Self-propelled enzyme-controlled IR-mesoporous silica Janus nanomotor for smart delivery.

Here, we report the preparation of a novel Janus nanoparticle with opposite Ir and mesoporous silica nanoparticles through a partial surface masking with toposelective modification method. This nanomaterial was employed to construct an enzyme-powered nanomachine with self-propulsion properties for on-command delivery. The cargo-loaded nanoparticle was provided with a pH-sensitive gate and unit control at the mesoporous face by first attaching boronic acid residues and further immobilization of glucose oxidase through reversible boronic acid esters with the carbohydrate residues of the glycoenzyme. Addition of glucose leads to the enzymatic production of H2O2 and gluconic acid, being the first compound catalytically decomposed at the Ir nanoparticle face producing O2 and causing the nanomachine propulsion. Gluconic acid leads to a pH reduction at the nanomachine microenvironment causing the disruption of the gating mechanism with the subsequent cargo release. This work demonstrates that enzyme-mediated self-propulsion improved release efficiency being this nanomotor successfully employed for the smart release of Doxorubicin in HeLa cancer cells.

Gelling properties of acid-induced tofu (soybean curd): Effects of acid type and nano-fish bone.

The effects of different types of acid coagulants and nano fish bone (NFB) additives on the characteristics of tofu were investigated using texture analyzers, SEM, FT-IR, and other techniques. The breaking force and penetration distance, in descending order, were found in the tofu induced by glucono-d-lactone (GDL) (180.27 g and 0.75 cm), citric acid (152.90 g and 0.74 cm), lactic acid (123.33 g and 0.73 cm), and acetic acid (69.84 g and 0.58 cm), respectively. The syneresis of these tofu samples was in the reverse order (35.00, 35.66, 39.66, and 44.50%). Lightness and whiteness were not significantly different among the different samples. Regardless of the acid type, the soluble calcium content in the soybean milk was significantly increased after adding NFB. As a result, the breaking force and penetration distance of all tofu samples increased significantly, but the syneresis decreased. Compared with tofu coagulated by other acids, GDL tofu formed a more uniform and dense gel network maintained by the highest intermolecular forces (especially hydrophobic interactions). Regarding the secondary structure, the lowest percentage of α-helix (22.72%) and, correspondingly, the highest ß-sheet (48.32%) and random coil (18.81%) were noticed in the GDL tofu. The effects of NFB on the tofu characteristics can be explained by the changes in the gel network, intermolecular forces, and secondary structure, which were in line with the acid type. The characteristics of acid-induced tofu can be most synergistically improved by coagulation with GDL and NFB.

Structural effects of oxidation on sugars: glucose as a precursor of gluconolactone and glucuronolactone.

Although structural information on sugars is wide, experimental studies on the oxidation products of sugars in the gas phase, free from solvent interactions, have been rarely reported. We present an experimental work on the changes in the structure and interactions of two products of glucose oxidation (D-glucono-1,5-lactone (GlcL) and D-glucurono-6,3-lactone (GlcurL)) with respect to their precursor. Features such as intramolecular interactions, ring puckering and tautomerism were observed.

Enabling High Activation of Glucose-6-Phosphate Dehydrogenase Activity Through Liquid Condensate Formation and Compression.

Droplet formation via liquid-liquid phase separation is thought to be involved in the regulation of various biological processes, including enzymatic reactions. We investigated a glycolytic enzymatic reaction, the conversion of glucose-6-phosphate to 6-phospho-D-glucono-1,5-lactone with concomitant reduction of NADP+ to NADPH both in the absence and presence of dynamically controlled liquid droplet formation. Here, the nucleotide serves as substrate as well as the scaffold required for the formation of liquid droplets. To further expand the process parameter space, temperature and pressure dependent measurements were performed. Incorporation of the reactants in the liquid droplet phase led to a boost in enzymatic activity, which was most pronounced at medium-high pressures. The crowded environment of the droplet phase induced a marked increase of the affinity of the enzyme and substrate. An increase in turnover number in the droplet phase at high pressure contributed to a further strong increase in catalytic efficiency. Enzyme systems that are dynamically coupled to liquid condensate formation may be the key to deciphering many biochemical reactions. Expanding the process parameter space by adjusting temperature and pressure conditions can be a means to further increase the efficiency of industrial enzyme utilization and help uncover regulatory mechanisms adopted by extremophiles.

Mechanism of the glucono-δ-lactone induced soymilk gelation: Enthalpy and entropy transformation in the cross-linking of protein molecules.

This study aimed to explore new techniques to regulate the quality of soy products. The glucono-δ-lactone (GDL) induced soymilk gelation process and the gel network structure characteristic were compared as a matter of temperature, and the role of reaction kinetics was discussed. Results showed that there were similarities in the development of G' curves under different temperatures, whereas the gel network structures and the energy requirements of cross-linking reactions were different. In the high-temperature region (70 °C-95 °C), the exposure and binding of reactive groups were promoted. The activation enthalpy (ΔH*) required by protein aggregates decreased and the effect of entropy reduction (-TΔS*) was enhanced, which led to shorten the preaggregation time (tg) and increase the gelation rate (k), resulting in the formation of rough, porous gel network with high stiffness. By contrast, in the low-temperature region (40 °C-70 °C), high enthalpy contributions and low entropy changes were required, then a fine, soft, and tender gel network formed. Besides, a funnel-shaped model of the enthalpy-entropy energy transformation mechanism of soymilk gelation was proposed. The results of this study revealed that adjusting the enthalpy-entropy energy requirements of the protein cross-linking reaction could be utilized to the regulation of the network structure and quality of soymilk gels, which could enrich the reaction kinetics theory and provide innovative ideas for food quality control technology from the perspective of energy requirement and energy input.

Design of glucono-δ-lactone-induced pinto bean protein isolate/κ-carrageenan mixed gels with various microstructures: fabrication, characterization, and release behavior.

BACKGROUND: Protein gels are used for different purposes, such as providing good texture, serving as fat replacers, and enhancing the nutritional and functional characteristics of foods. They can also deliver controlled release agents for sensitive drugs. The objective of this study was to investigate the impact of κ-carrageenan (kcr) concentration (0, 1.5, 3, and 4.5 mg g-1 ) on the morphological and physicochemical properties and release behavior of glucono-δ-lactone (GDL)-induced pinto bean protein aggregate (PBA) gels. RESULTS: When κ-carrageenan concentration increased from 0 to 1.5 and 3 mg.g-1 , the firmness of the samples increased significantly, by 2.04 and 3.7 fold, respectively (P < 0.05). A compact and homogenous network with considerable strength and maximum water-holding capacity (97.52 ± 1.17%) was obtained with the addition of 3 mg g-1 κ-carrageenan to the gel system. Further increasing the κ-carrageenan concentration to 4.5 mg g-1 produced a coarse gel structure with higher storage modulus (G'), firmness (6.30-fold), thermal stability, and entrapment efficiency (85.6%). Depending on the κ-carrageenan concentration, various microstructures from protein continuous phase to κ-carrageenan continuous phase were observed. The release test indicated that 70.25% of the loaded curcumin was released in the simulated gastrointestinal tract for pure PBA gels. In contrast, for binary gels containing 4.5 mg g-1 κ-carrageenan, curcumin was protected in the upper gastrointestinal tract, and 64.45% of loaded curcumin was delivered to the colon. CONCLUSION: Our study showed that κ-carrageenan/PBA gels had high entrapment efficiency and could protect curcumin in the upper gastrointestinal tract. The hydrogels are therefore very valuable for colon-targeting delivery purposes. © 2022 Society of Chemical Industry.

Characterization of 3 phylogenetically distinct membrane-bound d-gluconate dehydrogenases of Gluconobacter spp. and their biotechnological application for efficient 2-keto-d-gluconate production.

We identified a novel flavoprotein-cytochrome c complex d-gluconate dehydrogenase (GADH) encoded by gndXYZ of Gluconobacter oxydans NBRC 3293, which is phylogenetically distinct from previously reported GADHs encoded by gndFGH and gndSLC of Gluconobacter spp. To analyze the biochemical properties of respective GADHs, Gluconobacter japonicus NBRC 3271 mutant strain lacking membranous d-gluconate dehydrogenase activity was constructed. All GADHs (GndFGH, GndSLC, and GndXYZ) were successfully overexpressed in the constructed strain. The optimal pH and KM value at that pH of GndFGH, GndSLC, and GndXYZ were 5, 6, and 4, and 8.82 ± 1.15, 22.9 ± 5.0, and 11.3 ± 1.5 m m, respectively. When the mutants overexpressing respective GADHs were cultured in d-glucose-containing medium, all of them produced 2-keto-d-gluconate, revealing that GndXYZ converts d-gluconate to 2-keto-d-gluconate as well as other GADHs. Among the recombinants, the gndXYZ-overexpressing strain accumulated the highest level of 2-keto-d-gluconate, suggesting its potential for 2-keto-d-gluconate production.

Xylonolactonase from Caulobacter crescentus Is a Mononuclear Nonheme Iron Hydrolase.

Caulobacter crescentus xylonolactonase (Cc XylC, EC 3.1.1.68) catalyzes an intramolecular ester bond hydrolysis over a nonenzymatic acid/base catalysis. Cc XylC is a member of the SMP30 protein family, whose members have previously been reported to be active in the presence of bivalent metal ions, such as Ca2+, Zn2+, and Mg2+. By native mass spectrometry, we studied the binding of several bivalent metal ions to Cc XylC and observed that it binds only one of them, namely, the Fe2+ cation, specifically and with a high affinity (Kd = 0.5 µM), pointing out that Cc XylC is a mononuclear iron protein. We propose that bivalent metal cations also promote the reaction nonenzymatically by stabilizing a short-lived bicyclic intermediate on the lactone isomerization reaction. An analysis of the reaction kinetics showed that Cc XylC complexed with Fe2+ can speed up the hydrolysis of d-xylono-1,4-lactone by 100-fold and that of d-glucono-1,5-lactone by 10-fold as compared to the nonenzymatic reaction. To our knowledge, this is the first discovery of a nonheme mononuclear iron-binding enzyme that catalyzes an ester bond hydrolysis reaction.

Development of a pulse-induced electrochemical biosensor based on gluconamide for Gram-negative bacteria detection.

Pathogenic bacteria can cause the outbreaks of disease and threaten human health, which stimulates the development of advanced detection techniques. Herein, a specific and sensitive electrochemical biosensor for Gram-negative bacteria was established based on the conductive polymer with artificial muscle properties.  The effective recognition was achieved through the specific carbohydrate-carbohydrate interaction between gluconamide and lipopolysaccharide.  The application of impulse voltage enhances the efficiency of recognition and shortens the detection time through the temporary deformation of the electrode surface, with a limit of detection (LOD)  of 1 × 100 CFU/mL and a linear range of 1 × 100 - 1 × 106 CFU/mL for Escherichia coli (E. coli). In addition  to the merits of low cost, high efficiency, and rapidity,  the developed label-free electrochemical biosensor can also be applicable for other Gram-negative bacteria, owning promising potential in the application of portable devices and paving a potential way for the construction of electrochemical biosensors.

N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone.

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.

Modulation of the clinically accessible gelation time using glucono-d-lactone and pyridoxal 5'-phosphate for long-acting alginate in situ forming gel injectable.

The purpose of this study was to design alginate in situ forming gel (ISFG) injectable with clinically acceptable gelation time and controlled release of hydrophobic drug. Milled or unmilled paliperidone palmitate (PPP) was used. The gelation time was controlled by varying the ratios of glucono-d-lactone (GDL) and pyridoxal 5'-phosphate (PLP) in prefilled alginate solution mixtures (ASMs) containing PPP, CaCO3, GDL and PLP for clinically acceptable injectability. However, the gelation time was varied by the alginate type (M/G ratio), storage condition, and drug solubilizers. This ISFG exhibited 32.15 kPa of the maximal compressive stress without causing pain and stiffness. The ISFG containing conically milled PPP released PPP in a controlled manner without exhibiting any initial burst release for 4 weeks. The current alginate ISFG injectable using new combination of PLP and GDL could be used to deliver long-acting injectable drugs.

Differences in the physicochemical, digestion and microstructural characteristics of soy protein gel acidified with lactic acid bacteria, glucono-δ-lactone and organic acid.

This study evaluated the differences in the physicochemical, digestion and microstructure of soy protein gels acidified with Lactobacillus casei (L. casei), glucono-δ-lactone (GDL) and citric acid. The maximum acidification rate was as follows: citric acid > GDL > L. casei. The gelation points of L. casei-induced gel (LC gel) and GDL-induced gel (GDL gel) occurred at 74 min and 55 min; however, gelation point of citric acid-induced gel (CA gel) was not detected because acidification was too fast. LC gel showed the high gel hardness (20.40 ± 2.23 g) and water holding capacity (84.58 ± 0.59%). At the end of intestinal digestion, the average particle size of the LC gel was the largest, but there was no significant difference between GDL gel and CA gel. The microstructure of the GDL gel was found to be the densest. Acidification rate was the "key step" of acid-induced gels, while both the proteolytic and exopolysaccharide (EPS) production capacity were involved in LC gel.

Enzymatic Management of pH in White Wines.

This paper investigates the potential of the enzymatic management of high pH in white juice and wine using a combination of enzymes-glucose oxidase coupled with catalase. Catazyme® 25 L, a commercially available blend of the two enzymes, was added at different doses (0.2 g/L, 0.6 g/L, and 1g/L) to white grape juice and various parameters (glucose, gluconic acid, pH) were monitored over 24 h of treatment. Treated wines were fermented to dryness without any difficulty and the wines were chemically and sensorially evaluated. At the highest dose (1 g/L), pH was reduced from 3.9 to 3.2, with 20.5 g of gluconic acid produced, while at the lowest dose (0.2 g/L), pH decreased from 4.0 to 3.5 and 8.8 g of gluconic acid was produced. Flash profiling indicated that treated wines were lighter in color than the control and were described using terms such as floral, fruit, citrus, and sour while the control wine was described as being fermented, medicinal, pungent, and oxidized. In conclusion, glucose oxidase coupled with catalase was shown to be effective at significantly reducing juice and wine pH in a short amount of time and with a positive impact on the organoleptic profiles of the treated wines.

Cloning, purification, and characterization of the 6-phosphogluconate dehydrogenase (6 PGDH) from Giardia lamblia.

Giardia lamblia, due to the habitat in which it develops, requires a continuous supply of intermediate compounds that allow it to survive in the host. The pentose phosphate pathway (PPP) provides essential molecules such as NADPH and ribulose-5-phosphate during the oxidative phase of the pathway. One of the key enzymes during this stage is 6-phosphogluconate dehydrogenase (6 PGDH) for generating NADPH. Given the relevance of the enzyme, in the present work, the 6pgdh gene from G. lamblia was amplified and cloned to produce the recombinant protein (Gl-6 PGDH) and characterize it functionally and structurally after the purification of Gl-6 PGDH by affinity chromatography. The results of the characterization showed that the protein has a molecular mass of 54 kDa, with an optimal pH of 7.0 and a temperature of 36-42 °C. The kinetic parameters of Gl-6 PGDH were Km = 49.2 and 139.9 µM (for NADP+ and 6-PG, respectively), Vmax =26.27 µmol*min-1*mg-1, and Kcat = 24.0 s-1. Finally, computational modeling studies were performed to obtain a structural visualization of the Gl-6 PGDH protein. The generation of the model and the characterization assays will allow us to expand our knowledge for future studies of the function of the protein in the metabolism of the parasite.

Deep oxidization of glucose driven by 4-acetamido-TEMPO for a glucose fuel cell at room temperature.

Exploiting suitable oxidation catalysts is of great importance in the development of sugar-based fuel cells (SFCs). Herein, a novel room-temperature glucose/O2 fuel cell (GFC), which employs 4-acetamido-2,2,6,6-tetramethylpiperidin-1-oxyl (ACT) as an anodic electrocatalyst and air-breathing Pt-C as a cathode, is demonstrated. Under room temperature operation, the as-assembled GFCs are capable of delivering a maximum power density of 100 µW cm-2 in the presence of 50 mM glucose. Bulk electrolysis products of glucose identified by mass spectrum and Fourier transform infrared spectroscopy include gluconic acid and glucaric acid, suggesting that the aldehyde and primary hydroxy groups of glucose can be deeply oxidized into carboxyl groups through a 6e- pathway. The deep glucose oxidation capability makes ACT a promising anodic electrocatalyst for SFCs.

Synthesis, DNA binding, antibacterial and anticancer properties of two novel water-soluble copper(II) complexes containing gluconate.

In this paper, two new Cu(II) complexes, [Cu(Gluc)(HPB)(H2O)]Gluc (CuG1) and [Cu(Gluc)(HPBC)(H2O)]Gluc (CuG2) (where HPB = 2-(2'-pyridyl)benzimidazole, HPBC = 5-chloro-2-(2'-pyridyl)benzimidazole, Gluc = d-Gluconic acid), with good water solubility were synthesized and characterized. These complexes exhibited a five-coordinated tetragonal pyramidal geometry. The DNA binding and cleavage properties of the complexes were investigated using multi-spectroscopy, viscosity measurement, molecular docking and gel electrophoresis analysis methods. The results showed that the complexes could interact with DNA by insertion and groove binding, and cleave CT-DNA through a singlet oxygen-dependent pathway in the presence of ascorbic acid. The studies on antibacterial and anticancer activities in vitro demonstrated that both complexes had good inhibitory activity against three Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes) and one Gram-negative bacterium (Escherichia coli) and good cytotoxic activity toward the tested cancer cells (A549, HeLa and SGC-7901). CuG2 showed higher antimicrobial and cytotoxic activities than CuG1, which was consistent with their binding strength and cleavage ability to DNA, indicating that their antimicrobial and cytotoxic activities may be related to the DNA interaction. Moreover, the cell-based mechanism studies have indicated that CuG1 and CuG2 could arrest the cell cycle at G2/M phase, elevate the levels of intracellular reactive oxygen species (ROS) and decrease the mitochondrial membrane potential (MMP). The results showed that the complexes could induce apoptosis through DNA-damaged and ROS-mediated mitochondrial dysfunction pathways. Finally, the in vivo antitumor study revealed that CuG2 inhibited tumor growth by 50.44%, which is better than that of cisplatin (40.94%).

Colorimetric recognition of aromatic amino acid enantiomers by gluconic acid-capped gold nanoparticles.

In this work, we prepared gold nanoparticles (AuNPs) by employing gluconic acid (GlcA) as reducing-cum-stabilizing agent. The proposed GlcA-AuNPs successfully worked as a colorimetric sensor for visual chiral recognition of aromatic amino acid enantiomers, namely tyrosine (D/L-Tyr), phenylalanine (D/L-Phe), and tryptophan (D/L-Trp). After adding L-types to GlcA-AuNPs solution, the color of the mixture changed from red to purple (or gray), while no obvious color change occurred on the addition of D-types. The effect can be detected by naked eyes. The particles have been characterized by transmission electron microscopy, Fourier-transform infrared spectroscopy, zeta potential, the dynamic light scattering analysis as well as UV-Vis spectroscopy. This assay can be used to determine the enantiomeric excess of L-Trp in the range from 0 to + 100%. The method has advantages in simplicity, sensitivity, fast response, and low cost.

Concise Stereoselective Synthesis of ß-Hydroxy-γ-lactones: (4R,5R)-4-Hydroxy-γ-decalactone from the Japanese Orange Fly and Enantiomers of Arachnid Harvestmen Isolates.

The naturally occurring (4R,5R)-4-hydroxy-γ-decalactone from the Japanese orange fly and the antipode of (4S,5R)-4-hydroxy-γ-dodecalactone from the harvestmen arachnid and their stereoisomers are synthesized from the chiral pool material d-glucono-δ-lactone in a few steps. The one-pot conversion of the latter to γ-vinyl-ß-hydroxy-γ-lactone, cross-metathesis with requisite olefin, and hydrogenation enabled the synthesis of syn-lactones in just a two-pot operation. An additional efficient Pd-catalyzed allylic isomerization of γ-vinyl-ß-hydroxy-γ-lactone led to the anti-lactones in high yields.

Calcium, magnesium and aluminium ions as decontaminating agents against dermal fluoride absorption following hydrofluoric acid exposure.

The fluoride ions of the industrially largely irreplaceable, locally corrosive hydrofluoric acid (HF) can scavenge cations in biological tissues, which explains their high toxic potential, and also leads to local acidification through proton release. The influence of three complexing agents, calcium (Ca2+) gluconate (as 2.5% Ca2+gel and individually (2.84%) or commercially (10%) formulated Ca2+solution), magnesium (Mg2+) gluconate (2.84%) solution and aluminium (Al3+) solution (Hexafluorine®, pure and diluted) on the absorption of fluoride following HF exposure (1-3 min, 100 µl, 30%/0.64 cm2) through human skin was investigated in an ex-vivo diffusion cell model. Fluoride absorption was assessed over 6-24 h and analysed with a fluoride electrode. Decreasing the contamination time reduced the fluoride absorption distinctly which was further reduced by the application of fluoride-binding decontamination agents (Ca2+, Mg2+, Al3+) or water alone without being significantly different. Ca2+ appeared slightly more effective than Mg2+ in reducing fluoride absorption. Moreover, the addition of pH adjusting buffer promoted the decontamination efficacy. Fluoride-binding agents can facilitate the decontamination of dermal HF exposure. However, prompt decontamination appeared to be the key to successful limitation of fluoride absorption and pushes the choice of decontamination agent almost into the background.

The Usefulness of X-ray Diffraction and Thermal Analysis to Study Dietary Supplements Containing Iron.

X-ray powder diffraction (XRPD) and thermal analysis (differential scanning calorimetry/derivative of thermogravimetry (DSC/DTG)) are solid-state techniques that can be successfully used to identify and quantify various chemical compounds in polycrystalline mixtures, such as dietary supplements or drugs. In this work, 31 dietary supplements available on the Polish market that contain iron compounds, namely iron gluconate, fumarate, bisglycinate, citrate and pyrophosphate, were evaluated. The aim of the work was to identify iron compounds declared by the manufacturer as food supplements and to try to verify compliance with the manufacturer's claims. Studies performed by X-ray and thermal analysis confirmed that crystalline iron compounds (iron (II) gluconate, iron (II) fumarate), declared by the manufacturers, were present in the investigated dietary supplements. Iron (II) bisglycinate proved to be semi-crystalline. However, depending on the composition of the formulation, it was possible to identify this compound in the tested supplements. For amorphous iron compounds (iron (III) citrate and iron (III) pyrophosphate), the diffraction pattern does not have characteristic diffraction lines. Food supplements containing crystalline iron compounds have a melting point close to the melting point of pure iron compounds. The presence of excipients was found to affect the shapes and positions of the endothermic peaks significantly. Widening of endothermic peaks and changes in their position were observed, as well as exothermic peaks indicating crystallization of amorphous compounds. Weight loss was determined for all dietary supplements tested. Analysis of the DTG curves showed that the thermal decomposition of most food supplements takes place in several steps. The results obtained by a combination of both simple, relatively fast and reliable XRPD and DSC/DTG methods are helpful in determining phase composition, pharmaceutical abnormalities or by detecting the presence of the correct polymorphic form.