Ion-exchange Chromatography Research Articles

An up-scaled biotechnological approach for phosphorus-depleted rye bran as animal feed

Side streams from the milling industry offer excellent nutritional properties for animal feed; yet their use is constrained by the elevated phosphorus (P) content, mainly in the form of phytate. Biotechnological P recovery fosters sustainable P management, transforming these streams into P-depleted animal feed through enzymatic hydrolysis. The enzymatic P mobilization not only enables P recovery from milling by-products but also supports the valorization of these streams into P-depleted animal feeds. Our study presents the scalability and applicability of the process and characterizes the resulting P-depleted rye bran as animal feed component. Batch mode investigations were conducted to mobilize P from 100 g to 37.1 kg of rye bran using bioreactors up to 400 L. P reductions of 89% to 92% (reducing from 12.7 gP/kg to 1.41–1.28 gP/kg) were achieved. In addition, High Performance Ion Chromatography (HPIC) analysis showed complete depletion of phytate. The successful recovery of the enzymatically mobilized P from the process wastewater by precipitation as struvite and calcium hydrogen phosphate is presented as well, achieving up to 99% removal efficiency. Our study demonstrates a versatile process that is easily adaptable, allowing for a seamless implementation on a larger scale.Graphical

Ion chromatography‐based determination of lithium in human urine for monitoring medication compliance

AbstractContinuous Li monitoring is vital in ensuring the safety and treatment efficacy of probationers with psychotic disorders, such as bipolar disorder, depression, and mania. This study introduced a validated quantitative analysis method using ion chromatography (IC) to monitor Li+ concentration in urine. The methodology involved centrifuging the urine sample, diluting the supernatant with deionized water, and subsequent IC analysis using guard and analytical columns for precise Li+ concentration measurement. The method quantified Li+ within the range of 2–400 ng/mL, demonstrating its excellent linearity (r2 = 0.9983) with a weighting factor of 1/x. Precision levels below 12.8% were observed for both intraday and interday measurements with an accuracy of −5.9% to 15.2%. The detection limit was identified as 0.12 ng/mL. The method conformed to analytical standards in terms of selectivity, matrix effect, process efficiency, dilution integrity, and stability. Moreover, the method distinctly quantified Li+ while effectively eliminating interference from Na+, which had a similar retention time as Li+ with higher concentrations. Further, the developed method was successfully applied to analyze forensic urine samples of Li users, demonstrating its applicability for medication compliance monitoring using urine samples of mentally disordered probationers.

Aluminum dynamics in nitrogen‐saturated Andisols in Tokyo

AbstractThe debate over whether forests around the Tokyo metropolitan area are nitrogen (N) saturation persists, as atmospheric N deposition in throughfall has decreased. This decrease is evidenced by a notable decline in samples collected in the 1990s (especially 1991–1992 and 1995). This decline can be attributed to a reduction in nitrogen oxide (NOx) emissions from automobiles. The acidity derived from N deposition can increase aluminum (Al) mobility. We conducted a monitoring study from September 2010 to December 2021 to elucidate the effects of decreased N deposition on Al concentrations and flux in a forested Andisol. Throughfall and soil‐percolated water samples were collected under stands of Japanese cedar and Japanese cypress in Hachioji, Tokyo (Field Museum Tamakyuryo). Major inorganic ions were determined by ion chromatography. Total Al concentrations were determined using atomic absorption spectrometry after concentration under acidic conditions. Aluminum and nitrate () concentrations were significantly correlated in the both Japanese cedar and Japanese cypress stands. In the case of the Japanese cedar stand, Al concentrations tended to decrease over time from November 2010 to May 2015. Based on stepwise multiple regression analysis, acid load associated with N transformation ([H+]load) was chosen as the sole factor affecting Al mobilization in the Japanese cedar stand. Decreased N deposition affects Al dynamics via a decrease in [H+]load.

Impact of thermal soil treatment on heavy metal mobility in the context of waste management.

Thermal soil treatment is a well-established remediation method to remove organic contaminants from soils in waste management. The co-contamination with heavy metals raises the question if thermal soil treatment affects heavy metal mobility in soils. In this study, four contaminated soils and a reference sample were subjected to thermal treatment at 105°C, 300°C and 500°C for 7 day. Thermogravimetry and differential scanning calorimetry were used to understand the reactions, and resulting gases were identified by Fourier-transformed infrared spectroscopy. Treated and untreated samples were characterised by X-ray diffraction (XRD) and electron microprobe analysis and subjected to pH-dependent leaching tests, untreated samples additionally by X-ray-fluorescence (XRF) and inductively coupled plasma mass spectroscopy (ICP-MS). Leachates were analysed using ICP-MS and ion chromatography. Maximum available concentrations were used for hydrogeochemical modelling using LeachXS/Orchestra to predict leaching control mechanisms. Leaching experiments show that thermal treatment tends to decrease the mobility at alkaline pH of Pb, Zn, Cd, As and Cu, but to increase the mobility of Cr. In the acidic to neutral pH range, no clear trend is visible. Hydrogeochemical modelling suggests that adsorption processes play a key role in controlling leaching. It is suggested that the formation of minerals with a more negatively charged surface during thermal treatment are one reason why cations such as Pb2+, Zn2+, Cd2+ and Cu2+ are less mobile after treatment. Future research should focus on a more comprehensive mineralogical investigation of a larger number of samples, using higher resolution techniques such as nanoscale secondary ion mass spectrometry to identify surface phases formed during thermal treatment and/or leaching.

Relationship between dietary fiber content and prebiotic potential of polysaccharides from the seaweeds of the North west coast of India

The macroalgae are a sustainable bioresource that can be harnessed for their functional food and nutraceutical applications. This study characterized the biochemical composition and bioactive potential of natural biological macromolecules, such as macroalgal polysaccharides extracted using a green, aqueous extraction process. The in-vitro antioxidant and antiglycemic activity of these polysaccharides were evaluated using model, free radical and antiglycemic compounds. The prebiotic potential of macroalgal polysaccharides were analysed based on their ability to promote the growth of two potential probiotic bacteria Lactobacillus acidophilus and L. bulgaricus and suppress the growth of enteric bacteria, Escherichia coli. Among the polysaccharides studied, the brown algal polysaccharide MPS8 MPS9 and MPS10 exhibited good antioxidant, antiglycemic and prebiotic activity. Based on infrared spectroscopy, the functional groups sulfation and carboxylation were identified in potential polysaccharides. The monosaccharide composition in the bioactive polysaccharides was determined using High Performance Anion Exchange Chromatography Pulse Amperometric detector (HPAEC-PAD). These bioactive polysaccharides were fractionated using ion exchange chromatography to purify it and further characterized using gel permeation chromatography and NMR spectroscopy. The results these polysaccharides are mainly composed of fucose and glucose which is due to the fucoidan and laminarin, respectively. Such macromolecules with high dietary fiber content and bioactivity are in global demand as functional food, nutraceutical and pharmaceutical formulations.

Total and Class-Specific Determination of Fluorinated Compounds in Consumer and Food Packaging Samples Using Fluorine-19 Solid-State Nuclear Magnetic Resonance Spectroscopy.

Hamburger wrapping paper, coated with water-based barrier coatings, used in the food packaging industry was studied by using the total organic fluorine (TOF) method based on combustion ion chromatography and fluorine-19 solid-state nuclear magnetic resonance (19F ss-NMR) spectroscopy. Although the TOF method is a fast and affordable method used to screen for per- and polyfluoroalkyl substances (PFAS), the amount of fluorine it measures is heavily dependent on the extraction step and, therefore could lead to inaccurate results. Fluorine-19 ss-NMR spectroscopy can differentiate between organic and inorganic fluorinated sources, eliminating the need for sample clean up. To illustrate this, the 19F ss-NMR spectra of clean coated paper samples that contained naturally occurring F- ions from the talc raw material and spiked samples containing perfluorooctanoic acid were compared. A range of experimental conditions was explored to improve sensitivity for low PFAS concentrations (in the order of 10-20 mg/kg). Despite the disadvantages of ss-NMR spectroscopy, such as the low limit of detection and resolution, the results demonstrate it can be a viable tool to directly detect PFAS moieties in consumer and food packaging. Therefore, 19F solid-state NMR spectroscopy challenges and complements current methods, which only provide indirect evidence of the presence of PFAS.

Optimization of Extraction and Analysis of Inulin in Extracts of Roasted Açaí (Euterpe oleracea Mart.) Seeds by Ion Chromatography

Optimization of Extraction and Analysis of Inulin in Extracts of Roasted Açaí (Euterpe oleracea Mart.) Seeds by Ion Chromatography

Towards bioprocess engineering of cable bacteria: Establishment of a synthetic sediment.

Cable bacteria, characterized by their multicellular filamentous growth, are prevalent in both freshwater and marine sediments. They possess the unique ability to transport electrons over distances of centimeters. Coupled with their capacity to fix CO2 and their record-breaking conductivity for biological materials, these bacteria present promising prospects for bioprocess engineering, including potential electrochemical applications. However, the cultivation of cable bacteria has been limited to their natural sediment, constraining their utility in production processes. To address this, our study designs synthetic sediment, drawing on ion exchange chromatography data from natural sediments and existing literature on the requirements of cable bacteria. We examined the effects of varying bentonite concentrations on water retention and the impacts of different sands. For the first time, we cultivated cable bacteria on synthetic sediment, specifically the freshwater strain Electronema aureum GS. This cultivation was conducted over 10 weeks in a specially developed sediment bioreactor, resulting in an increased density of cable bacteria in the sediment and growth up to a depth of 5 cm. The creation of this synthetic sediment paves the way for the reproducible cultivation of cable bacteria. It also opens up possibilities for future process scale-up using readily available components. This advancement holds significant implications for the broader field of bioprocess engineering.

Analytical methods for quantitating sulfate in plasma and serum.

Circulating sulfate needs to be maintained at sufficiently high levels for healthy growth and development. Animal studies have shown the adverse physiological consequences of low circulating sulfate level on the skeletal, neurological and reproductive systems. However, sulfate is not routinely measured in clinical investigations, despite the importance of sulfate being documented over the past several decades. Several methods have been developed for measuring serum and plasma sulfate level in animals and humans, including a range of barium sulfate precipitation techniques that have been a major focus of sulfate analytics since the 1960s. Evaluation of an ion chromatography method demonstrated its utility for investigation of sulfate levels in human health. More recently, liquid chromatography-tandem mass spectrometry has been used to show hyposulfatemia in a human case of mild skeletal dysplasia. This article provides an overview of analytical methods for measuring sulfate in serum and plasma, highlighting the strengths and limitations of each method.

Towards elevated perfluorooctanoic acid (PFOA) enrichment in water: Sequential liquid-liquid extraction pretreatment for ion chromatography detection

The widespread detection of perfluorooctanoic acid (PFOA) in the environment has raised significant concerns. The standard PFOA analytical method relies on expensive solid-phase extraction (SPE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) instruments, making routine use prohibitive. We herein proposed a cost-effective yet novel enrichment method for determining PFOA at ng L−1 level. This method entailed a two-step sample preparation process: firstly, PFOA was extracted and enriched using a forward-extraction under acidic conditions, followed by a backward-extraction and enrichment step utilizing alkaline water. The enriched samples were subsequently subjected to a common ion chromatography (IC). Results reveal that maintaining a forward-extraction pH below its pKa value (2.8) is essential, as protonated PFOA proves effective in enhancing the enrichment factor (EF). The challenge lied in driving PFOA from forward-extractant to aqueous backward-extractant due to the decreased hydrophobicity of deprotonated PFOA (log Kow2 = 1.0). In addition, we found that evaporating forward-extractant with alkaline backward-extractant (containing 5% methanol) reduced potential analytical uncertainties associated with PFOA evaporation and sorption. Under optimal conditions, the method achieved a detection limit of 9.2 ng L−1 and an impressive EF value of 719. Comparison with SPE-LC-MS/MS confirmed the proposed method as a promising alternative for PFOA determination. Although initially targeted for PFOA, the novel methodology is likely applicable to preconcentration of other poly-fluoroalkyl substances.

Abstract PO1-26-04: Patient-derived organotypic tissue cultures for organoid isolation and probing unique metabolic features of individual breast cancer patients

Abstract Patient-derived organotypic tissue cultures (PD-OTC) are unique models for probing how individual patient’s tumor microenvironment (TME) influences cancer development and treatment responses. They retain the patient tumor architecture and TME, that are difficult to recapitulate in other models, while affording flexible treatment options and comparison of cancer (CA) versus matched non-cancer (NC) tissue responses to treatments. We have developed a culturing method for establishing PD-OTC of breast cancer patients that exhibit CA tissue growth and extensive metabolic reprogramming between CA versus matched NC tissues while enabling patient-derived organoids (PDO) and fibroblasts to be isolated. Pairs of CA and NC tissues freshly resected from six patients bearing early-stage estrogen receptor/progesterone receptor positive ductal carcinoma were sliced at 750 µm, embedded in Matrigel, and cultured in a 6-well plate with a Biopore membrane insert and custom medium at 37°C/5% CO2 with gentle rocking (16-40 mg wet weight; n=3). Excess tissue slices were cryopreserved and kept at -196 °C. Culture media were sampled periodically for metabolite analysis by NMR. Microscopic examination revealed significant tissue, organoid-like, and fibroblast outgrowth after 1-1.5 month of culturing. Two to three days prior to the harvest of two patients’ (CZ16 and 17) OTC, the culture medium was replaced with [U-2H]-glucose + [U-13C,15N]-glutamine-containing medium to allow the two stable isotope tracers to be metabolized, followed by Stable Isotope-Resolved Metabolomic (SIRM) analysis by NMR and ion chromatography coupled with ultra high-resolution Fourier transform mass spectrometry (IC-UHRFTMS). Two separate pieces of the OTC were cut and each was assayed for glucose uptake and mitochondrial membrane potential by live fluorescence spectroscopy via a portable custom-built fluorescence microscope, and by histochemical analysis. Histological analysis showed that both CA and NC OTC of all patients maintained their structural integrity. SIRM analysis of two patients’ OTC revealed active catabolic and anabolic metabolism including glucose uptake/glycolysis, the Krebs cycle/mitochondrial membrane potential, the pentose phosphate pathway (PPP), gluconeogenesis, glycogen synthesis, and purine/pyrimidine/sugar nucleotides synthesis. The CA OTC of both patients showed enhanced glucose uptake, glycolysis, mitochondrial membrane potential/Krebs cycle, PPP, glycogen synthesis, and purine nucleotide synthesis, when compared with the matched NC OTC. Treatment-naïve CA OTC of CZ16 showed much more significant metabolic reprogramming (NAD+ metabolism, in particular) than CA OTC of CZ17 who has undergone neoadjuvant treatment, chemotherapy, and hormone therapy prior to surgery. Distinct metabolic features of breast CA tissues can be exploited as therapeutic targets. Organoids and fibroblasts were isolated from CA and/or NC OTC cultures and their characterization is in progress. When revived, cryopreserved OTC exhibited active metabolism and tissue/organoid/fibroblast outgrowth similarly to fresh OTC. In conclusion, our culturing method enables PD-OTC models to be established efficiently for early-stage breast cancer patients for downstream functional studies including live cell imaging and SIRM analysis. Organoids and fibroblasts can also be derived from < 20 mg of tissues for further studies. Future direction includes therapeutic studies on these patient-derived models to better understand and predict individual patient’s responses to therapy. Citation Format: Teresa Fan, Carlos Goncalves, Jing Yan, Jahid Islam, Penghui Lin, Mohamed Kaddah, Richard Higashi, Andrew Lane, Caigang Zhu. Patient-derived organotypic tissue cultures for organoid isolation and probing unique metabolic features of individual breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-26-04.

Minimally Modified Human Blood Coagulation Factor X to Bypass Direct Factor Xa Inhibitors

Direct oral factor Xa inhibitors are widely used as alternatives to conventional vitamin K antagonists in managing venous thromboembolism and nonvalvular atrial fibrillation. Unfortunately, bleeding-related adverse events remain a major concern in clinical practice. In case of bleeding or emergency surgery, rapid-onset reversal agents may be required to counteract the anticoagulant activity. The ability of factor Xa variants to bypass the direct oral factor Xa inhibitors was assessed. Human factor Xa variants were generated through substitution of phenylalanine 174 (F174) for either alanine, isoleucine, or serine. Factor Xa variants were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography. F174-substituted human factor X variants demonstrated efficacy in restoring thrombin generation in plasma containing direct factor Xa inhibitors (apixaban, rivaroxaban, edoxaban). Their ability to bypass the anticoagulant effects stems from a significant reduced sensitivity for the direct factor Xa inhibitors, due to a decrease in binding affinity determined using molecular dynamics simulations and free energy computation. Furthermore, F174 modification resulted in a partial loss of inhibition by tissue factor pathway inhibitor, enhancing the procoagulant effect of F174-substituted factor X. Consequently, the F174A- and F174S-substituted factor X variants effectively counteracted the effects of two widely used anticoagulants, apixaban and rivaxoraban, in plasma of atrial fibrillation and venous thromboembolism patients. These human factor X variants have the potential to serve as a rescue reversal strategy to overcome the effect of direct factor Xa inhibitors in case of life-threatening bleeding events or emergency surgical interventions.

Arsenic and fluoride in groundwater triggering a high risk: Probabilistic results using Monte Carlo Simulation and Species Sensitivity Distribution

The widespread presence of arsenic (As) and fluoride (F-) in groundwater poses substantial risks to human health on a global scale. These elements have been identified as the most prevalent geogenic contaminants in groundwater in northern Mexico. Consequently, this study aimed to evaluate the human health and ecological risks associated with the content of As and F- in the Meoqui-Delicias aquifer, which is in one of Mexico's most emblematic irrigation districts. Concentrations of As and F- were measured in 38 groundwater samples using ICP-MS and ion chromatography, respectively. Overall, these elements showed a similar trend across the aquifer, revealing a positive correlation between them and pH. The concentration of As and F- in the groundwater ranged from 5.3 μg/L to 303 μg/L and from 0.5 mg/L to 8.8 mg/L, respectively. Additionally, the levels of As and F- surpassed the established national standards for safe drinking water in 92% and 97% of samples, respectively. Given that groundwater is used for both agricultural purposes and human activities, this study also assessed the associated human health and ecological risks posed by these elements using Monte Carlo simulation and Species Sensitivity Distribution. The findings disclosed a significant noncarcinogenic health risk associated with exposure to As and F-, as well as an unacceptable carcinogenic health risk to As through water consumption for both adults and children. Furthermore, a high ecological risk to aquatic species was identified for F- and high to medium risks for As in the sampling sites. Therefore, the findings in this study provide valuable information for Mexican authorities and international organizations (e.g., WHO) about the adverse effects that any exposure without treatment to groundwater from this region represents for human health.

Remediation of 3,4,3′,4′-tetrachlorobiphenyl (PCB77) contaminated soil via a fluidized bed dielectric barrier discharge

In this study, 3,4,3′,4′-tetrachlorobiphenyl (PCB77) contaminated soil was remediated by a fluidization bed dielectric barrier discharge (DBD) reactor and a fixed bed DBD reactor. The fluidized bed reactor could attain superior removal efficiency of PCB77 under same experimental parameters. In-situ discharge mode was more conducive to the degradation of PCB77 than ex-situ discharge mode due to short-lived active species existing in in-situ discharge. The influence of experimental parameters in the fluidized bed DBD reactor on the degradation of PCB77 were discussed such as electric features, gas features, soil features and initial PCB77 concentration. PCB77 removal efficiency in air discharge could reach 88.5 % after 8 min under the alkaline condition. Optical emission spectroscopy (OES) and quench tests showed that reactive oxygen species (ROS) and reactive nitrogen species (RNS) were generated in the discharge system and they both played a vital role in the degradation of PCB77. Scanning electron microscopy (SEM) results demonstrated that discharge had little effect on the morphology of soil particles. Energy dispersive spectrometer (EDS), ion chromatography (IC), and total organic carbon (TOC) results showed that the DBD could effectively mineralize and dechlorinate PCB77. The possible degradation pathway of PCB77 was inferred at the end based on the degradation products determined by gas chromatography–mass spectrometry (GC–MS).

Large-scale separation and purification of exosomes using ionexchange chromatography

Large-scale separation and purification of exosomes using ionexchange chromatography

Production of heat-resistant blastospores by Cordyceps javanica IF-1106 through optimizing metal ions composition

Heat tolerance of fungi is critical for field stability and efficacy of fungal insecticides. Components of the medium, such as carbon source, nitrogen source and metal ions, contribute to the thermotolerance of fungi. In this study, several metal ions were detected to establish their effects on the sporulation and thermotolerance of the blastospores of Cordyceps javanica IF-1106 when cultured in liquid medium. The results showed that metal ions play different roles in sporulation and thermotolerance of the fungi. Mn2+ greatly increased the thermotolerance of the blastospores, and Fe2+ increased the thermotolerance slightly. However, both has no or negative effects on sporulation of the fungi. In the case of Cu2+ and Fe3+, sporulation yield and thermotolerance of the blastospores increased significantly. In addition, intracellular trehalose and mannitol involved in thermotolerance of fungi were extracted and then quantified by ion chromatography. We find that the trehalose and mannitol are higher in the presence of Mn2+, implying that Mn2+ could adjust the intracellular trehalose and mannitol level confers the blastospores with higher tolerance to thermal stress. Further, Response Surface Methodology (RSM) was applied to screen the optimal composition of metal ions. When the formula was MnSO4·H2O 8 mg/L, CuSO4·5H2O 5 mg/L, FeSO4·7H2O 11 mg/L, FeCl3·6H2O 8 mg/L, maximum sporulation yield and GT50 were obtained. Lastly, the virulence of the blastospores to Acyrthosiphon pisum was validated, a higher virulence is achieved with decreased LT50 (2 d) and LC50 (2.37 × 104 blastospores mL−1). In conclusion, thermotolerant blastospores of C. javanica IF-1106 and higher sporulation yield could be achieved by optimizing the metal ions composition, which helps to determine the formula for larger scale fermentation process.

Zinc and Silicon Nano-Fertilizers Influence Ionomic and Metabolite Profiles in Maize to Overcome Salt Stress.

Salinity stress is a major factor affecting the nutritional and metabolic profiles of crops, thus hindering optimal yield and productivity. Recent advances in nanotechnology propose an avenue for the use of nano-fertilizers as a potential solution for better nutrient management and stress mitigation. This study aimed to evaluate the benefits of conventional and nano-fertilizers (nano-Zn/nano-Si) on maize and subcellular level changes in its ionomic and metabolic profiles under salt stress conditions. Zinc and silicon were applied both in conventional and nano-fertilizer-using farms under stress (100 mM NaCl) and normal conditions. Different ions, sugars, and organic acids (OAs) were determined using ion chromatography and inductively coupled plasma mass spectroscopy (ICP-MS). The results revealed significant improvements in different ions, sugars, OAs, and other metabolic profiles of maize. Nanoparticles boosted sugar metabolism, as evidenced by increased glucose, fructose, and sucrose concentrations, and improved nutrient uptake, indicated by higher nitrate, sulfate, and phosphate levels. Particularly, nano-fertilizers effectively limited Na accumulation under saline conditions and enhanced maize's salt stress tolerance. Furthermore, nano-treatments optimized the potassium-to-sodium ratio, a critical factor in maintaining ionic homeostasis under stress conditions. With the growing threat of salinity stress on global food security, these findings highlight the urgent need for further development and implementation of effective solutions like the application of nano-fertilizers in mitigating the negative impact of salinity on plant growth and productivity. However, this controlled environment limits the direct applicability to field conditions and needs future research, particularly long-term field trials, to confirm such results of nano-fertilizers against salinity stress and their economic viability towards sustainable agriculture.

КОМПЛЕКСНЫЙ ПОДХОД В ИССЛЕДОВАНИИ ОГНЕТУШАЩИХ ПОРОШКОВЫХ СРЕДСТВ ПРИ ЭКСПЕРТИЗЕ ПОЖАРОВ

The article discusses the possibility of using a set of instrumental methods, such as morphological analysis, elemental analysis, X-ray phase analysis, infrared spectroscopy, ion chromatography or capillary electrophoresis to study the chemical composition of fire extinguishing powders during fire-technical examinations. Examples of the use of these methods in the investigation of real fires are given. It is described in detail how studies of fire extinguishing agents are carried out to answer questions related to establishing their composition, as well as identifying the compliance of the fire extinguishing powder submitted for study with technical documentation. It is shown that in order to obtain complete information about the chemical composition of the fire extinguishing agent, it is necessary to use a set of methods, since each of the considered methods provides additional information during the study.

Purified Pyropia yezoensis Pigment Extract-Based Tandem Dye Synthesis

Red phycoerythrin (R-PE) is a highly valuable protein found in an edible seaweed, Pyropia yezoensis. It is used extensively in biotechnological applications due to its strong fluorescence and stability in diverse environments. However, the current methods for extracting and purifying R-PE are costly and unsustainable. The aim of the present study was to enhance the financial viability of the process by improving the extraction and purification of R-PE from dried P. yezoensis and to further enhance R-PE value by incorporating it into a tandem dye for molecular biology applications. A combination of ultrafiltration, ion exchange chromatography, and gel filtration yielded concentrated (1 mg·mL–1) R-PE at 99% purity. Using purified PE and Cyanine5 (Cy5), an organic tandem dye, phycoerythrin-Cy5 (PE-Cy5), was subsequently established. In comparison to a commercially available tandem dye, PE-Cy5 exhibited 202.3% stronger fluorescence, rendering it suitable for imaging and analyzes that require high sensitivity, enhanced signal-to-noise ratio, broad dynamic range, or shorter exposure times to minimize potential damage to samples. The techno-economic analysis confirmed the financial feasibility of the innovative technique for the extraction and purification of R-PE and PE-Cy5 production.

Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Culture Supernatant of Coprinus comatus.

A novel fibrinolytic enzyme was produced by the liquid fermentation of Coprinus comatus. The enzyme was purified from the culture supernatant by hydrophobic interactions, gel filtration, and ion exchange chromatographies. It was purified by 241.02-fold, with a specific activity of 3619 U/mg and a final yield of 10.02%. SDS-PAGE analysis confirmed the purity of the enzyme, showing a single band with a molecular weight of 19.5 kDa. The first nine amino acids of the N-terminal of the purified enzyme were A-T-Y-T-G-G-S-Q-T. The enzyme exhibited optimal activity at a temperature of 42 °C and pH 7.6. Its activity was significantly improved by Zn2+, K+, Ca2+, Mn2+, and Mg2+ while being inhibited by Fe2+, Fe3+, Al2+, and Ba2+. The activity of the enzyme was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and it was also dose-dependently inhibited by phenylmethylsulfonyl fluoride (PMSF) and soy trypsin inhibitor (SBTI). However, inhibitors such as N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK), aprotinin, and pepstatin did not significantly affect its activity, suggesting that the enzyme was a serine-like metalloproteinase. The enzyme acted as both a plasmin-like fibrinolytic enzyme and a plasminogen activator, and it also exhibited the capability to hydrolyze fibrinogen and fibrin. In vitro, it demonstrated the ability to dissolve blood clots and exhibit anticoagulant properties. Furthermore, it was found that the enzyme prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), and reduced the levels of fibrinogen (FIB) and prothrombin activity (PA). Based on these studies, the enzyme has great potential to be developed as a natural agent for the prevention and treatment of thrombotic diseases.