Citrate-phosphate Buffer Research Articles

Antibiotics in honey: a comprehensive review on occurrence and analytical methodologies.

Honey is a food of great nutritional importance and has always been used for human consumption. The production of honey and other beekeeping products depends on the proper functioning of this extremely important sector, as it has a direct impact on other sectors such as agriculture. The decline in bee colony numbers has been linked, among other factors, to bacterial diseases affecting bees, including American and European foulbrood, and Nosema spp. disease. In this matter, prophylactic or therapeutic use of veterinary drugs in apiculture is common but can lead to their accumulation in bees and in honey. Consumption of contaminated honey can have adverse effects such as allergic or hypersensitivity reactions, carcinogenicity, reproductive effects, and teratogenicity. Commission Regulation (EU) N⍛ 37/2010 sets MRLs for antibiotics in various foods, but these limits are not set for api-products. The lack of harmonized rules has led some countries to set recommended concentrations and minimum performance limits. Nonetheless, to achieve this goal, development of accurate and precise analytical methodologies is crucial. In recent years, the analysis of antibiotics in honey has led to the development of methods in an extensive range of families, including aminoglycosides, amphenicols, lincosamides, macrolides, nitroimidazoles, quinolones, sulfonamides, tetracyclines and nitrofurans. This review work entails an in-depth exploration of occurrence studies, extraction methodologies, and analytical techniques for the determination of antibiotics in apiculture products. It was found that the most used extraction methods include solid-phase extraction, dispersed solid or liquid phase extraction and QuEChERS. Due to the complexity of the honey matrix, samples are often diluted or acidified using McIlvaine buffer, H2O, MeOH, acidified ACN and TCA solution. This is usually followed by a purification step using SPE cartridges or PSA. Golden analytical methodologies include high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer (MS/MS) with Orbitrap or Q-ToF detectors.

Comparison of dissolution profiles of brand name and generic drugs of mycophenolate mofetil in the presence of concomitant drugs

Generic drugs have become popular because they reduce medical costs. However, switching from a brand name drug to a generic drug of mycophenolate mofetil (MMF) in immunosuppressive therapy after kidney transplantation resulted in an insufficient increase in its blood concentration in some cases. Immunosuppressive therapy after transplantation typically involves a multidrug strategy. Therefore, changes in efficacy due to switching to a generic drug of MMF may be due to its combination with other drugs. Since the first barrier to the absorption of an orally administered formulation is dissolution, the present study examined the effects of prednisolone (PDN) and tacrolimus (TAC), which are used in immunosuppressive therapy, on the dissolution behavior of MMF. A dissolution experiment was performed in the 1st liquid for dissolution testing (pH 1.2), McIlvaine buffer (pH 4.0), and a 2nd liquid for dissolution testing (pH 6.8) under the single condition of the brand name drug and two generic drugs of MMF as well as under combined conditions with PDN and TAC. Although the initial dissolution rate of MMF differed between the brand name and generic drugs, no significant difference was observed in the final dissolution rate. Furthermore, the f2 value, which evaluates the equivalence of dissolution behavior, was within the standard range. In the 2nd liquid for dissolution testing (pH 6.8), the dissolution rate and ratio slightly decreased under the combined condition with PDN or TAC; however, in comparisons with the single conditions, the f2 value was within the standard range. Therefore, a reduced blood concentration due to switching to a generic drug of MMF may not be due to a change in dissolution behavior.

Simultaneous determination of macrolide and quinolone antibiotics in animal tissues for human consumption validated by Regulation (EU) 2021/808

In this work, an analytical method for the simultaneous detection and quantification of 11 macrolides (erythromycin A, gamithromycin, lincomycin, neospiramycin, pirlimycin, spiramycin, tildipirosin, tilmicosin, tulathromycin A and its metabolite CP-60300, and tylosin A) and 10 quinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, oxolinic acid, and sarafloxacin) in muscle (bovine, ovine, porcine, equine, rabbit, poultry including laying hens, and fish) and kidney (bovine, ovine, porcine, equine) for human consumption was developed and validated according to Commission Implementing Regulation (EU) 2021/808. Sample preparation consisted of liquid extraction using a citrate–phosphate/EDTA buffer solution (McIlvaine buffer) followed by solid phase extraction for cleanup and UHPLC-MS/MS analysis. The developed method was comprehensively validated with regard to relative matrix effects (CVMF ≤ 20 %), linearity (R2 ≥ 0.98), accuracy (84–119 %), trueness (−16 to + 19 %), repeatability (2.1–20.0 %), reproducibility (3.0–25.0 %), selectivity, and robustness. The method limit of quantification values ranged from 2 to 40 µg kg−1, established as 0.1 × the lowest maximum residue limit (MRL) by species for the authorized antibiotics. Method applicability was demonstrated in six interlaboratory proficiency tests, and in the official control analyses of 1200 field samples of muscle and kidney.

Gene cloning, IPTG-independent auto-induction and characterization of a novel hyperstable S9 prolyl oligopeptidase having lipolytic activity from Thermotoga naphthophila RKU-10T with applications

A hyperstable lipase from Thermotoga naphthophila (TnLip) was cloned and overexpressed as a soluble and active monomeric protein in an effectual mesophilic host system. Sequence study revealed that TnLip is a peptidase S9 prolyl oligopeptidase domain (acetyl esterase/lipase-like protein), belongs to alpha/beta (α/β)-hydrolase superfamily containing a well-conserved α/β-hydrolase fold and penta-peptide (GLSAG) motif. Various cultivation and induction strategies were applied to improve the heterologous expression and bacterial biomass, but TnLip intracellular activity was enhanced by 14.25- fold with IPTG-independent auto-induction approach after 16 h (26 °C, 150 rev min−1) incubation. Purified TnLip (35 kDa) showed peak activity at 85 °C in McIlvaine buffer (pH 7.0–8.0), and has great stability over a broad range of pH (5.0–10.0), and temperature (40–85 °C) for 8 h. TnLip exhibited prodigious resistance toward various commercial detergents, chemical additives, and salt. TnLip activity was improved by 170.51 %, 130.67 %, 127.42 %, 126.54 %, 126.61 %, 120.32 %, and 116.31 % with 50 % (v/v) of methanol, ethanol, n-butanol, isopropanol, acetone, glycerol, and acetic acid, respectively. Moreover, with 3.0 M of NaCl, and 10 mM of Ca2+, Mn2+, and Mg2+ TnLip activity was augmented by 210 %, 185.64 %, 152.03 %, and 116.26 %, respectively. TnLip has an affinity with various substrates (p-nitrophenyl ester and natural oils) but maximal hydrolytic activity was perceived with p-nitrophenyl palmitate (pNPP, 3600 U mg−1) and olive oil (1182.05 U mg−1). The values of Km (0.576 mM), Vmax (4216 μmol mg−1 min−1), VmaxKm−1 (7319.44 min−1), kcat (1106.74 s−1), and kcatKm−1 (1921.42 mM−1 s−1) were calculated using pNPP substrate. Additionally, TnLip degraded animals' fats and removed oil stains within 3 h and 5 min, respectively. All these features make halo-alkali-thermophilic TnLip as an auspicious contender for laundry detergents (cleaning bio-additive), fat degradation, wastewater treatment and endorse eco-friendly stewardship along with various other biotechnological applications.

Development and performance evaluation of a microbiological method for screening and LC-MS/MS for conformation of sulfonamides in animal-derived foods

This study developed a highly sensitive microbiological method utilizing a novel microtiter plate to screen 10 sulfonamides in chicken muscles, eggs, and prawns. This plate was fabricated from agar incorporating trimethoprim and spread with Bacillus megaterium. After residue detection by bioassay, the same test solutions were analyzed by LC-MS/MS for accurate identification and quantification. It also proved eco-friendly compared to using other quantitative methods. The residual drugs were extracted with McIlvaine buffer and purified using an Oasis® MCX cartridge. A triethylamine/methanol/water (0.5:75:24.5, v/v/v) mixture was used as the eluate. The obtained LOD values of the bioassay ranged from 5 to 25 µg kg−1 allowing the detection of the target drugs at the MRLs established in Japan. Adhering to ISO/IEC 17025 standards, the performance of the bioassay was evaluated. Based on the inhibition zone size in bioassay results, quality control yielded a Z score within ±2, indicating reasonable control over the screening process. Proficiency testing of a chicken muscle sample spiked with sulfadimidine demonstrated the inhibition zone detection of the bioassay and quantified value alignment of LC-MS/MS with reference values. In a surveillance study of 91 samples, sulfamethoxazole was detected in one prawn sample.

Antibiotics in honey: a comprehensive review on occurrence and analytical methodologies.

Honey is a food of great nutritional importance and has always been used for human consumption. The production of honey and other beekeeping products depends on the proper functioning of this extremely important sector, as it has a direct impact on other sectors such as agriculture. The decline in bee colony numbers has been linked, among other factors, to bacterial diseases affecting bees, including American and European foulbrood, and Nosema spp. disease. In this matter, prophylactic or therapeutic use of veterinary drugs in apiculture is common but can lead to their accumulation in bees and in honey. Consumption of contaminated honey can have adverse effects such as allergic or hypersensitivity reactions, carcinogenicity, reproductive effects, and teratogenicity. Commission Regulation (EU) N⍛ 37/2010 sets MRLs for antibiotics in various foods, but these limits are not set for api-products. The lack of harmonized rules has led some countries to set recommended concentrations and minimum performance limits. Nonetheless, to achieve this goal, development of accurate and precise analytical methodologies is crucial. In recent years, the analysis of antibiotics in honey has led to the development of methods in an extensive range of families, including aminoglycosides, amphenicols, lincosamides, macrolides, nitroimidazoles, quinolones, sulfonamides, tetracyclines and nitrofurans. This review work entails an in-depth exploration of occurrence studies, extraction methodologies, and analytical techniques for the determination of antibiotics in apiculture products. It was found that the most used extraction methods include solid-phase extraction, dispersed solid or liquid phase extraction and QuEChERS. Due to the complexity of the honey matrix, samples are often diluted or acidified using McIlvaine buffer, H2O, MeOH, acidified ACN and TCA solution. This is usually followed by a purification step using SPE cartridges or PSA. Golden analytical methodologies include high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer (MS/MS) with Orbitrap or Q-ToF detectors.

Evaluation of strain variability of food microorganisms in response to decontamination by pulsed electric fields and thermal treatments

The effect of pulsed electric fields (PEF) and thermal treatments on the inactivation of the population of 40 strains of 4 model microorganisms (Escherichia coli, Listeria monocytogenes, Lactiplantibacillus plantarum, Saccharomyces cerevisiae) were investigated. Microbial samples of McIlvaine buffer pH 7.0 were subjected to pulses with electric field strength 20 kV/cm and total specific energies (88, 136, and 184 kJ/kg). Depending on the species and strain, microorganisms exhibited various resistances. PEF microbial resistance and strain variability data were correlated to the total specific energy used. E. coli strains showed statistical log10 inactivation differences under the 88 and 136 kJ/kg but not under the 184 kJ/kg PEF treatment. In contrast, L. monocytogenes strains showed statistical log10 inactivation differences only under the 184 kJ/kg treatment. L. monocytogenes L6 strain was identified as the most resistant strain at PEF treatment (184 kJ/kg). This result was in accordance with the resistance under thermal treatment (62.8 °C, 30 min). Industrial relevanceThe identification of target microorganisms related to their resistance in one or more technologies can help at establishing treatment conditions that reassure food safety. Data obtained in this research show that species and strain behaviours vary and are dependent on the technology and the applied treatment conditions. Thus, the resistance exhibited by microorganisms of public health importance may be dependent on the used technology and the applied treatment.

Optimisation of Saccharomyces cerevisiae BRYC 501 ascospore formation and recovery for heat inactivation experiments

Why was the work done: Ascospores from Saccharomyces cerevisiae BRYC 501 are useful biological indicators for validating the pasteurisation of beer. Ascospores are formed by yeast in response to low nutrient conditions and are characterised by greater resistance to stressors including desiccation, freezing and, during pasteurisation, high temperatures. Accordingly, the sporulation temperature of yeast ascospores, their shelf life, heat resistance and recovery need to be investigated. How was the work done: The role of temperature in sporulation was investigated at 25, 27 and 30°C by measurement of the rate of sporulation over ten days. Shelf life of ascospores was established in deionised water at 1-5°C over 120 days by the measurement of heat resistance in McIlvaine's buffer. The recovery conditions of ascospores were determined using six agars after heat inactivation experiments in McIlvaine's buffer, alcohol free and lager beer. What are the main findings: The sporulation rate of S. cerevisiae ascospores was highest at 27°C, and lowest at 30°C. Heat resistance of ascospores formed at 30°C was low with a D60 value of <3 minutes, whereas at 25 and 27°C the D60 was 8.8 and 9.2 minutes. The shelf life of ascospores was 60 days after an initial 14 days of maturation at both 25 and 27°C. The variation of heat resistance for ascospores grown at 25°C was shown to be lower than those grown at 27°C. Use of yeast extract-peptone-glucose agar to recover heat injured cells was > 200% higher than with the control agar. Why is the work important: Sporulation should be performed at 25°C to produce the maximum number of spores for heat inactivation experiments. Spores can be maintained for 60 days, enabling several experiments to be performed. After heat inactivation experiments, ascospores should be recovered on YEPG agar for 10 days at 27°C

Electrolyte Influence on Properties of Ultra-Thin Anodic Memristors on Titanium

Titanium anodic memristors were prepared in phosphate buffer (PB) and citrate buffer (CB) electrolytes. Studying their I-U sweeps, the memristors presented self-rectifying and volatile behaviors. Transmission electron microscopic analysis revealed crystalline protrusions inside a semi-crystalline Ti oxide. Grounded in this, a hybrid interfacial memristive switching mechanism relaying on partial filaments was proposed. Moreover, both analyzed memristor types demonstrated multilevel switching capabilities. The memristors anodized in the PB and CB showed high-to-low resistance ratios of 4 × 104 and 1.6 × 102, respectively. The observed (more than two order of magnitude) ratio improvement of the PB memristors suggests their better performance, in spite of their modestly high resistive state instabilities, attributed to the thermal stress caused by consecutive switching. The endurance and retention of both the PB and CB memristors was measured over up to 106 cycles, indicating very good lifetimes. Phosphate incorporation into the anodic oxide was confirmed by photoelectron spectroscopy analysis and was related to the improved memristive behavior of the PB sample. The presence of phosphate inside the memristively active layer modifies the availability of free O species (vacancies and ions) in the oxide. Taking all this into consideration, Ti anodic memristors anodized in PB are emphasized as candidates for neuromorphic computing.

Pea and soy protein isolate fractal gels: The role of protein composition, structure and solubility on their gelation behaviour

The gelation behaviour of two different pea protein isolates and one soy protein isolate were investigated with a focus on the role of the protein properties. Protein solubility was the lowest in pH 3 citrate-phosphate buffer (<10% w/w), increased in pH 7.4 phosphate-buffered saline (12–21% w/w), and was the highest in pH 7.6 MilliQ water (∼20–40% w/w). Heat-induced gelation conditions for the protein sources were sensitive to both the soluble and the insoluble fractions as obtained during extraction. At low protein concentrations (≤5% w/v), the proteins started to lose their viscoelastic behaviour and exhibited predominantly viscous properties. Fitting of the fractional Kelvin-Voigt model to the frequency sweeps showed an increase in the fractal gel strength with increasing protein concentration. Secondary structures of the soluble species showed mostly unordered proteins, suggesting that the proteins were denatured during the commercial extraction process although gelation has to date been suggested to be highly dependent on the denaturation of soluble proteins. Synchrotron Radiation Circular Dichroism measurements of the insoluble proteins showed a significant amount of ordered protein structures. SEM imaging of the gels also suggested a new gelation pathway in which insoluble proteins act as dispersed fillers within a continuous matrix of soluble proteins. This research elucidated the role of different protein fractions, globulins and albumins, and their secondary structure in the formation of a gel network and how this affects their viscoelastic behaviour.

Кислородный биокатод на основе laccase Pleurotus ostreatus HK-35 для биотопливного элемента

The main reasons that make mankind look for new sources of energy are decreasing dependence on fossil fuels and reducing pollution. Wastewater treatment with the help of microbial fuel cells is an area where these two goals can be combined. Microbial fuel cells, in which microorganisms catalyze the oxidation of organic substances, represent a new and promising alternative for electricity generation. The creation of an efficient cathode in such systems is the important problem. It is evident that in these applications an oxygen (air) electrode is a promising cathode. In this study, the oxygen biocathode based on the laccase enzyme Pleurotus ostreatus HK-35 was developed and its electrochemical properties were studied depending on the immobilization method of the enzyme on the surface of a carbon-graphite electrode and the type of the electrolyte. It was experimentally established that laccase injection using a sol-gel matrix was the effective method for immobilizing laccase on the surface of a carbon-graphite electrode. It was shown that the more efficient operation of the laccase-based biocathode was observed in a phosphate-citrate (pH 4.0) buffer solution, i.e., in an acidic environment.

A simple and reliable electrochemical method employing an unmodified boron-doped diamond electrode for the determination of triclocarban

Since the advent of the COVID-19 pandemic, triclocarban-containing cleaning products have been increasingly consumed. As triclocarban is an emerging compound, it is necessary to develop new sensors for its monitoring. So, a simple and reliable electroanalytical method for the determination of triclocarban using an unmodified boron-doped diamond electrode was developed. The application of a cathodic pretreatment (−2.0 V, 30 min, 0.5 mol L−1 H2SO4) on the electrode surface provided higher current intensities and lower resistance to charge transfer in contrast to the non-treated surface. On the diamond electrode surface, the triclocarban showed an irreversible behavior with two oxidation peaks at +1.10 and +1.40 V vs. Ag/AgCl, KCl(sat). The hypothesis for removing hydrogens of both amide groups of the triclocarban structure was supported by computational methodology which also explained the broadened oxidation peaks and the distance between them. The calibration curve for triclocarban, obtained by using differential pulse voltammetry in a supporting electrolyte composed of McIlvaine buffer (pH 3.0) and ethanol (80:20 v/v), covered a concentration range of 1.0 to 50 μmol L−1, and it exhibited a detection limit of 0.31 μmol L−1 under optimized experimental conditions. Using an accumulation time of 120 s at open circuit potential, a calibration curve by differential pulse adsorptive stripping voltammetry was constructed in a range of 0.05 to 20 µmol L−1 of triclocarban, with a detection limit of 18 nmol L−1. Triclocarban quantification assays were carried out on samples of antibacterial soaps and river water, and data were recorded with acceptable precision and accuracy.

Application of Immobilized β-Glucosidase from Candida boidinii in the Hydrolysis of Delignified Sugarcane Bagasse.

Candida boidinii is a methylotrophic yeast with wide geographical distribution. In the present study, the microorganism was isolated from the Bahian semiarid and the enzymatic extract containing β-glucosidase was obtained through submerged fermentation. Response surface methodology was employed to optimize of fermentation medium. The higher production of β-glucosidase was obtained after 71h of fermentation in an optimized medium composed of 3.35% glucose, 1.78% yeast extract and 1% peptone. The optimum pH and temperature of enzymatic activity were 6.8 (citrate-phosphate buffer) and 71.7°C, respectively. Salts tested (10mM) CaCl2, Na2SO4 and ZnSO4 promotes the increase of 91%, 45% and 80% of activity, respectively. The enzyme retained 64% ± 2.3 of its initial activity after 1h heating at 90°C. The production of reducing sugars was 95.94% after 24h of hydrolysis and, with the addition of metal ions, this value increased more than 2 times. Among the supports analyzed for immobilization, chitosan showed higher residual activity during reuse. The immobilized enzyme showed higher activity at 60°C with pH 6 and preserved almost 100% of the initial activity after 30min at 70°C.

Determination of 55 Veterinary Antibiotics in Chicken Manure Using Ultra-High-Performance Liquid Chromatography – Tandem Mass Spectrometry (UPLC-MS/MS)

A highly efficient method for the detection and quantification of 55 veterinary antibiotics (VAs) in chicken manure belonging to six drug classes using solid-phase extraction (SPE) and ultrahigh-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed. The mixture of EDTA–McIlvaine buffer (pH 3.0) and the organic extractant (methanol–acetonitrile, 1:3, v/v) was used to isolate the target VAs from freeze-dried chicken manure. The extract was purified by a hydrophile–lipophile balance cartridge. All antibiotics showed excellent linear relationships in the range between 1 and 100 µg/kg with the coefficients of determination of the standard curves above 0.990. The recoveries of antibiotics were between 35.66% and 103.54% using matrix-matched calibration for quantification. The limits of detection and quantification were from 0.01 to 1.59 µg/kg and 0.04 to 4.76 µg/kg, respectively. The method was demployed to determine VAs in chicken manure from 12 farms, revealing contamination in all samples. A total of 13 VAs belonging to six classes were detected with concentrations up to 41.47 mg/kg. The presence of high concentrations of antibiotic residues in poultry manure poses a potential risk for environmental contamination. This work has identified significant differences in the types of VAs compared to literature reports. Additionally, it detected pleuromutilin antibiotics in chicken manure for the first time.

Recovery of filter paperase from mouldy rice husk in solid state fermentation by Aspergillus protuberus

Recovery of enzymes such as FPase (filter paperase) or exoglucanase from fermented substrate is a sustainable approach in enzyme production; however, there is a scarcity of optimization studies in this field. The present study was aimed to standardize number of parameters (selection of solvent, solvent volume, soaking time, leaching conditions and number of washes) to extract maximum amount of FPase from fermented rice husk by Aspergillus protuberus. Novel Aspergillus protuberus was first report from our lab on cellulases production in solid state fermentation (SSF). Among the tested solvents, citrate phosphate buffer (0.02 M, pH 5.0) proved best solvent for maximum recovery of FPase. Consequent experimental parameters were further optimized with citrate phosphate buffer. Two washes with citrate phosphate buffer each by shaking (60 min) in a ratio of 1 g of rice husk: 5 ml of citrate phosphate buffer together attained higher recovery efficiency (88 %) of FPase from the fermented rice husk.

Proteolitic Activity of Marine Strain Bacillus sp. 051

The main interest in the study of marine microorganisms is due to their ability to produce a wide range of unique enzymes, including peptidases with different specificities. In recent years, interest has increased in peptidases that are able to cleave elastin as a specific substrate. Streptomyces fradiae and Bacillus thermoproteolyticus elastases are among the most potent elastolytic proteinases discovered to date because they are 4-8-fold more effective than pancreatic elastases. The disadvantages of these producers include the fact that most of them are pathogenic for humans, and the elastase enzyme secreted from them is directly involved in the initiation of the pathogenetic process. All this significantly limits the scope of their practical application. Therefore, the search for new, more effective, safe for humans’ producers continues to be an urgent question, taking into account the fact that there are no highly active elastase producers in Ukraine. Previously we found elastase activity in only 4 of the 10 studied isolates of bacteria from the Black Sea. Since among them, the elastase activity of the Bacillus sp. 051 was the highest, the aim of this work was to study the physicochemical properties and substrate specificity of the enzyme. Methods. We used methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic) activity. Protein concentration was determined by the Lowry method. The study of the effect of temperature on the enzymatic activity was carried out in the range from 4 to 70 °C and pH values from 2.0 to 12.0, created using 0.01 M phosphate-citrate buffer. Results. It has been shown that the growing temperature of 12°C is the most optimal for biosynthesis of enzyme by the culture of Bacillus sp. 051. The complex enzyme preparation capable of hydrolyzing elastin, casein and fibrinogen. The enzyme showed maximum activity in relation to elastin (3.65 U/mg). The optimum pH of the enzyme action is 8.0, the thermal optimum is 40°C. The rate of casein hydrolysis compared to elastin was 2.7 times lower and amounted to 1.35 U/mg. The complex enzyme preparation also hydrolyzed fibrinogen (1.16 U/mg). Conclusions. According to its physicochemical and catalytic properties, the representative of the Black Sea, Bacillus sp. 051 is promising for further research as an enzyme producer with elastolytic activity.

Fluorometric investigation of boric acid-pyridoxal 5′ phosphate interaction: A turn-on-fluorescence assay for the detection of boric acid in water bodies and fetal bovine serum

Boron is a trace element that is beneficial to plants as well as human and animal health. Boron supplementation is beneficial for absorbing vitamin D and magnesium, activating antioxidant enzymes and anticancer treatments. However, environmental accumulation of boron exceeding the permissible limit harms plants, animals and humans. Boric acid (BA), a form of boron, is known to interact with biomolecules such as NAD+/NADH, vitamin C, vitamin D, vitamin B1 and vitamin B6 determined through techniques like NMR, mass spectrometry, and FTIR. The fluorescence interaction of BA with the biologically active form of vitamin B6, Pyridoxal 5′ phosphate (PLP), has not been explored to date. Therefore, this study reports the interaction mechanism of BA with PLP via fluorescence spectroscopy analysis. The absorption, excitation and fluorescence lifetime measurements indicate that BA may form a complex through the mono or di-esterification of the phenolic hydroxyl group and aldehyde of PLP at different pH, forming the boronate esters. BA addition enhanced the fluorescence intensity of PLP by 12–22-fold in phosphate buffer (at acidic, neutral, and basic pH conditions). On the other hand, in citrate buffer at pH 4.0, BA enhanced PLP fluorescence by 28-fold. The binding constant for BA in phosphate buffer and citrate buffer was found to be 173 mol−1·L and 623 mol−1·L, respectively. At the same time, the detection limit for BA was found to be 445 μg/L and 212 μg/L in respective buffers. Therefore, PLP was used as a turn-on fluorescent probe to detect and quantify BA in water bodies and fetal bovine serum (FBS). The developed method successfully detected BA in environmental water bodies, achieving an average recovery rate of 99.84% (with a relative standard deviation (RSD) of 0.38%) in phosphate buffer and 81.59% (RSD, 4.56%) in citrate buffer. Furthermore, detecting BA in PLP-spiked FBS has been demonstrated in citrate buffer at pH 4.0, yielding an average recovery rate of 108.25% (RSD, 3.19%). Therefore, this study provides a basis for understanding the interaction of biomolecules like vitamins with boronates which aid in development of novel boronate based therapeutics and also helps in understanding the metabolic reactions of vitamin derivatives using fluorescence spectroscopy.

Production of chitosan complexes with 3ʹ,5ʹ-сyclic diadenosine monophosphate

To solve the problem of delivering pharmacologically promising 3ʹ,5ʹ-cyclic diadenosine monophosphate (cyclo-diAMP) to target cells in humans and animals, the complexes of the above-mentioned dinucleotide with natural polymer – chitosan were originally synthesized by ionotropic gelation technique. It was found that the binding degree of cyclo-diAMP to this biopolymeric carrier reaches 60 %; wherein the capacity of the obtained complexes with respect to the dinucleotide is 800–860 µg/mg of the produced complex. Cyclo-diAMP has also been shown to elute from the chitosan complex to the citrate-phosphate buffer (pH 7.4) up to 36 % by 21 hours. The obtained results testify in favor of potential application of cyclodiAMP complex with chitosan for prolonged delivery of the studied cyclic dinucleotide to target cells.

Extraction, purification and characterization of papain enzyme from papaya

Papain is one of the widely used enzymes in the industry. This study aimed to extract papain enzyme from various parts (i.e. latex, leaves, peels and seeds) of papaya using distilled water and citric phosphate buffer and subsequently purified with a two-step salt precipitation method. The optimum protein concentration of purified papain was obtained with citric phosphate buffer (0.159-0.319 mg/mL) and distilled water (0.119-0.347 mg/ mL). The protein concentration was almost similar for crude extracted with both the solvent. The purification procedure enhanced up to 1.56 fold when extracted with distilled water and 0.83 fold when extracted with citric phosphate buffer. Latex, leaves, peels and seeds showed maximum enzyme activity at 60oC and pH 8.0 as well as better thermal stability at 60oC for 30 mins. Findings highlighted that extracted papain enzyme from waste parts of papaya could be utilized as a potential source of industrial application in terms of waste to value-added products.

β-Xylosidase mutant immobilization on UiO-66-NH2 for continuous production of ginsenoside Rg1 and selective production of furfural from notoginsenoside R1

In order to improve the activity and reusability of β-xylosidase, the β-xylosidase mutant S160N-R333H (R333H) which could convert notoginsenoside R1 to ginsenoside Rg1 and xylose was immobilized on the metal-organic framework (MOF) UiO-66-NH2. The physical and biochemical properties of immobilized β-xylosidase (R333H@UiO-66-NH2) were studied in detail. The results showed improved performances, i.e. the high immobilization yield of 93% and recovery activity of 192.8%, better stability under acidic or trypsin-containing conditions, the higher activity in the reaction containing ions, and the improved catalytic efficiency (was 1.37-fold than that of free R333H). Importantly, the ginsenoside Rg1 yields remained more than 80% after six recycles in McIlvaine buffer and pure water. The UiO-66-NH2 could catalyze xylose dehydration to produce furfural and the yield reached 69%. Thus this study provides a promising method for production of bioactive or platform chemicals via continuous conversion of xyloylated substrates using nanocatalyst synthesized by β-xylosidase and MOF.