Heterologous codA Gene Expression Leads to Mitigation of Salt Stress Effects and Modulates Developmental Processes.

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.

Immobilization of a Broad Range of Polypeptides on the Frustule of the Diatom Thalassiosira pseudonana.

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. IMPORTANCE Proteins immobilized on biosilica which have superior reactivity and specificity and are innocuous to natural environments could be useful biological materials in industrial processes. Living diatom silica immobilization (LiDSI) is a recently developed technique for in vivo protein immobilization on the diatom frustule. We aimed to explore the possibility of using LiDSI for industrial applications by successfully immobilizing six polypeptides: (i) protamine (Oncorhynchus keta), a stable antibacterial agent; (ii) metallothionein (Saccharomyces cerevisiae), a metal adsorption molecule useful for bioremediation; (iii) phosphotriesterase (Sulfolobus solfataricus), a scavenger for toxic organic phosphates; (iv) choline oxidase (Arthrobacter globiformis), an enhancer for photosynthetic activity and yield of plants; (v) laccase (Bacillus subtilis), a phenol oxidase utilized for delignification of lignocellulosic materials; and (vi) branched-chain polyamine synthase (Thermococcus kodakarensis), which produces branched-chain polyamines important for DNA and RNA stabilization at high temperatures. This study provides new insights into the field of applied biological materials.

A sensitive amperometric detection of neurotransmitter acetylcholine using carbon dot-modified carbon paste electrode.

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 H2 O2 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.

A Crosstalk- and Interferent-Free Dual Electrode Amperometric Biosensor for the Simultaneous Determination of Choline and Phosphocholine.

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.

Comparative effects of glycinebetaine on the thermotolerance in codA- and BADH-transgenic tomato plants under high temperature stress.

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.

Correction to: Magnetic-core@dual-functional-shell nanocomposites with peroxidase mimicking properties for use in colorimetric and electrochemical sensing of hydrogen peroxide.

A self-sacrificing catalytic method is described for the preparation of magnetic core/dual-functional-shell nanocomposites composed of magnetite, gold and Prussian blue (type Fe3O4@Au-PB). Two reaction pathways are integrated. The first involves chemical dissolution of Fe3O4 (the self-sacrificing step) by acid to release ferrous ions which then reacts with hexacyanoferrate(IV) to generate PB in the proximity of the magntic nanoparticles (MNPs). The second involves the reduction of tetrachloroaurate by hydroxylamine to generate gold under the catalytic effect of the MNPs. At the end, the MNP@Au-PB nanocomposite is formed. This method exploits both the chemical reactivity and catalytic effect of the MNPs in a single step. The multi-function material was applied (a) in an optical assay for H2O2; (b) in an amperometric assay for H2O2; (c) in an enzymatic choline assay using immobilized choline oxidase. The limit of electrochemical detection of H2O2 (at a potential as low as 50 mV) is 1.1 µM which is comparable or better than most analogous methods. The sensors display superior performance compared to the use of conventional core@single-shell (MNP@Au-PB) nanomaterials. Graphical abstract A self-sacrificing catalytic method is described to prepare magnetic core/dual-functional-shell nanocomposites composed of magnetic nanoparticle, gold and Prussian blue (type MNP@Au-PB). The nanocomposites worded well as candidates to develop colorimetric and electrochemical sensors of H2O2 with superior performance to analogues.

Magnetic-core@dual-functional-shell nanocomposites with peroxidase mimicking properties for use in colorimetric and electrochemical sensing of hydrogen peroxide.

A self-sacrificing catalytic method is described for the preparation of magnetic core/dual-functional-shell nanocomposites composed of magnetite, gold and Prussian Blue (type Fe3O4@Au-PB). Two reaction pathways are integrated. The first involves chemical dissolution of Fe3O4 (the self-sacrificing step) by acid to release ferrous ions which then reacts with hexacyanoferrate(IV) to generate PB in the proximity of the magntic nanoparticles (MNPs). The second involves the reduction of tetrachloroaurate by hydroxylamine to generate gold under the catalytic effect of the MNPs. At the end, the MNPs@Au-PB nanocomposite is formed. This method exploits both the chemical reactivity and catalytic effect of the MNPs in a single step. The multi-function material was applied (a) in an optical assay for H2O2; (b) in an amperometric assay for H2O2; (c) in an enzymatic choline assay using immobilized choline oxidase. The limit of electrochemical detection of H2O2 (at a potential as low as 50 mV) is 1.1 µM which is comparable or better than most analogous methods. The sensors display superior performance compared to the use of conventional core@single-shell (MNPs@PB) nanomaterials. Graphical abstract A self-sacrificing catalytic method is described to prepare magnetic core/dual-functional-shell nanocomposites composed of magnetic nanoparticle, gold and Prussian Blue (type MNP@Au-PB). The nanocomposites work well as candidates to develop colorimetric and electrochemical sensors of H2O2 with superior performance to analogues.

An Amperometric Biosensor for the Determination of Bacterial Sepsis Biomarker, Secretory Phospholipase Group 2-IIA Using a Tri-Enzyme System.

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.

Evidence for proton tunneling and a transient covalent flavin-substrate adduct in choline oxidase S101A.

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-[2H4]-choline as a substrate. The limiting rate constants klim1 and klim2 at saturating substrate were well separated (klim1/klim2>9), and were >15-fold slower than for wild-type choline oxidase. Solvent deuterium kinetic isotope effects (KIEs) ~4 established that klim1 probes the proton transfer from the substrate hydroxyl to a catalytic base. Primary substrate deuterium KIEs ≥7 demonstrated that klim2 reports on hydride transfer from the choline alkoxide to the flavin. Between 15°C and 39°C the klim1 and klim2 values increased with increasing temperature, allowing for the analyses of H+ and H- transfers using Eyring and Arrhenius formalisms. Temperature-independent KIE on the klim1 value (H2Oklim1/D2Oklim1) suggests that proton transfer occurs within a highly reorganized tunneling-ready-state with a narrow distribution of donor-acceptor distances. Eyring analysis of the klim2 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.

Substitutions of S101 decrease proton and hydride transfers in the oxidation of betaine aldehyde by choline oxidase.

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.

Enzymatic methods for choline-containing water soluble phospholipids based on fluorescence of choline oxidase: Application to lyso-PAF.

In this paper we present methods to determine water soluble phospholipids containing choline (wCh-PL). The analytes were hydrolyzed by the enzyme phospholipase D and the choline formed was oxidized by the enzyme Choline Oxidase (ChOx); the fluorescence changes of the ChOx are followed during the enzymatic reaction, avoiding the necessity of an indicating step. Both reactions (hydrolysis and oxidation) can be combined in two different ways: 1) a two-step process (TSP) in which the hydrolysis reaction takes place during an incubation time and then the oxidation reaction is carried out, the analytical signal being provided by the intrinsic fluorescence of ChOx due to tryptophan; 2) a one-step process (OSP) in which both enzymatic reactions are carried out simultaneously in the same test; in this case the analytical signal is provided by the ChOx extrinsic fluorescence due to a fluorescent probe (Ru (II) chelate) linked to the enzyme (ChOx-RuC). The analytical capabilities of these methods were studied using 1,2-dioctanoyl-sn-glycero-3-phosphocholine (C8PC), a water soluble short alkyl chain Ch-PL as a substrate, and 1-O-hexadecyl-sn-glyceryl-3-phosphorylcholine (lyso-PAF). The analytical features of merit for both analytes using both methods were obtained. The TSP gave a 10-fold sensitivity and lower quantification limit (1.0*10-5 M for lyso-PAF), but OSP reduced the determination time and permitted to use the same enzyme aliquot for several measurements. Both methods gave similar precision (RSD 7%, n = 5). The TSP was applied to the determination of C8PC and lyso-PAF in spiked synthetic serum matrix using the standard addition method. The application of this methodology to PLD activity determination is also discussed.

Structure of choline oxidase in complex with the reaction product glycine betaine.

Choline oxidase from Arthrobacter globiformis, which is involved in the biosynthesis of glycine betaine from choline, has been extensively characterized in its mechanistic and structural properties. Despite the knowledge gained on the enzyme, the details of substrate access to the active site are not fully understood. The `loop-and-lid' mechanism described for the glucose-methanol-choline enzyme superfamily has not been confirmed for choline oxidase. Instead, a hydrophobic cluster on the solvent-accessible surface of the enzyme has been proposed by molecular dynamics to control substrate access to the active site. Here, the crystal structure of the enzyme was solved in complex with glycine betaine at pH 6.0 at 1.95 Šresolution, allowing a structural description of the ligand-enzyme interactions in the active site. This structure is the first of choline oxidase in complex with a physiologically relevant ligand. The protein structures with and without ligand are virtually identical, with the exception of a loop at the dimer interface, which assumes two distinct conformations. The different conformations of loop 250-255 define different accessibilities of the proposed active-site entrance delimited by the hydrophobic cluster on the other subunit of the dimer, suggesting a role in regulating substrate access to the active site.

Sensitive detection of acetylcholine based on a novel boronate intramolecular charge transfer fluorescence probe.

A highly sensitive and selective fluorescence method for the detection of acetylcholine (ACh) based on enzyme-generated hydrogen peroxide (H2O2) and a new boronate intramolecular charge transfer (ICT) fluorescence probe, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide (BN), was developed. This strategy involves the reaction of ACh with acetylcholinesterase (AChE) to produce choline, which is further oxidized by choline oxidase (ChOx) to obtain betaine and H2O2. The enzyme-generated H2O2 reacts with BN and results in hydrolytic deprotection of BN to generate fluorescent product (4-hydroxyl-N-butyl-1,8-naphthalimide, ON). Two consecutive linear response ranges allow determining ACh in a wide concentration range with a low detection limit of 2.7 nM (signal/noise=3). Compared with other fluorescent probes based on the mechanism of nonspecific oxidation, this reported boronate probe has the advantage of no interference from other biologically relevant reactive oxygen species (ROS) on the detection of ACh. This study provides a new method for the detection of ACh with high selectivity and sensitivity.

Enzyme engineering of choline oxidase for improving stability.

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.

The in-silico study of the structural changes in the Arthrobacter globiformis choline oxidase induced by high temperature.

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.

An Amperometric Acetylcholine Biosensor Based on Co-Immobilization of Enzyme Nanoparticles onto Nanocomposite.

An electrochemical biosensor was fabricated using nanoparticles of acetylcholinesterase (AChE) and choline oxidase (ChO)/Pt nanoparticles (PtNPs)/porous graphene oxide nanosheet (GONS) composite. A pencil graphite electrode (PGE) was used for the electrodeposition of nanocomposite and the determination of acetylcholine (ACh), a neurotransmitter. Various techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectra and cyclic voltammetry (CV) were used for characterization. This biosensor (AChENPs-ChONPs/GONS/PtNPs/PGE) indicated a very short response time (3 s), a lower limit of detection (0.001 µM), good linearity (0.001-200 µM), longer storage stability (6 months) and better reproducibility. The percent analytical recoveries of added acetylcholine in serum (5.0 and 10 µM) were found to be 97.6 ± 0.7 and 96.5 ± 0.3 for the present biosensor. The coefficients of variation were obtained to be 8% and 3.25%, correspondingly. The biosensor was applied to measure the ACh amount in the serum of healthy individuals and patients with Alzheimer's disease. The number of interferents had no effect on the biosensor at their physiological concentrations.

Simplifying the complexity: Single enzyme (choline oxidase) inhibition-based biosensor with dual-readout method for organophosphorus pesticide detection.

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 H2O2 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 H2O2 formed, and oxTMB could quench the fluorescence of Fe-CDs. In light of the fact that OPs exhibited activity in inhibiting ChOx, less H2O2 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.

Choline Oxidase-Integrated Copper Metal-Organic Frameworks as Cascade Nanozymes for One-Step Colorimetric Choline Detection.

Choline is an important factor for regulating human health and is widely present in various foods. In this work, a sensor strategy based on a choline oxidase-integrated copper(II) metal-organic framework with peroxidase-like activity is constructed for one-step cascade detection of choline. The one-step cascade strategy can avoid intermediate product transferring in general multi-step reactions, and the multi-enzyme activities can be well exerted under one condition, thus exhibiting excellent catalytic activity and enhanced stability. In the integrated system, choline is catalyzed by ChOx to produce betaine and H2O2, which eventually got converted to hydroxyl radicals by the peroxidase nanozyme, oxidized the chromogenic substrate ABTS, and produced an observable absorption peak at 420 nm. A new choline detection method was thus established and showed a satisfactory linear relationship at 6-300 µM, which has been used for the choline analysis in milk.

Production of recombinant choline oxidase and its application in betaine production.

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02960-z.

Freeform 3D Bioprinting Involving Ink Gelation by Cascade Reaction of Oxidase and Peroxidase: A Feasibility Study Using Hyaluronic Acid-Based Ink.

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.