphite surface, on electrochemical responses of the polymer-enzyme sensor constructs and kinetic parameters of enzymatic oxidation of choline by ChO integrated into the polymer-enzyme films was revealed. Stimuli-sensitivity of PDMAEMA was found to control the properties of the formed polymer coatings (surface coverage, film thickness and roughness), which decisively determine the amount of the ChO bound by the polymer film and, consequently, the resultant electrochemical characteristics of the polymer-enzyme sensor constructs. [ABSTRACT FROM AUTHOR]

Choline oxidase catalyzes the oxidation of choline to glycine betaine via betaine aldehyde in glycine betaine biosynthesis and betaine acts as an osmolyte. Choline oxidase has attracted a great deal of attention because of its wide application in clinical and its potential use in enzymatic betaine production. Therefore, the development of efficient methods for overexpression of choline oxidase will be very valuable. In the present study, the choline oxidase gene was amplified from a newly isolated Gram-positive soil Arthrobacter globiformis strain HYJE003 and was cloned into a pET expression vector. Furthermore, the culture conditions were optimized for overexpression of cloned choline oxidase gene in different hosts for periplasmic expression of the enzyme. Expression host system Rosetta-gami2(DE3)pLysS yielded more cell-free protein and 20 fold higher active enzyme compared to any other reported studies. Terrific Broth media were found to be yielding the highest cell biomass, by applying the optimized culture conditions and purification strategy 20,902 U of choline oxidase was produced with a specific activity of 95 U/mg. The optimum pH and temperature for the enzyme activity were found to be 7 and 37 °C, respectively. Finally, we have demonstrated efficient bioconversion of betaine using overexpressed and purified choline oxidase enzyme. The enzymatically produced betaine was estimated by the formation of betaine reineckate and we were able to produce 0.83 molar of betaine from one molar of choline chloride. [ABSTRACT FROM AUTHOR]

Choline oxidase, a flavoprotein, is an enzyme that catalyzes the reaction which converts choline into glycine betaine. Choline oxidase started its journey way back in 1933. However, the impact of the high temperature on its structure has not been explored despite the long history and availability of its crystal structure. Both choline oxidase and its product, glycine betaine, have enormous applications spanning across multiple industries. Understanding how the 3D structure of the enzyme will change with the temperature change can open new ways to make it more stable and useful for industry. This research paper presents the in-silico study and analysis of the structural changes of A. globiformis choline oxidase at temperatures from 25 °C to 60 °C. A step-wise process is depicted in Fig. 1. Multiple sequence alignment (MSA) of 11 choline oxidase sequences from different bacteria vs Arthrobacter globiformis choline oxidase showed that active site residues are highly conserved. The available crystal structure of A. globiformis choline oxidase with cofactor Flavin Adenine Dinucleotide (FAD) in the dimeric state (PDB ID: 4MJW) 1 was considered for molecular dynamics simulations. A simulated annealing option was used to gradually increase the temperature of the system from 25 °C to 60 °C. Analysis of the conserved residues, as well as residues involved in Flavin Adenine Dinucleotide (FAD) binding, substrate binding, substate gating, and dimer formationwas done. At high temperatures, the formation of the inter-chain salt bridge between Arg50 and Glu63 was a significant observation near the active site of choline oxidase. Molecular dynamics studies suggest that an increase in temperature has a significant impact on the extended Flavin Adenine Dinucleotide (FAD) binding region. These changes interfere with the entry of substrate to the active site of the enzyme and make the enzyme inactive. [ABSTRACT FROM AUTHOR]

A novel metal composite material based on zirconium dioxide decorated gold nanoparticles (ZrO2@AuNPs), copper (I) oxide at manganese (IV) oxide (Cu2O@MnO2) and immobilized choline oxidase (ChOx) onto a glassy carbon electrode (GCE) (ChOx/Cu2O@MnO2‐ZrO2@AuNPs/GCE) has been developed for enhancing the electro‐catalytic property, sensitivity and stability of the amperometric choline biosensor. The ChOx/Cu2O@MnO2‐ZrO2@AuNPs/GCE displayed an excellent electrocatalytic response to the oxidation of the byproduct H2O2 from the choline catalyzed reaction, which exhibited a charge transfer rate constant (Ks) of 0.97 s−1, a diffusion coefficient value (D) of 4.50×10−6 cm2 s−1, an electroactive surface area (Ae) of 0.97 cm2 and a surface concentration (γ) of 0.54×10−8 mol cm−2. The modified electrode also provided a wide linear range of choline concentration from 0.5 to 1,000.0 μM with good sensitivity (97.4 μA cm−2 mM−1) and low detection limit (0.3 μM). The apparent Michaelis‐Menten constant was found to be 0.08 mM with Imax of 0.67 μA. This choline biosensor presented high repeatability (%RSD=2.9, n=5), excellent reproducibility (%RSD=2.9, n=5), long time of use (n=28 with %I>50.0 %) and good selectivity without interfering effects from possible electroactive species such as ascorbic acid, aspirin, amoxicillin, caffeine, dopamine, glucose, sucrose and uric acid. This optimal method was successfully applied for choline measurement in prepared human blood samples which demonstrated accurate and excellent reliability in the recovery range from 96.7 to 102.0 %. [ABSTRACT FROM AUTHOR]

Acetylcholine is a neurotransmitter, which is located at the intersections of the nerve and muscles in the lymph nodes of the internal organs motor systems and in various parts of the central nervous system. A decrease of acetylcholine in brain is associated with Alzheimer's disease. That is why it is an important agent for this disease. In this study, a bienzymatic biosensor system with acetylcholine esterase and choline oxidase was prepared with carbon paste electrode modified with carbon nano Dot‐(3‐Aminopropyl) triethoxysilane (CDs‐APTES) for determination of the amount of acetylcholine. Acetylcholine esterase and choline oxidase enzymes were immobilized onto a modified carbon paste electrode by cross‐linking with glutaraldehyde. Determination of acetylcholine was carried out by the oxidation of enzymatically produced H2O2 at 0.4 V versus Ag/AgCl. The effect of temperature, pH, and substrate concentration on the acetylcholine response of the prepared biosensor was investigated. In addition, the optimum CDs‐APTES amount, the linear operating range of the biosensor, and the interference effect were also investigated. [ABSTRACT FROM AUTHOR]

Freeform bioprinting, realized by extruding ink-containing cells into supporting materials to provide physical support during printing, has fostered significant advances toward the fabrication of cell-laden soft hydrogel constructs with desired spatial control. For further advancement of freeform bioprinting, we aimed to propose a method in which the ink embedded in supporting materials gelate through a cytocompatible and rapid cascade reaction between oxidase and peroxidase. To demonstrate the feasibility of the proposed method, we extruded ink containing choline, horseradish peroxidase (HRP), and a hyaluronic acid derivative, cross-linkable by HRP-catalyzed reaction, into a supporting material containing choline oxidase and successfully obtained three-dimensional hyaluronic acid-based hydrogel constructs with good shape fidelity to blueprints. Cytocompatibility of the bioprinting method was confirmed by the comparable growth of mouse fibroblast cells, released from the printed hydrogels through degradation on cell culture dishes, with those not exposed to the printing process, and considering more than 85% viability of the enclosed cells during 10 days of culture. Owing to the presence of derivatives of the various biocompatible polymers that are cross-linkable through HRP-mediated cross-linking, our results demonstrate that the novel 3D bioprinting method has great potential in tissue engineering applications.

Key message: We propose that codA tomato plants exhibited higher degrees of enhanced thermotolerance than BADH tomato plants, and H2O2 as a signaling molecule also plays an important role in heat resistance. Betaine aldehyde dehydrogenase (BADH) and choline oxidase (COD) are key enzymes in glycinebetaine (GB) synthesis. In this study, two kinds of transgenic tomato plants, which were transformed with BADH gene and codA gene, respectively, were used to explore their thermotolerance. Our results showed that the levels of GB in leaves of the fourteen independent transgenic lines ranged from 1.9 μmol g−1 fresh weight to 3.4 μmol g−1 fresh weight, while GB was almost undetectable in leaves of WT plants. CO2 assimilation and photosystem II (PSII) photochemical activity in transgenic plants were more thermotolerant than WT plants, especially the codA-transgenic plants showed the most. Significant accumulation of hydrogen peroxide (H2O2), superoxide anion radical (O2·−), and malondialdehyde (MDA) were more in WT plants than transgenic plants, while this accumulation in codA-transgenic plant was the least. Furthermore, the expression of the heat response genes and the accumulation of heat shock protein 70 (HSP70) were found to be more in transgenic plants than that in WT plants during heat stress, as well as showing the most expression and accumulation of HSP70 in the codA-transgenic plants. Taken together, our results suggest that the enhanced thermotolerance in transgenic plants is due to the positive role of GB in response to heat stress. And interestingly, in addition to the major role of GB in codA-transgenic plants, H2O2 as a signaling molecule may also play an important role in heat resistance, leading to higher thermotolerance compared to BADH-transgenic plants. [ABSTRACT FROM AUTHOR]

This article is a report on a novel and high-stability biosensor with minor interference effects of surface modification of working electrode with β-Ga2O3 nanowires (NWs) on choline oxidase biosensor were investigated in an electrochemical detection system. β-Ga2O3 NWs were materialized on the silicon substrate in a catalyst-free growth mechanism. The β-Ga2O3 NWs were in string form with 10 μm in length and uniformly 30 nm in diameter. Then the enzyme choline oxidase (ChOx) immobilized on the β-Ga2O3 NW/CB arrays on the working electrode. The Cyclic voltammetry (CV), Impedance spectroscopy and differential pulse voltammetry (DPV) measurements were performed for bio-sensing detection with choline chloride as substrate. One of the most prominent features of this surface modification with Ga2O3 NWs/CB is that the peak intensity, which is often of the order of μA, is greatly increased and reaches about mA. Chronoamperometry amplification well confirms the performance of this surface modification. The current with the CB modified electrode is about three times more than the non-modified electrode, and Ga2O3 NWs/CB modified electrode twice more than the CB modified electrode. The maximum current in the DPV data is modelled linearity iap= 0.159 (C)+2.28 and R² = 0.982. The limit of detection (LOD) of the electrode for the choline measurement was reached to 8.29 μM. The Sensitivity of the electrode was around 0.0397 mA mM-¹ mm-². The stability of this biosensor has been well studied over a period of 6 months, and more than 80% of the permanency of its response has been confirmed. [ABSTRACT FROM AUTHOR]

Natural deep eutectic solvents (NADES) are proposed as alternative solvents for peroxygenase‐catalysed oxyfunctionalization reactions. Choline chloride‐based NADES are of particular interest as they can serve as solvent, enzyme‐stabiliser and sacrificial electron donor for the in situ H2O2 generation. This report provides the first proof‐of‐concept and basic characterisation of this new reaction system. Highly promising turnover numbers for the biocatalysts of up to 200,000 have been achieved. [ABSTRACT FROM AUTHOR]

Organophosphorus pesticides (OPs) are widely used in agricultural production, but their residues could cause pollution to the environment and living organisms. In this paper, a simple dual-readout method for OPs detection was proposed based on ChOx single enzyme inhibition. Firstly, ChOx can catalyze the production of H 2 O 2 from choline chloride (Ch-Cl). Bifunctional iron-doped carbon dots (Fe-CDs) with good peroxidase-like activity and superior fluorescence properties can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidized TMB (oxTMB) by H 2 O 2 formed, and oxTMB could quench the fluorescence of Fe-CDs. In light of the fact that OPs exhibited activity in inhibiting ChOx, less H 2 O 2 and the decreasing oxTMB led to a result that the fluorescence of the system recovered and the solution became lighter in blue color. Moreover, the process of ChOx inhibition by OPs was analyzed by molecular docking technique and it was found that OPs interact with key amino acid residues catalyzed by ChOx (Asn510, His466, Ser101, His351, Phe357, Trp331, Glu312). Finally, a dual-mode (colorimetry and fluorescence) sensor was created for the detection of OPs with the detection limit of 6 ng/L, and was successfully used in the quantitative determination of OPs in actual samples with satisfactory results. [Display omitted] •The mechanism of ChOx inhibition by OPs was analyzed through molecular docking. •The single enzyme method based on ChOx inhibition was proposed to assay OPs. •A simple dual-readout method for OPs detection was built cobined with Fe-CDs. •It offers a clear version for the inhibition mechanism and fluorescence quenching mechanism. [ABSTRACT FROM AUTHOR]

In this paper, we constructed a photoelectrochemical biosensor with ITO electrode as the reaction platform, g-C 3 N 4 and TiO 2 as the reaction substrate, Nafion solution as the cross-linking agent, dropwise addition of choline oxidase (ChOx) and optimized its preparation and detection conditions, and finally used this photoelectrochemical biosensor for the detection of Phosphatidylcholine (PC) in actual soybean gross oil. The experiments showed that the PC concentration showed a good linear relationship with the photocurrent response value in the range of 0.05–280 mg/L, and the limit of detection was 0.05 mg/L (S/N = 3). Under the optimized conditions, the sensor had the highest photocurrent response value and the best sensitivity for the artificially simulated gross oil; Phosphatidyl ethandamine (PE), Phosphatidic acid (PA) and Phosphatidyl inositol (PI) showed no significant interference for the detection of PC, and the sensor showed high selectivity for PC detection. The results showed that the relative photocurrent value of the sensor remained at a high level during the storage time of about 32 days, which proved that the electrode storage stability was good, and provided a theoretical basis for the establishment of a fast, accurate and low-cost method for the determination of the PC content, which could more accurately reflect the rule of change of PC in the catalytic reaction of soybean oil degumming, and further realise the precise and moderate processing of fats and oils. [Display omitted] • g-C 3 N 4 and TiO 2 were utilized to construct the heterojunction. • Heterojunction loaded with ChOx enabled excellent optoelectronic properties. • The PEC biosensor can be used for the detection of phospholipids in actual samples. • The sensor had a low detection limit (0.05 mg/L). [ABSTRACT FROM AUTHOR]

Functioning as a flavoprotein, choline oxidase facilitates the transformation of choline into glycine betaine. Notably, choline oxidase and its resultant product, glycine betaine, find extensive applications across various industries and fields of study. However, its high sensitivity and tendency to lose functional activity at high temperatures reduces its industrial usage. MD simulation and mutation studies have revealed the role of certain residues responsible for the enzyme's thermal instability. This study focuses on inducing thermal stability to choline oxidase of A. globiformis through computational approaches at a maximum temperature of 60 °C. MD simulation analysis showed that Trp 331, Val 464 and Ser 101 contribute to structural instability, leading to the instability at 60 °C. Mutation of these residues with phenylalanine residues and simulation of the mutated enzyme at 60 °C exhibited thermostability and insignificant residual fluctuation. The re-docking and MM/GBSA analyses further validated the mutated enzyme's binding affinity and catalytic activity.Communicated by Ramaswamy H. Sarma.

Choline (Ch) and phosphocholine (PCh) levels in tissues are associated to tissue growth and so to carcinogenesis. Till now, only highly sophisticated and expensive techniques like those based on NMR spectroscopy or GC/LC- high resolution mass spectrometry permitted Ch and PCh analysis but very few of them were capable of a simultaneous determination of these analytes. Thus, a never reported before amperometric biosensor for PCh analysis based on choline oxidase and alkaline phosphatase co-immobilized onto a Pt electrode by co-crosslinking has been developed. Coupling the developed biosensor with a parallel sensor but specific to Ch, a crosstalk-free dual electrode biosensor was also developed, permitting the simultaneous determination of Ch and PCh in flow injection analysis. This novel sensing device performed remarkably in terms of sensitivity, linear range, and limit of detection so to exceed in most cases the more complex analytical instrumentations. Further, electrode modification by overoxidized polypyrrole permitted the development of a fouling- and interferent-free dual electrode biosensor which appeared promising for the simultaneous determination of Ch and PCh in a real sample.

A simple, selective and stable biosensor with the enzymatic reactor based on choline oxidase (ChOx) was developed and applied for the determination of choline (Ch) in flow injection analysis with amperometric detection. The enzyme ChOx was covalently immobilized with glutaraldehyde to mesoporous silica powder (SBA‐15) previously covered by NH2‐groups. This powder was found as an optimal filling of the reactor. The detection of Ch is based on amperometric monitoring of consumed oxygen during the enzymatic reaction, which is directly proportional to Ch concentration. Two arrangements of an electrolytic cell in FIA, namely wall‐jet cell with working silver solid amalgam electrode covered by mercury film and flow‐through cell with tubular detector of polished silver solid amalgam were compared. The experimental parameters affecting the sensitivity and stability of the biosensor (i. e. pH of the carrier solution, volume of reactor, amount of the immobilized enzyme, the detection potential, flow rate, etc.) were optimized. Under the optimized conditions, the limit of detection was found to be 9.0×10−6 mol L−1. The Michaelis‐Menten constant for covalently immobilized ChOx on SBA‐15 was calculated. The proposed amperometric biosensor with the developed ChOx‐based reactor exhibits good repeatability, reproducibility, long‐term stability, and reusability. Its efficiency has been confirmed by the successful application for the determination of Ch in two commercial pharmaceuticals. [ABSTRACT FROM AUTHOR]

A new and highly selective amperometric biosensor able to analyse choline in clinical samples from patients suffering from renal diseases and receiving repetitive haemodialysis treatment is described. The proposed biosensor is based on choline oxidase immobilized by co-crosslinking onto a novel anti-fouling and anti-interferent membrane. Between the several polymeric films electrosynthesized on a Pt electrode whose permselective behaviours were here investigated, those based on overoxidized polypyrrole/poly(o -aminophenol) bilayer revealed the most effective in rejecting common interferents usually present in biological fluids. The so realized biosensor showed notably analytical performances, displaying linear choline responses up to 100 μM, a sensitivity of 156 nA mM−1 mm−2 and a limit of detection, calculated at a signal-to-noise ratio equal to 3, of 1 μM; further, the within-a-day coefficients of variation for replicate (n = 3) were 2.7% and 1.2% at 100 μM and 10 μM choline levels, respectively. The remarkable performances and anti-interference behaviour allowed us the use of the proposed biosensor for the selective and fouling-free detection of choline in dialysate coming from patients on haemodialysis and even in their unpretreated human sera. Preliminary results gave choline levels in good agreement with the expected values. • A biosensor for choline analysis in untreated human serum and dialysate is reported. • A novel bilayer made of overoxidized poly(pyrrole)/poly(o-aminophenol) was realized. • The morphology and permselective behaviour of the bilayer were studied. • Choline biosensing was performed with high sensitivity and selectivity. • Biological fluids from renal diseases patients on haemodialysis were analysed. [ABSTRACT FROM AUTHOR]

Acetylcholine (ACh) and its precursor choline (Ch) play important roles in many biological processes. It is expected that Alzheimer's disease is occurred due to the reduction in synthesis of ACh. On the other hand, the increase in the level of ACh results in a depression of heart rate and over production of saliva. Therefore, the quantitative determination of Ch and ACh is very important in biological media. In the current work, sensitive and selective biosensors composed of choline oxidase (ChO) and/or acetylcholine esterase (AChE) on graphene oxide-ionic liquid (GO-IL)/ glassy carbon electrode (GCE) hyphenated with anodic differential pulse stripping voltammetry (ADPSV) were firstly established for the determination of ACh and Ch in human serum samples. The molecular bond of ionic liquid 1-allyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (AMIM TFSI) with GO was investigated by FT-IR and UV–Vis techniques. Furthermore, the surface topography of ChO/GO–IL and AChE-ChO/GO–IL composites was investigated by SEM and XRD. Then, the electron transfer features of biosensors ChO/GO-IL/GCE and AChE-ChO/GO-IL/GCE were characterized by the electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. The ADPSV was further used for the determination of Ch and ACh. The experimental parameters such as differential pulse working potential, differential pulse scan rate, equilibrium time and long-term stability were further optimized. Detection limits of 0.885 and 1.352 nmol L−1 with excellent linearity (R2 = 0.9996) over the range of 5–1000 nmol L−1 were obtained for Ch and ACh, respectively. The developed analytical methods showed excellent accuracy and precision for the determination of Ch and ACh in human serum samples avoiding their pretreatment or purification. fx1 • 1-allyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ionic liquid is first used along with graphene oxide. • Hyphenation of enzyme-based biosensors with anodic differential pulse stripping voltammetry. • Reliable detection of acetylcholine and its precursor choline in human serum. [ABSTRACT FROM AUTHOR]

• Constructed a highly sensitive Pd nano /MoS 2 based interface for detection of Ach. • Facile preparation of Pd nano /MoS 2 nanocomposite modified Au electrode. • Co-immobilization of AchE and Cho enzymes over nanocomposite modified Au electrode. • The linear response from 1.0 nM to 1000 μM for Ach with a detection limit of 1.0 nM. • Improved analytical performance of sensor with feasibility in real samples analysis. Acetylcholine (ACh) present inside the brain has ability to alter neuronal activation, influencing transmission of synapse by inducing neurons causing the enhancement in the ability of neuronal networks inside the brain. In result, ACh modifies the response of internal and external environment and perform function as a neuromodulator. This empowers the plan of a sensor particular for ACh that is effectively brought into biological frameworks. The work described here, an acetylcholinesterase (AChe) based sensor which is the most promising tool for analysis of neuromodulator level in biological samples. The prepared biosensor interface is based on co-immobilization of the AChe and choline oxidase (Cho) onto palladium nanoparticles (Pd nano) adsorbed over molybdenum disulfide (MoS 2) nanostructures electrodeposited on the surface of gold electrode (Au-ET). The sensor had excellent linear range of 1.0 nM to 10 mM with 1.0 nM as limit of detection. The novel AChe-Cho/Pd nano /MoS 2 @Au-ET shows a tremendous potential for applications in medical research and clinical diagnosis. The novel biosensor formed has good accuracy, precision and reliability hence, it might provide a new outlook for the detection and recognition of ACh in real serum samples. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Platinum nanoparticles (PtNPs) encapsulated metallic organic framework (MOF) modified Au electrode was fabricated. • Acetylcholine esterase (AChE) and choline oxidase (ChO) were co-immobilized onto modified Au electrode. • Fabricated electrochemical biosensor showed excellent detection limit and storage stability. • The developed sensor was tested in real serum samples and correlated with the standard HPLC method. Abstract Acetylcholinesterase (AChE) and choline oxidase (ChO) were co-immobilized on platinum nanoparticles (PtNPs) and metallic organic framework (MOF) modified Au electrode to construct an acetylcholine (ACh) biosensor. The morphology and structure of the nanoparticles modified working electrode were characterized by Scanning Electron Microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDX), Cyclic voltammetry (CVs) and Electrochemical Impedance Spectroscopy (EIS) techniques. The prepared biosensor exhibit a wide linear range of 0.01–500 μM for acetylthiocholine chloride (ATCl) and a low detection limit of 0.01 μM. No interferences were observed for other possible compounds/substances present in the serum samples. For detection of ACh in clinical (serum) samples the results of the nanoparticle based biosensor were well correlate with the results obtained from HPLC method. The half-life of the working electrode was 4 months, when stored at 4 °C. [ABSTRACT FROM AUTHOR]

The use of natural deep eutectic solvents (NADES) as multifunctional solvents for limonene bioprocessing was reported. NADES were used for the extraction of limonene from orange peel wastes, as solvent for the chemoenzymatic epoxidation of limonene, and as sacrificial electron donor for the in situ generation of H2O2 to promote the epoxidation reaction. The proof‐of‐concept for this multifunctional use was provided, and the scope and current limitations of the concept were outlined. Biocatalytic cascade epoxidation: Peracids are formed through lipase catalysis with H2O2, which is produced through choline oxidase‐catalyzed choline chloride oxidation in natural deep eutectic solvents (NADES) and O2 reduction. This enables direct oxidation of limonene into limonene epoxide. [ABSTRACT FROM AUTHOR]

Abstract Choline is an essential nutrient that is crucial for normal cellular function and its daily intake is recommended by the U.S. National Academy of Sciences. Choline and its derivatives play important roles in such events as neurotransmitter acetylcholine synthesis, cell-membrane signaling, liver and muscle function. Choline deficiency is now thought to have an impact on disorders such as Alzheimer's and Parkinson's diseases and also increases the risk of multiple cancers. Monitoring of choline level or its metabolism allows its application as a potential biomarker for early stage diagnosis. In comparison to the expensive, time-consuming and relatively complicated traditional methods, biosensors provide an easy, cost-effective, disposable, sensitive and specific sensing platform. In this manuscript, the innovative strategies used to develop electrochemical and optical enzyme-based biosensors for choline are reviewed. We believe that this review provides an insight for the development of novel biocompatible nanocomposites-based biosensors for the detection of choline and its metabolites covering its future prospects and commercialization. Highlights • Choline: an essential nutrient and a potential biomarker for early diagnosis of multiple cancers and neurological disorders. • Recent progress and up-to-date findings on various enzyme-based biosensors for detecting of choline. • Application of enzymatic biosensors as on-line analytical techniques for in vivo monitoring of choline. • Current challenges and the future perspectives of the biosensors based on smart nanocomposites in point-of-care devices. [ABSTRACT FROM AUTHOR]

A low-cost bio-enzyme screen-printed electrode (SPE) was produced by immobilizing choline oxidase (ChOx) and horseradish peroxidase (HRP) on an SPE with MWCNTs/SnO2/CS as modified material. The conditions of ChOx were as follows: concentration of 3 U, HRP concentration of 4 U, and pH 7.5. The bio-enzyme SPE had a significant electrochemical response to phosphatidylcholine (PC); the linear relationship between the peak current and PC content ranged from 30 to 270 mg/L, and the detection limit was 3 mg/L (S/N = 3). Bio-enzyme SPE was used to detect the content of PC in soybean crude oil. The spiked recovery of the samples ranged from 96.68 to 106.21%. The detection results obtained using high-performance liquid chromatography (HPLC) confirmed the results of the bio-enzyme SPE with a high correlation (r2 = 0.9978). After 45 days of storage of the bio-enzyme SPE, the current value remained 90% of initial current. The reproducibility and stability of the PC content detected by the bio-enzyme SPE in soybean crude oil exhibited a high performance. [ABSTRACT FROM AUTHOR]

In this work a novel bioassay for mustard agent detection was proposed. The bioassay is based on the capability of these compounds to inhibit the enzyme choline oxidase. The enzymatic activity, which is correlated to the mustard agents, was electrochemically monitored measuring the enzymatic product, hydrogen peroxide, by means of a screen-printed electrode modified with Prussian Blue nanoparticles. Prussian Blue nanoparticles are able to electrocatalyse the hydrogen peroxide concentration reduction at low applied potential (−50 mV vs. Ag/AgCl), thus allowing the detection of the mustard agents with no electrochemical interferences. The suitability of this novel bioassay was tested with the nitrogen mustard simulant bis(2-chloroethyl)amine and the sulfur mustard simulants 2-chloroethyl ethyl sulfide and 2-chloroethyl phenyl sulfide. The bioassay proposed in this work allowed the detection of mustard agent simulants with good sensitivity and fast response, which are excellent premises for the development of a miniaturised sensor well suited for an alarm system in case of terrorist attacks.

In this study, the pesticide sensor by graphene quantum dots (GQDs) and active enzyme, GQDs/enzyme platform, has been developed for monitoring the organophosphate pesticides. In this concept, H 2 O 2 generated from the active enzymatic reaction of acetylcholinesterase (AChE) and choline oxidase (CHOx) enables to react with GQDs resulting in a “turn-off” photoluminescence of GQDs. A “turn-on” photoluminescence of GQDs at 467 nm was recovered in the presence of organophosphate. Definitely, the photoluminescence changes of GQDs/AChE/CHOx biosensor reasonably correspond to the amount of pesticide. The detection limit of GQDs/AChE/CHOx biosensor towards dichlorvos was 0.172 ppm (0.778 μM). In this approach, this biosensor offers the promising determination of the organophosphate pesticides and a benefit for easy checking of organophosphate pesticides in food, water and environment with low cost, easy-to-prepare and less toxic to environment. [ABSTRACT FROM AUTHOR]

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A tri-enzyme system consisting of choline kinase/choline oxidase/horseradish peroxidase was used in the rapid and specific determination of the biomarker for bacterial sepsis infection, secretory phospholipase Group 2-IIA (sPLA2-IIA). These enzymes were individually immobilized onto the acrylic microspheres via succinimide groups for the preparation of an electrochemical biosensor. The reaction of sPLA2-IIA with its substrate initiated a cascading enzymatic reaction in the tri-enzyme system that led to the final production of hydrogen peroxide, which presence was indicated by the redox characteristics of potassium ferricyanide, K3Fe(CN)6 . An amperometric biosensor based on enzyme conjugated acrylic microspheres and gold nanoparticles composite coated onto a carbon-paste screen printed electrode (SPE) was fabricated and the current measurement was performed at a low potential of 0.20 V. This enzymatic biosensor gave a linear range 0.01-100 ng/mL (R2 = 0.98304) with a detection limit recorded at 5 × 10-3 ng/mL towards sPLA2-IIA. Moreover, the biosensor showed good reproducibility (relative standard deviation (RSD) of 3.04% (n = 5). The biosensor response was reliable up to 25 days of storage at 4°C. Analysis of human serum samples for sPLA2-IIA indicated that the biosensor has potential for rapid bacterial sepsis diagnosis in hospital emergency department. [ABSTRACT FROM AUTHOR]

The plastids involved in photosynthesis and other essential metabolic processes are the cellular compartments most susceptible to abiotic stress factors with adverse effects in plants. In order to protect the chloroplast compartment, the plants of the Belyi Naliv tomato cultivar and the Darenka potato cultivar were transformed with genes for Fe-dependent superoxide dismutase and choline oxidase with the use of the method for plant genetic engineering. These target genes were equipped with signal sequences directing the proteins into the plastid compartment. The experimental results have demonstrated the optimization of the agrobacterium-mediated transformation method for the analyzed tomato and potato cultivars. The naturally growing leaves and stems of the mature plants proved to be the most effective explants to obtain the regenerants. The highest regeneration frequency in the explants was reached with the use of a medium containing 5 mg L-1 BAP, 0.1 mg L-1 NAA, and 1 mg L-1 zeatin. Augmentin and timentin were equally efficient among the antibiotics tested for the inhibition of the agrobacterium; the concentration of 200 mg L-1 in the culture medium was sufficient to eliminate the agrobacterium. In addition, they had no inhibitory effects on the shoot regeneration process. However, the cefotaxime concentration required to inhibit the agrobacterium should be 500 mg L-1 and higher. Moreover, this antibiotic at the concentration of 100 mg L-1 was proven to inhibit the regenerant growth. [ABSTRACT FROM AUTHOR]

Choline oxidase oxidizes choline to glycine betaine, with two flavin-mediated reactions to convert the alcohol substrate to the carbon acid product. Proton abstraction from choline or hydrated betaine aldehyde in the wild-type enzyme occurs in the mixing time of the stopped-flow spectrophotometer, thereby precluding a mechanistic investigation. Mutagenesis of S101 rendered the proton transfer reaction amenable to study. Here, we have investigated the aldehyde oxidation reaction catalyzed by the mutant enzymes using steady-state and rapid kinetics with betaine aldehyde. Stopped-flow traces for the reductive half-reaction of the S101T/V/C variants were biphasic, corresponding to the reactions of proton abstraction and hydride transfer. In contrast, the S101A enzyme yielded monophasic traces like wild-type choline oxidase. The rate constants for proton transfer in the S101T/C/V variants decreased logarithmically with increasing hydrophobicity of residue 101, indicating a behavior different from that seen previously with choline for which no correlation was determined. The rate constants for hydride transfer also showed a logarithmic decrease with increasing hydrophobicity at position 101, which was similar to previous results with choline as a substrate for the enzyme. Thus, the hydrophilic character of S101 is necessary not only for efficient hydride transfer but also for the proton abstraction reaction. [ABSTRACT FROM AUTHOR]

The effect of temperature on the reaction of alcohol oxidation catalyzed by choline oxidase was investigated with the S101A variant of choline oxidase. Anaerobic enzyme reduction in a stopped-flow spectrophotometer was biphasic using either choline or 1,2-[ 2 H 4 ]-choline as a substrate. The limiting rate constants k lim1 and k lim2 at saturating substrate were well separated ( k lim1 / k lim2 > 9), and were > 15-fold slower than for wild-type choline oxidase. Solvent deuterium kinetic isotope effects (KIEs) ~ 4 established that k lim1 probes the proton transfer from the substrate hydroxyl to a catalytic base. Primary substrate deuterium KIEs ≥ 7 demonstrated that k lim2 reports on hydride transfer from the choline alkoxide to the flavin. Between 15 °C and 39 °C the k lim1 and k lim2 values increased with increasing temperature, allowing for the analyses of H + and H – transfers using Eyring and Arrhenius formalisms. Temperature-independent KIE on the k lim1 value ( H2O k lim1 / D2O k lim1 ) suggests that proton transfer occurs within a highly reorganized tunneling-ready-state with a narrow distribution of donor-acceptor distances. Eyring analysis of the k lim2 value gave lines with the slope (choline) > slope (D-choline) , suggesting kinetic complexity. Spectral evidence for the transient occurrence of a covalent flavin-substrate adduct during the first phase of the anaerobic reaction of S101A CHO with choline is presented, supporting the notion that an important role of amino acid residues in the active site of flavin-dependent enzymes is to eliminate alternative reactions of the versatile enzyme-bound flavin for the reaction that needs to be catalyzed. [ABSTRACT FROM AUTHOR]

The determination of lead ions by inhibition of choline oxidase enzyme has been evaluated for the first time using an amperometric choline biosensor. Choline oxidase (ChOx) was immobilized on a glassy carbon electrode (GCE) modified with multiwalled carbon nanotubes (MWCNT) through cross-linking with glutaraldehyde. In the presence of ChOx, choline was enzymatically oxidized into betaine at -0.3 V versus Ag/AgCl reference electrode, lead ion inhibition of enzyme activity causing a decrease in the choline oxidation current. The experimental conditions were optimised regarding applied potential, buffer pH, enzyme and substrate concentration and incubation time. Under the best conditions for measurement of the lowest concentrations of lead ions, the ChOx/MWCNT/GCE gave a linear response from 0.1 to 1.0 nM Pb2+ and a detection limit of 0.04 nM. The inhibition of ChOx by lead ions was also studied by electrochemical impedance spectroscopy, but had a narrower linear response range and low sensitivity. The inhibition biosensor exhibited high selectivity towards lead ions and was successfully applied to their determination in tap water samples. [ABSTRACT FROM AUTHOR]