Enzyme Reaction Mixture Research Articles

Production Physiology and Biochemical properties of Crude Thermophile Protease by Mild - Halophilic Bacillus thuringiensis aizawai HD 865

Objective: The current study aims to enrich the research library of Bacillus thuringensis, the most famous bacteria as a biopesticide, with a product that may enhance its ability as a biopesticide as well as its many other applications, namely the heat-tolerant alkaline protease. Methods: The strain Bacillus thuringiensis aizawai HD 865, obtained from HD Culture Collection was grown on a liquid growth medium of nutrient broth and yeast extract, and colonies were counted on agar plates. The zygote is then transferred to a skim milk environment to estimate the enzymatic activity under the different growth conditions of the growth environment (sodium chloride concentration, pH, aeration, fermentation duration), then testing the optimal conditions for enzymatic activity in the enzymatic reaction mixture (sodium chloride concentration, salinity resistance, optimum temperature, thermal stability, optimum pH and stability). Then analyze the data statistically. Results: Optimum alkaline protease medium production conditions were, 2 % NaCl, so Bacillus thuringiensis aizawai HD865 a mild - halophilic microorganism, aeration level 80%, pH 7 and 8 days of fermentation period. On the other hand, crude alkaline protease properties were found that, the optimum temperature for its activity was relatively high (70ºC), while heat-stabilities after 5 min of heating crude enzyme in boiling water bath then incubated for 15min on 30, 40, 50, 60, 70ºC, were 184.23, 178.07, 164.29, 165.51 and 121.46 Units, respectively; whereas the optimum pH was at pH 7.6 (151.76 Units) with tris buffer then at pH 9.6 with glycine - NaOH buffer; While pH stability was at pH 7.6 with tris buffer then at pH 8.4 with glycine - NaOH buffer. Thus, activities have decreased at different concentrations (0, 1, 2, 3, 4, 5 %) of sodium chloride in reaction mixture with enzyme activities (151.25, 137.35, 135.12, 135, 135.89, 114.38 Units) respectively, also decreased when rinse crude enzyme for 5 min in NaCl (0, 1, 2, 3, 4, 5 %), with (151.04, 175.57, 168.69, 154.61, 155.39 and 143.39 Units) respectively. Conclusion: Bacillus thuringiensis aizawai HD 865 strain is considered a low - salt tolerant strain, while the alkaline protease extracted from it is heat - resistant.

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

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

Numerical modeling of metal-dielectric metasurface as an element of microwave sensors for biomedical applications

This paper reports the results of numerical modeling of wave reflection coefficient behavior of the metal-dielectric metasurface at microwaves that can be used for biomedical applications. The study includes optimization of the working parameters of the metasurface-based structure as a sensitive element of a microwave sensoring systems for determination of proteins concentration in different solutions. In the current research a unit with the geometry, which is similar to the geometry of one well of the standard 96-multiwell laboratory microplate, is used as a resonant metasurface unit cell and liquid-holding structure. Human serum albumin (HSA) is used as a protein specimen in our model study. The present numerical modeling is based on the results of our previous experimental measurements of complex permittivity values of HSA water solutions and its biochemical reaction mixtures using microwave dielectrometry method and the developed setup. The unit cell approach calculations are performed by COMSOL Multiphysics software. The optimization of working parameters of the metal-dielectric metasurface structure with tested solutions allow us to observe the resonance effects of the wave reflection coefficient in the microwave range. We can determine the HSA concentration changes in water solutions and enzymatic reaction mixtures by the resonance frequency shift of the wave reflection coefficient of the metal-dielectric metasurface. Developed metal-dielectric metasurface-based structure demonstrates prospects to be used as a sensitive element of microwave sensors for proteins concentration determination with biomedical purposes.

Microwell fluoride assay screening for enzymatic defluorination.

There is intense interest in removing fluorinated compounds from the environment, environments are most efficiently remediated by microbial enzymes, and defluorinating enzymes are readily monitored by fluoride determination. Fluorine is the most electronegative element. Consequently, all mechanisms of enzymatic C-F bond cleavage produce fluoride anion, F-. Therefore, methods for the determination of fluoride are critical for C-F enzymology and apply to any fluorinated organic compounds, including PFAS, or per- and polyfluorinated alkyl substances. The biodegradation of most PFAS chemicals is rare or unknown. Accordingly, identifying new enzymes, or re-engineering the known defluorinases, will require rapid and sensitive methods for measuring fluoride in aqueous media. Most studies currently use ion chromatography or fluoride specific electrodes which are relatively sensitive but low throughput. The methods here describe refashioning a drinking water test to efficiently determine fluoride in enzyme and cell culture reaction mixtures. The method is based on lanthanum alizarin complexone binding of fluoride. Reworking the method to a microtiter well plate format allows detection of as little as 4nmol of fluoride in 200μL of assay buffer. The method is amenable to color imaging, spectrophotometric plate reading and automated liquid handling to expedite assays with thousands of enzymes and/or substrates for discovering and improving enzymatic defluorination.

A Facile Fluorometric Assay of Orotate Phosphoribosyltransferase Activity Using a Selective Fluorogenic Reaction for Orotic Acid

Orotate phosphoribosyltransferase (OPRT) exists as a bifunctional enzyme, uridine 5'-monophosphate synthase, in mammalian cells and plays an important role in pyrimidine biosynthesis. Measuring OPRT activity has been considered important for understanding biological events and development of molecular-targeting drugs. In this study, we demonstrate a novel fluorescence method for measuring OPRT activity in living cells. The technique utilizes 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent, which produces selective fluorescence for orotic acid. To perform the OPRT reaction, orotic acid was added to HeLa cell lysate, and a portion of the enzyme reaction mixture was heated at 80 °C for 4 min in the presence of 4-TFMBAO under basic conditions. The resulting fluorescence was measured using a spectrofluorometer, which reflects the consumption of orotic acid by the OPRT. After optimization of the reaction conditions, the OPRT activity was successfully determined in 15 min of enzyme reaction time without further procedures such as purification of OPRT or deproteination for the analysis. The activity obtained was compatible with the value measured by the radiometric method with [3H]-5-FU as the substrate. The present method provides a reliable and facile measurement of OPRT activity and could be useful for a variety of research fields targeting pyrimidine metabolism.

Isoquinoline Alkaloid Contents in Macleaya cordata Extracts and Their Acetylcholinesterase and Butyrylcholinesterase Inhibition.

An important strategy for treating neurodegenerative disorders is to maintain the levels of acetylcholine in the synaptic cleft by blocking the cholinesterases. Searching for new effective compounds with inhibited acetylcholinesterase and butyrylcholinesterase activity is one of the most significant challenges of the modern scientific research. The aim of this study was the optimization of the condition for cholinesterase activity determination by high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) in terms of concentrations of enzymatic reaction mixture components, temperature of incubation, and incubation time. In vitro investigation of acetylcholinesterase and butyrylcholinesterase activity inhibition by some isoquinoline alkaloids and extracts obtained from the aerial part and roots of Macleaya cordata collected in May, July, and September. Acetylcholinesterase and butyrylcholinesterase activity inhibition of the extracts obtained from the plant had not been tested previously. The application of the HPLC method allowed eliminating absorption of interfering components, for example, alkaloids such as sanguinarine and berberine. The HPLC method was successfully applied for the evaluation of the acetylcholinesterase inhibitory activity in samples such as plant extracts, especially those containing colored components adsorbing at the same wavelength as the adsorption wavelength of 5-thio-2-nitro-benzoic acid, which is the product of the reaction between thiocholine (product of the hydrolysis of acetyl/butyrylthiocholine reaction) with Ellman’s reagent. Moreover, liquid chromatography coupled with a triple quadrupole mass spectrometer (LC–QqQ–ESI–MS/MS) analysis allowed evaluating the identification of relevant bioactive compounds in the obtained plant extracts. The investigated alkaloids, especially sanguinarine and chelerythrine, and all the Macleaya cordata extracts, especially the extract obtained from the aerial part collected in May, exhibited very high cholinesterase activity inhibition. HPLC-DAD was also applied for the kinetics study of the most active alkaloids sanguinarine and chelerythrine. Our investigations demonstrated that these plant extracts can be recommended for further in vivo experiments to confirm their cholinesterase inhibition activity.

Sulfated Phenolic Substances: Preparation and Optimized HPLC Analysis.

Sulfation is an important reaction in nature, and sulfated phenolic compounds are of interest as standards of mammalian phase II metabolites or pro-drugs. Such standards can be prepared using chemoenzymatic methods with aryl sulfotransferases. The aim of the present work was to obtain a large library of sulfated phenols, phenolic acids, flavonoids, and flavonolignans and optimize their HPLC (high performance liquid chromatography) analysis. Four new sulfates of 2,3,4-trihydroxybenzoic acid, catechol, 4-methylcatechol, and phloroglucinol were prepared and fully characterized using MS (mass spectrometry), 1H, and 13C NMR. The separation was investigated using HPLC with PDA (photodiode-array) detection and a total of 38 standards of phenolics and their sulfates. Different stationary (monolithic C18, C18 Polar, pentafluorophenyl, ZICpHILIC) and mobile phases with or without ammonium acetate buffer were compared. The separation results were strongly dependent on the pH and buffer capacity of the mobile phase. The developed robust HPLC method is suitable for the separation of enzymatic sulfation reaction mixtures of flavonoids, flavonolignans, 2,3-dehydroflavonolignans, phenolic acids, and phenols with PDA detection. Moreover, the method is directly applicable in conjunction with mass detection due to the low flow rate and the absence of phosphate buffer and/or ion-pairing reagents in the mobile phase.

An effect of choline lactate based low transition temperature mixtures on the lipase catalytic properties

A new series of low transition temperature mixures (LTTM) based on choline lactate quaternary ammonium salt and various hydrogen bond donors was prepared and characterized towards their physicochemical properties and usability as an enzymatic reaction mixture for lipase-catalyzed transesterification reactions. Studies of low transition temperature mixtures have shown a long-term stabilizing effect for lipase as well as a positive influence on lipase thermal stability. In the case of Ch[Lac]:Gly: EthGly increasing the stability of lipase by 8 °C (up to 55.2 °C) compared to the control sample. Conducted transesterification reactions were characterized by high yields – up to 98% – and high purity of the obtained products.

Bioremoval and Detoxification of Anthracene by a Halophilic Laccase from Alkalibacillus salilacus.

Polycyclic Aromatic Hydrocarbons (PAHs) as resistant compounds in the environment has received much attention in recent years due to their adverse effects on ecological health. Among the various methods studying the removal of PAHs, enzyme biotechnology is one of the most effective and appropriate method. In the present study, a halophilic laccase was used for bioremoval of anthracene in the presence of 1-Hydroxybenzotriazole (HBT). Halophilic laccase from Alkalibacillus salilacus was tested for anthracene degradation. Residual concentration of anthracene at various concentrations of NaCl (0‒4 M), incubation time, pH, solvent, and surfactants in the enzymatic reaction mixtures was determined by HPLC. The maximum removal of substrate was achieved after 72 h at 40 °C, pH 8, and NaCl concentration 1.5 M. Besides, the addition of 1% (v/v) ionic and non-ionic surfactants and 25% (v/v) of various organic solvents increased removal efficiency. The kinetic parameters Km and Vmax of the laccase for removing of anthracene were 0.114 μM and 0.546 μmoL. h.-1 mg-1, respectively. Laccase showed the maximum removal efficiency of anthracene in the presence of 1-Hydroxybenzotriazole (HBT).

The Effect of Single-Walled Carbon Nanotubes on UDP-Glucuronosyltransferase 1A Activity in Human Liver

Single-walled carbon nanotubes (SWCNTs) are made from rolled single graphene sheets with a diameter in the nanometer range and are potential carriers for drug delivery systems. However, their effects on uridine 5'-diphosphate-glucuronosyltransferase (UGT) 1A activities remain unclear. The present study aimed to investigate the effect of two kinds of SWCNTs (EC1.5-P- and FH-P-SWCNTs) and other nanocarbons on human UGT1A activity due to the proposed application of SWCNTs in drug and gene delivery. β-Estradiol 3-glucuronidation, which is catalyzed mainly by UGT1A1, was inhibited by 99 and 76% in the presence of 0.1 mg/mL EC1.5-P- and FH-P-SWCNTs in human liver microsomes, respectively. The observed decrease of free UGT1A1 protein in the enzyme reaction mixture suggests a higher interaction with SWCNTs, and indicates the inhibition of β-estradiol 3-glucuronidation. Imipramine N-glucuronidation, which is formed mainly by UGT1A4, was also decreased by SWCNTs. Serotonin glucuronidation, which is mainly responsible for UGT1A6, was only influenced by specific nanocarbons in human liver microsomes. The attenuation of free UGT1A6 protein was observed with SWCNTs and carbon black, indicating that UGT1A6 activity was not influenced by the direct interaction of SWCNTs. We also observed a 127% increase by FH-P-SWCNTs for propofol glucuronidation in human liver microsomes, which is catalyzed mainly by UGT1A9. The values of maximum velocity and intrinsic clearance for propofol glucuronidation in the presence of FH-P-SWCNT were 1.8- and 2.0-fold higher than those of the control in human liver microsomes. These results suggest that the effects of SWCNTs on UGT1A are different among isoforms.

Glycogen debranching pathway deduced from substrate specificity of glycogen debranching enzyme

Glycogen debranching enzyme (GDE) is bifunctional in that it exhibits both 4-α-glucanotransferase and amylo-α-1,6-glucosidase activity at two distinct catalytic sites. GDE converts the phosphorylase-limit biantennary branch [G-G-G-G-(G-G-G-G↔)G-G- residue, where G = D-glucose, hyphens represent α-1,4-glycosidic bonds, and the double-headed arrow represents an α-1,6-glycosidic bond] into a linear maltooligosyl residue, which is then subjected to phosphorylase, and glycogen degradation continues. The prevailing hypothesis regarding the glycogen debranching pathway was that 4-α-glucanotransferase converts the phosphorylase-limit biantennary branch into the G-G-G-G-G-G-G-(G↔)G-G- residue and amylo-α-1,6-glucosidase cleaves the remaining α-1,6-linked G residue. In the present study, we analyzed the substrate specificities of 4-α-glucanotransferase and amylo-α-1,6-glucosidase using fluorogenic biantennary dextrins such as G-G-G-G-(G-G-G-G↔)G-G-GPA (F4/4/2; where GPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol), G-(G-G-G-G↔)G-G-GPA (F1/4/2), and G-G-G-G-G-G-G-(G↔)G-G-GPA (F7/1/2). Contrary to the prevailing hypothesis, the main branch of F4/4/2 was an important donor substrate component of 4-α-glucanotransferase and did not serve as an acceptor substrate. However, when G-G-G-G-G-GPA was added to the mixture, it successfully accepted a maltotriosyl (G3-) residue from F4/4/2. In addition, amylo-α-1,6-glucosidase exhibited strong activity towards G-G-G-G-(G↔)G-G-GPA but weak activity towards F7/1/2. Furthermore, the debranching activity of GDE towards phosphorylase-limit glycogen substantially increased when methyl α-maltooligosides with lengths equal to or greater than that of methyl α-maltopentaoside (G5-OCH3) were added to the enzyme reaction mixture. Based on these results, we propose the following macroscopic debranching pathway: Via 4-α-glucanotransferase, the G3- residue of the donor branch is transferred to a long (n ≥ 5) linear Gn- residue linked to a different branching G residue.

Acceleration of lactose hydrolysis using beta-galactosidase and deep eutectic solvents

A series of new deep eutectic solvents based on choline levulinate and various hydrogen bond donors were prepared and characterized by1H NMR, FT-IR, TG, and DSC. In particular, their physicochemical properties (density, viscosity, conductivity, and thermal stability) were determined and their usability as an enhancing additive to the enzymatic reaction mixture, for the enzyme was checked. It has been shown, that prepared DES, exhibits low viscosity (at 40 °C within the range 0.1–0.8 Pa·s), high thermal stability (in almost all cases above 150 °C), and density within the range 1.1–1.17 g cm−3. Also, it has been shown, that obtained mixtures can stabilize the enzymes, and positively influence on its activity. The addition of up to 15% (v/v) of DES mixture composed of choline levulinate: ethylene glycol, enhanced more than threefold lactose hydrolysis yield by β-galactosidase. The present study shows the relevance of the newly designed DES series for improving enzymes properties with the potential to apply in the effective conversion of food processing origin substrates.

2D and 3D maximum-quantum NMR and diffusion spectroscopy for the characterization of enzymatic reaction mixtures.

1D 1H NMR spectroscopy has been widely used to monitor enzymatic activity by recording the evolution of the spectra of substrates and/or products, thanks to the linear response of NMR. For complex systems involving the coexistence of multiple compounds (substrate, final product and various intermediates), the identification and quantification can be a more arduous task. Here, we present a simple analytical method for the rapid characterization of reaction mixtures involving enzymatic complexes using Maximum Quantum (MaxQ) NMR, accelerated with the Non-Uniform Sampling (NUS) acquisition procedure. Specifically, this approach enables, in the first analytical step, the counting of the molecules present in the samples. We also show, using two different enzymatic systems, that the implementation of these pulse sequences implies precautions related to the short relaxation times due to the presence of metallo-enzymes or paramagnetic catalysts. Finally, the combination of MaxQ and diffusion experiments, which leads to a 3D chart, greatly improves the resolution and offers an extreme simplification of the spectra while giving valuable indications on the affinity of the enzymes to the different compounds present in the reaction mixture.

A direct spectropolarimetric assay of arabinose 5-phosphate isomerase

Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M−1cm−1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1–3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API.

Development of Sars-Cov-2 Isothermal Amplification Detection Kits

The SARS-CoV-2 isothermal amplification detection kits based on loop-mediated isothermal amplification (LAMP) were developed and evaluated on three types samples of SARS-CoV-2 The kits included enzyme reaction mixtures and chromogenic agents After the isothermal amplification reactions were completed, the reaction results were judged by using the chromogenic agents to determine whether SARS-CoV-2 exists in the samples to be tested The detection kits have the advantages of convenient operation, fast detection speed and high sensitivity up to 1 copy of virus particles per reaction, which can speed up the detection speed of suspected cases, and avoid the missing detection problems caused by the low detection sensitivity

Screening acetylcholinesterase inhibitors from traditional Chinese medicines by paper-immobilized enzyme combined with capillary electrophoresis analysis

Discovering acetylcholinesterase (AChE) inhibitors is one of the important ways to develop new drugs for the treatment of Alzheimer's disease. In this work, a simple strategy was developed for screening AChE inhibitors from traditional Chinese medicines (TCMs) by capillary electrophoresis (CE) analysis combined with enzymatic assay, in which immobilized AChE was employed. For AChE immobilization, cellulose filter paper (CFP) was used as the carrier material and physically coated with chitosan owing to moderate viscosity of chitosan to be introduced into amino groups, and then AChE was covalently bonded to the amino-functionalized CFP through a Schiff base reaction using glutaraldehyde (GA) as a cross-linking agent. The CFP-immobilized AChE exhibited enhanced endurance to pH and temperature, improved storage stability, excellent repeatability and reusability. More remarkably, CFP-immobilized AChE can be instantly separated from enzyme reaction mixture thus greatly simplifying the operational process. For immobilized AChE, the Michaelis-Menten constant, inhibition constant and IC50 were determined using huperzine A as a model inhibitor. Finally, CFP-immobilized AChE was applied to inhibitor screening from 17 TCMs, among which Chebulae Fructus (ripe fruits of Terminalia chebula) exhibited the strongest inhibitory effect on AChE. The positive results indicated that such a screening strategy may open up a new avenue to discover active components from TCMs.

Enzymatic hydrolysis of cellulose recovered from ionic liquid-salt aqueous two-phase system

Regenerated cellulose can be prepared by treatment with an ionic liquid (IL) and an anti-solvent such as water, which significantly enhances the enzymatic hydrolysis in comparison to crystalline cellulose. The IL-aqueous two-phase system (IL-ATPS) is consisted of IL-condensed top phase and salt-condensed bottom phase, which could be suitable to produce regenerated cellulose with smaller amount of IL. Using IL-ATPS with different pH, the enzymatic saccharification efficiency of crystalline cellulose was determined. The use of 1-allyl-3-methylimidazolium chloride resulted in relatively higher yield of glucose production as compared to 1-butyl-3-methylimidazolium chloride. The IL-ATPS showing optimal pH for cellulase was prepared with mixed salt (NaH2PO4/Na2HPO4=5/1 (wt/wt)), which provide a regenerated cellulose with the pH range of 4.8-4.9 in enzymatic reaction mixture. Using such regenerated cellulose as feed of saccharification, the final yield of glucose was about 70%.

Feasibility of synthesizing γ-[Glu](n≥1)-Gln using high solid concentrations and glutaminase from Bacillus amyloliquefaciens as the catalyst

Effects of using high solid concentrations on the synthesis of γ-[Glu](n≥1)-Gln catalyzed by glutaminase from Bacillus amyloliquefaciens were examined in this study. An increment in solid concentration from 10% to 50% (w/w) increased the extent of synthesis of γ-[Glu](n≥1)-Gln, based on the analyses of amino acid composition and amino nitrogen content. Size-exclusion high-performance liquid chromatography analysis revealed an increase in molecular mass of γ-[Glu](n≥1)-Gln resulting from the increase of solid concentration from 10% to 50% (w/w). UPLC-Q-TOF-MS/MS analysis showed that the enzymatic reaction mixtures post γ-glutamyl transpeptidation contained γ-Glu-Gln, γ-Glu-Glu-Gln, γ-Glu-Glu-Glu-Gln, γ-Glu-Glu-Glu-Glu-Gln and γ-Glu-Glu-Glu-Glu-Glu-Gln. The intensity of each γ-[Glu](n=1,2,3,4,5)-Gln produced at a solid concentration of 50% (w/w) was higher than that at 10% (w/w). These findings indicated the potential of such an energy- and water-efficient approach for synthesizing γ-[Glu](n≥1)-Gln at high solid concentrations.

Salivary vitamin E and uric acid in patients with OLP and healthy individuals

Background: Oral Lichen planus (OLP) is a T-cell mediated chronic inflammatory oral mucosal disease of unknown etiology. Recent studies have reported an increased oxidative stress and lipid peroxidation in such patients. This suggests that reactive oxygen species may have a role in the pathogenesis of lichen planus. Oxidative stress in OLP release molecules consisting of granzymes resulting in local tissue damage in the effectors. Antioxidants that can defend against oxidative stress in the body cells include enzymes, as well as non- enzymatic antioxidants, such as melatonin, uric acid, vitamin A and E. Purpose: To study the level of salivary vitamin E and uric acid as antioxidant agents in patients with OLP and compared with healthy control. Methods: Twenty five patients with OLP were enrolled in this study. Age, gender, occupation, smoking status (smokers or non-smokers), lesion types, duration, location and size were recorded for each patient. After an oral examination, salivary samples were collected and flow rates (ml/min) were recorded. The collected samples were centrifuged at 3000 rpm for 10 minutes; the clear supernatants were separated and stored frozen at (-20 c) until analysis. Then salivary vitamin E was investigated using ELISA kit based on bioten double antibody sandwich technology. Uric acid was analyzed using a proprietary enzymatic reaction mixture that enables the detection of uric acid by the production of a red chromogen, which is quantitatively measured at a wavelength of 515 or 520 nm. Results: The mean age of OLP patients was 48.3 years with a range of 30-60 years. Control group consisted of 35 healthy subjects who were age matched with OLP patients. Fourteen (56%) patients were with reticular and 11 (44%) were with erosive form, with the buccal mucosa was the most commonly affected site (88%), followed by tongue (8%) then gingiva (4%). A significantly lower salivary flow-rate, lower salivary vitamin E and uric acid level in OLP patients compared to control; while, no significant difference was seen between reticular and erosive type of OLP for both vitamin E and uric acid level. Regarding gender, no differences were found between males and females in salivary vitamin E. No significant correlation was found between vitamin E /uric acid and age. Similarly, no difference was found between males and females in uric acid. Conclusion: Salivary anti-oxidant markers represented by vitamin E and uric acid decreased in OLP patients due to increase oxidative stress which may have an important role in the pathogenesis. Thus, it is recommended to give OLP patients anti-oxidant agents that may either help in healing process or decreased the severity.

A comprehensive study of oleuropein aglycone isomers in olive oil by enzymatic/chemical processes and liquid chromatography-Fourier transform mass spectrometry integrated by H/D exchange

A comprehensive structural characterization of the complex family of isomeric forms related to Oleuropein aglycone (OA) detected in virgin olive oil (VOO) was performed by reverse phase liquid chromatography with electrospray ionization and Fourier-transform mass spectrometry (RPLC-ESI-FTMS), integrated by enzymatic/chemical reactions performed on Oleuropein, the natural precursor of OA. First, some of the OA-related isomers typically observed in VOO extracts were generated upon enzymatic hydrolysis of the glycosidic linkage of Oleuropein. This step mimicked the process occurring during olive drupes crushing in the first stage of oil production. The incubation of the enzymatic reaction mixture at a more acidic pH was subsequently performed, to simulate the conditions of olive paste malaxation during oil production. As a result, further isomeric forms were generated and the complex chromatographic profile typically observed for OA in olive oil extracts, including at least 13 different peaks/bands/groups of peaks, was carefully reproduced. Each of those chromatographic features could be subsequently assigned to specific types of OA-related isomers, belonging to one of four structurally different classes. Specifically, diastereoisomers/geometrical isomers corresponding to two different types of open-structure forms and to as many types of closed-structure, di-hydropyranic forms of OA, characterized by the presence of one or two carbonyl groups, according to the case, were evidenced. In addition, the presence of stable enolic/dienolic tautomers, providing an indirect structural confirmation for some OA isomers, was ascertained through RPLC-ESI-FTMS analyses performed under H/D exchange conditions, i.e. in the presence of deuterated water as one of the mobile phase solvents.