Gel Filtration Research Articles

Purification, characterization and molecular docking studies of analogous alpha amylase inhibitor compounds

BackgroundA large number of protein inhibitors are found in higher plants, cereals and legumes. These inhibitors are helpful in the prevention as well as medical treatment of metabolic syndromes such as type 2 diabetes. Basically, the diabetes mellitus is a chronic disorder that occurs either due to inadequate insulin secretions or when body fails to utilize the produced insulin. The alpha amylase inhibitors are termed as starch blockers. They catalyze the hydrolysis of α-(1,4)-D-glycosidic linkages of starch and other glucose polymer. They play a significant role in inhibition of the activity of salivary and pancreatic amylase in vitro and in vivo. MethodThe present study was based on the purification, characterization and molecular docking studies of the alpha amylase inhibitor isolated from the kidney bean sample. The seed sample was collected from G.B. Pant Nagar University. The crude extract was prepared, the in-vitro studies and heat stability were determined. Following it, purification was carried out using ammonium sulphate precipitation and size exclusion chromatography was done. Later, characterization and molecular docking studies of the purified sample was done after obtaining GC–MS results. ResultThe in-vitro analysis was done and noted that the inhibitory activity was 96.5 ± 0.84 % post size exclusion chromatography. The molecular weight was about 54 kDa. The molecular docking studies revealed that there were interactions between the ligand molecules (the constituents that were selected from the GC–MS chromatogram peaks based on the height and area) and the human pancreatic alpha amylase (1HNY). The hydrogen bonding as affinity binding capacity was also been observed in each of the ligand–protein interaction. ConclusionFrom the tests, it has been elucidated that the alpha amylase inhibitor isolated from the sample has a great potential to serve as an anti-diabetic drug. However, in order to check the potential effects and to explore the optimization and development of the anti-diabetic drug the in-vivo studies can also be done further.

The quick screening of binding compounds and proteins for drug discovery and pharmacological research

In drug discovery and pharmacological research, early identification of target molecules for compounds with pharmacological effects is crucial. However, this process often requires significant effort and can be rate-limiting, thereby slowing down research progress. This paper introduces a simplified and rapid method for quick screening of binding compounds or proteins. Utilizing Affinity Selection Mass Spectrometry (ASMS), this technique efficiently detects compound-target binding through size-exclusion chromatography and mass spectrometry. ASMS offers high sensitivity and specificity, making it ideal for accurate identification of binding interactions. We have further enhanced ASMS to handle membrane proteins without solubilization, creating Binder Selection Technology (BST). BST allows screening against both soluble and challenging membrane proteins such as GPCRs and SLC transporters. By using cell membrane fractions or organelle fractions with high target molecule expression, BST efficiently identifies potential binding compounds. This innovative method constructs a comprehensive database of binding compounds for various targets, facilitating rapid hypothesis testing and pharmacological evaluation. Additionally, BST screens 17,000 proteins, including membrane proteins, using wheat germ cell-free and animal cell expression systems. This approach allows exploration of binding interactions without labeling compounds or immobilizing proteins, preserving their native state. BST is powerful for identifying targets of compounds with known pharmacological effects but unknown targets in animal or cell-based assays. By utilizing BST, researchers can overcome bottlenecks in early drug discovery, significantly enhancing research speed and success rates. This method represents a major advancement, providing an efficient and effective way to identify and validate target molecules in drug discovery.

Research Progress of Maillard Reaction and its Application in Processing of Traditional Chinese Medicine

The Maillard reaction, also known as the ‘non-enzymatic browning reaction’, involves a reaction between reducing sugars and amino compounds, including amino acids and proteins. The Maillard reaction has been increasingly used in the production of traditional Chinese medicine and food. This study thoroughly examines the mechanism of the reaction, the factors that influence it and the techniques used for extracting, separating and purifying Maillard reaction products (MRPs) in food and Chinese medicine processing. Additionally, the article explores the physiological functions of MRPs, highlighting their antioxidant, hepato-protective, antibacterial, immune-boosting, anti-mutagenic, anti-inflammatory, health-promoting, detoxifying and antiviral effects. MRPs have diverse outcomes because of various influencing factors; they can contain trace amounts of potentially harmful constituents, such as acrylamide, 5-hydroxymethylfurfural and advanced glycosylation products. The study also includes a comparative analysis of common methods used for the separation and purification of concentrated MRPs. These methods consist of ultrafiltration, macroporous resin, gel chromatography, ion exchange chromatography and dialysis. Results indicate that each method has advantages and disadvantages, and thus careful consideration should be placed on specific applications. Furthermore, a future outlook for the research and development of Maillard reaction and its products is proposed. Given that the physiological activities of MRPs are diverse, thorough qualitative and quantitative analysis of the reaction mechanisms, influencing factors and pharmacologically active ingredients is needed. This study offers valuable insights into the advancement of MRPs for industries involved in food and traditional Chinese medicinal material processing. These insights can be effectively utilised in healthcare and clinical practice, offering innovative ideas for scientific and technological professionals involved in research and development.

Improving separation efficiency of various micropollutants from water by polymer-enhanced ultrafiltration using oxidized alginate featuring less viscous and low filtration resistance

Polymer-enhanced ultrafiltration (PEUF) is an economical and energy-efficient alternative using a versatile water-soluble polymer (WSP) to remove micropollutants by electrostatic interactions, allowing size exclusion through ultrafiltration (UF) membranes due to the WSP being bigger than the pores of UF membranes. However, the WSP used in PEUF is so viscous that it inevitably causes significant filtration resistance and membrane fouling, reducing filtration and energy efficiency. To overcome this problem, in this study, sodium alginate (Alg), which significantly performs in removing cationic pollutants, was oxidized using sodium periodate to produce oxidized alginate (OxAlg). The retention and water flux profiles of OxAlg for three micropollutants, methylene blue (MB), ciprofloxacin, and tetramethylammonium hydroxide, using the PEUF method were investigated in terms of pH, ionic strength, polymer and pollutant amount. A comparison between Alg and OxAlg demonstrated 1.19% to 1.8% higher MB retention rates (99.31%-99.97%) and up to 4.07 times higher water flux depending on the amount of polymer added. Furthermore, OxAlg displayed excellent retention rates of the NF level and 20-60 times higher water flux than typical NF. This outstanding OxAlg performance was mainly due to OxAlg’s properties, such as the low viscosity, flexibility and the resulting accessibility, and appropriate size from the perspective of filtration. Lastly, OxAlg was found to have the potential to surpass the existing WSP in that it showed a higher maximum retention capacity than about 560 to 1250mg/g in the real-world pH enrichment test even before the saturation.

Development of an optimized SEC method for characterization of genome DNA leakage from adeno-associated virus products.

Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes due to their superior safety, relatively low immunogenicity, and ability to target diverse tissues. AAV gene therapy products are typically formulated as frozen liquid and stored below - 60 °C, and therefore are subjected to multiple freeze/thaw cycles during manufacturing and administration. Recent studies have shown that genome DNA leakage could be induced by freeze/thaw stress. DNA leakage from AAV capsids has been reported to potentially impact product stability, induce immune responses, and compromise product efficacy. Thus, further characterization to improve the understanding of genome DNA leakage is necessary for mitigating the risks associated with genome DNA leakage during AAV product development. In this work, we developed an optimized size-exclusion chromatography (SEC) method for quantifying the leakage of genome DNA across multiple different AAV serotypes and demonstrated satisfactory assay performance in sensitivity, precision, and linearity. Furthermore, we showed that this method could also be applied to quantifying additional quality attributes of AAV, including the percentage of full capsids and quantification of AAV dimers. By using this optimized SEC method, we demonstrated that significantly increased free DNA was observed with increasing freeze/thaw cycles or at a temperature approaching the onset temperature for genome DNA ejection, which was effectively mitigated by the addition of 1.5% w/v sucrose in the AAV formulation. Thus, this optimized SEC method can serve as an invaluable tool for AAV formulation, product, and process development in ensuring the quality and stability of AAV gene therapy products.

High-yield production of recombinant human myelin oligodendrocyte glycoprotein in SHuffle bacteria without a refolding step

Experimental autoimmune encephalomyelitis (EAE) is a model for central nervous system (CNS) autoimmune demyelinating diseases such as multiple sclerosis (MS) and MOG antibody-associated disease (MOGAD). Immunization with the extracellular domain of recombinant human MOG (rhMOG), which contains pathogenic antibody and T cell epitopes, induces B cell-dependent EAE for studies in mice. However, these studies have been hampered by rhMOG availability due to its insolubility when overexpressed in bacterial cells, and the requirement for inefficient denaturation and refolding. Here, we describe a new protocol for the high-yield production of soluble rhMOG in SHuffle cells, a commercially available E. coli strain engineered to facilitate disulfide bond formation in the cytoplasm. SHuffle cells can produce a soluble fraction of rhMOG yielding >100 mg/L. Analytical size exclusion chromatography multi-angle light scattering (SEC-MALS) and differential scanning fluorimetry of purified rhMOG reveals a homogeneous monomer with a high melting temperature, indicative of a well-folded protein. An in vitro proliferation assay establishes that purified rhMOG can be processed and recognized by T cells expressing a T cell receptor (TCR) specific for the immunodominant MOG35–55 peptide epitope. Lastly, immunization of wild-type, but not B cell deficient, mice with rhMOG resulted in robust induction of EAE, indicating a B cell-dependent induction. Our SHuffle cell method greatly simplifies rhMOG production by combining the high yield and speed of bacterial cell expression with enhanced disulfide bond formation and folding, which will enable further investigation of B cell-dependent EAE and expand human research of MOG in CNS demyelinating diseases.

Poxylated stereocomplexes from PEtOx-b-PLA diblock copolymers

An ω-hydroxyl terminated poly(2-ethyl-2-oxazoline) (PEtOx) was obtained by termination of the cationic ring-opening polymerization of 2-ethyl-2-oxazoline with acetate and subsequent transesterification with methanol. The resulting PEtOx-OH featuring a degree of polymerization (DP) of 20 was used as a macroinitiator for the ring-opening polymerization of l-lactide as well as d-lactide. The resulting PEtOx-b-PLA block copolymers featured DP values of 25, 50, 100 and 150, respectively, and were characterized by means of size exclusion chromatography, 1H NMR spectroscopy as well as matrix-assisted laser desorption ionization mass spectrometry. Differential scanning calorimetry, wide-angle x-ray scattering and polarized light microscopy indicated the formation of stereocomplexes in racemic blends of PEtOx-b-PLA with opposing chirality. Thereby, the amorphous PEtOx block did not affect the modification of the stereocomplex crystallites, as shown by comparison with data obtained from PLA homopolymer stereocomplexes. Similar to those, the degree of crystallinity and melting temperature decreased with increasing molar mass of the PLA in the stereocomplexes formed from PEtOx-b-PLA block copolymers.

Expression, purification, and biophysical analysis of a part of the C-terminal domain of human hypoxia inducible factor-2α (HIF-2α)

Hypoxia inducible factor 2α (HIF-2α) is a member of the basic helix-loop-helix(bHLH)-Per-Arnt-Sim (PAS) family of transcription factors. It is overexpressed in several cancers, associated with poor prognosis of the patients and resistance to treatment. Here, we study the residues 366-704 of the C-terminal end of human HIF-2α, which contains the N-transcriptional activation domain (NTAD), the oxygen-dependent degradation domain (ODD), and a part of the inhibitory domain (IH). An efficient protocol was developed to produce the 366-704 domain of human HIF-2α protein. Subsequently, we analyzed its biophysical characteristics using circular dichroism spectroscopy and size exclusion chromatography showing that the protein forms an antiparallel beta sheet conformation, and a computational model of the HIF-2α structure was produced. Our data offer new structural information for the unique biological properties of HIF-2α.

Structure-function relationship of PE11 esterase of Mycobacterium tuberculosis with respect to its role in virulence

The lipolytic enzymes of Mycobacterium tuberculosis play a critical role in immunomodulation and virulence. Among these proteins, PE11 which also belongs to the PE/PPE family, is the smallest (∼10.8 kDa) and play a significant role in cell wall remodelling and virulence. PE11 is established to be an esterase, but its enzymatic and structural properties are not yet characterized. In this study, using homology modelling we deduced the putative structure which shows the presence of both α-helix and β-sheet structures which is in close agreement with that observed by CD spectra of the purified protein. PE11 was found to contain a Gx3Sx4G motif homologous to canonical ‘GxSxG’ motif present in many serin hydrolases. The catalytic triad appears to be located within this motif as substitution of Serine26 and Glycine31 residues abrogated its enzymatic activity. Gel-filtration chromatography data indicate that PE11 possibly exists as dimer and tetramer showing positive cooperativity for binding its substrates. In addition, PE11 esterase activity was found to be critical for cell wall remodelling, antibiotic resistance and conferring survival advantages to M. tuberculosis. Our data suggest that PE11 can be targeted for designing potential therapeutic strategies.

Chromatographic analysis of branched and debranched starch structure: Variability of their results

Starch structure determination is crucial for understanding its properties and applications. However, method-dependent variations in size determination can lead to ambiguous interpretations. This study investigates the ambiguities in starch structure determination by evaluating the variation in size of four commercial branched starches as determined by average molar mass, gyration radius, hydrodynamic radius, and chain-length distribution. The starches were analyzed using high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS) and refractive index detector (RI), and after debranching by HPSEC-RI and high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detector (PAD). MALLS-derived MW values for amylose and amylopectin were higher than those obtained using the hydrodynamic volume method. The molecular weight of amylopectin chains, determined by the degree of polymerization (DP), ranged between 2.1 and 2.9 × 103 g/mol for short chains, and between 4.1 × 103 and 1.1 × 104 g/mol for long chains. Differences in amylopectin chain content were observed between HPSEC-RI and HPAEC-PAD, highlighting the complementary nature of these techniques. The study underscores the need for standardizing chromatography-based methodologies in starch research, particularly with the advent of new technologies.

An improved synthesis of alkyl AMP esters

Enzymes belonging to the Acyl-CoA/NRPS/Luciferase (ANL) superfamily of enzymes, are significant targets in drug development. One member of this superfamily is Acetyl-CoA Synthetase (ACS). This enzyme is an emerging target for the treatment of various infectious diseases and cancer. Alkyl AMP esters have emerged as potent inhibitors of ACS. Previous methods for synthesizing these esters often involved water-based reactions or necessitate purification through reverse phase prep-HPLC or ion-exchange chromatography. To address these challenges, we developed a new approach utilizing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) to couple 5′-adenylic acid to the corresponding alcohol, utilizing triethylamine as the base. This method yielded primary, secondary, unsaturated, and cyclic alcohols in excellent yields. Importantly, we optimized the reaction conditions to achieve excellent yield and purity without the need for reverse phase prep-HPLC or ion exchange chromatography. Instead, purification was achieved through silica gel chromatography using Biotage ® Sfar spherical silica gel.

Antibacterial Polyketides from the Plant Endophytic Fungus Fusarium sp.

Background: Endophytic fungi have been recognized as new sources of natural products with a variety of biological activities, providing lead compounds for drug discovery and development. Objective: The objective of this study is to isolate and identify the secondary metabolites from the plant endophytic fungus Fusarium sp. HJY2 and evaluate their antibacterial activities. Methods: The compounds were isolated and purified by the methods of silica gel column chromatography, Sephadex LH-20 gel chromatography, and semi-preparative high performance liquid chromatography (HPLC). The structures of the isolated compounds were elucidated by comparing the NMR and MS spectroscopic data with those of pieces of literature. The antibacterial activities were evaluated by the broth microdilution method. Results: Seven polyketides were isolated from the fermented extracts of the fungus Fusarium sp. HJY2 and identified as sydowinol (1), dihydrolateropyrone (2), 13-oxo-9Z,11E-octadecadienoic acid (3), (E)-ferulic acid (4), 4-hydroxyphenylacetic acid (5), methyl 2-(2-hydroxyphenyl)acetate (6) and 4-hydroxy-3-methoxyacetophenone (7). Compound 3 exhibited moderate antibacterial activities against Bacillus subtilis, Mycobacterium smegmatis, Ralstonia solanacearum, and Xanthomonas campestris pv. campestris with MIC values of 40, 40, 80 and 40 μg/mL, respectively. Conclusion: Seven compounds were isolated from the plant endophytic fungus Fusarium sp. HJY2. Compound 1 was isolated from the Fusarium genus for the first time. Compound 3 showed moderate antibacterial activities.

Degradation of (1→3)(1→6)-α-D-dextran by ultrasound: Molecular weight, viscosity and kinetics

The (1→3)(1→6)-α-D-dextran (alternating dextran) produced by Leuconostoc citreum SK24.002 is a novel functional exopolysaccharide, and its low molecular weight derivatives have potential applications in the food, pharmaceutical, and chemical industries. In this work, we used sonication, a green polysaccharide disruption method, to study the degradation process of this dextran by changing the intensity and duration of the sonication treatment and the concentration of the dextran solution. The molecular weight and viscosity of the dextran products were measured with a high-performance size exclusion column chromatography–multi-angle laser light scattering–refractive index system and by rheometry, respectively. The degradation efficiency of dextran was directly affected by the duration and intensity of the ultrasonic treatment and the concentration of the dextran solution. The polydispersity index fluctuated as the duration of the sonication treatment increased. The combination of a high intensity (672 W/cm2) and long (120 min) sonication treatment and a low solution concentration (3 g dextran/100 mL) was most effective for reducing the apparent and complex viscosities of dextran. The storage modulus of dextran was always slightly larger than its loss modulus, indicating that it formed a gel-like structure. The second-order kinetic model (1/Mwt - 1/Mw0 = kt) was the best fit to explain the degradation dynamics of dextran by sonication at intensities of 168 W/cm2–834 W/cm2 and with dextran solution concentrations of 1 g/100 mL – 7 g/100 mL. Our findings show that sonication is an effective way to reduce the molecular weight of alternating dextran.

Identification of fibrinogen as a plasma protein binding partner for lecanemab biosimilar IgG.

Recombinant monoclonal therapeutic antibodies like lecanemab, which target amyloid beta in Alzheimer's disease, offer a promising approach for modifying the disease progression. Due to its relatively short half-life, lecanemab administered as a bi-monthly infusion (typically 10 mg/kg) has a relatively brief half-life. Interaction with abundant plasma proteins binder in the bloodstream can affect pharmacokinetics of drugs, including their half-life. In this study, we investigated potential plasma protein binding (PPB) interaction to lecanemab using lecanemab biosimilar. Lecanemab biosimilar used in this study was based on publicly available sequences. ELISA and western blotting were used to assess lecanemab biosimilar immunoreactivity in the fractions of human plasma obtained through size exclusion chromatography. The binding of lecanemab biosimilar to candidate plasma binders was confirmed by western blotting, ELISA, and surface plasmon resonance analysis. Using a combination of equilibrium dialysis, ELISA, and western blotting in human plasma, we first describe the presence of likely PPB partners to lecanemab biosimilar and then identify fibrinogen as one of them. Utilizing surface plasmon resonance, we confirmed that lecanemab biosimilar does bind to fibrinogen, although with lower affinity than to monomeric amyloid beta. In the context of lecanemab therapy, these results imply that fibrinogen levels could impact the levels of free antibodies in the bloodstream and that fibrinogen might serve as a reservoir for lecanemab. More broadly, these results indicate that PPB may be an important consideration when clinically utilizing therapeutic antibodies in neurodegenerative disease.

Fabrication of model ultrafiltration membranes with uniform, high aspect ratio pores

In this manuscript, we report the facile fabrication of large-area model membranes with highly uniform and high aspect ratio pores with diameters <20 nm. These membranes are useful for fundamental investigations of separation by size exclusion in the ultrafiltration regime, where species to be separated from solution have dimensions of 1–100 nm. Such investigations require membranes with narrow pores and high aspect ratios such that the Hagen–Poiseuille equation is followed, enabling well-known models such as the hindered transport model to be evaluated and other affecting factors to be ignored. We demonstrate that the sub-20 nm pores in the membrane are of sufficiently high aspect ratio such that water flux through the membrane is consistent with the Hagen–Poiseuille equation. The fabrication relies on self-assembling block copolymers to form uniform, densely packed patterns with sub-20 nm resolution, sequential infiltration synthesis to convert the block copolymer in situ into a mask with adequate contrast to etch pores with an aspect ratio >5, and low-resolution photolithography to transfer the pattern over a large area into a silicon nitride membrane. Model membranes with narrow pore-size distribution fabricated in this way provide the means to investigate parameters that impact size-selective ultrafiltration separations such as the relationships between solute or particle size and pore size, their distributions, and rejection profiles, and, therefore, test the validity or limits of separation models.

Structural, molecular, and physicochemical properties of starch in high-amylose durum wheat lines

There is growing interest in the development of high-amylose cereals such as wheat due to their functional properties and nutritional value in foods. The increase in amylose content is associated with significant changes in the physicochemical, molecular, and structural characteristics of wheat starch which could affect its processing quality. The present study aimed to analyze the physicochemical, molecular, and structural characteristics of high-amylose starch from three durum wheat near isogenic lines (NILs), each obtained using three different recurrent parents. Morphology, granule size distribution, pasting and thermal properties, X-ray diffraction, and structural features were determined. The NILs showed the highest amylose content (65.3 and 69.8%), granules with an elongated shape, size between the wild-type and the mutant, restricted swelling in the pasting profile, the highest average and final gelatinization temperature, and B-polymorphism. The starches from the NILs showed the highest molar mass of amylopectin (AP) (7.0–7.6 × 107 Da) and the lowest amylose (AM) (1.1–1.8 × 106 Da), an issue associated with the biosynthesis of both components. The study on the debranched sample by high-performance size-exclusion chromatography showed that the high-amylose starch had the highest content of long B2 and B3 chains of AP (23.9–26.6%), which was corroborated with the high-performance anion-exchange chromatography analysis where the chains with degree of polymerization >37 had the highest content (18.3–19.1%). The PC1 is highly associated with the MwAM and the AM fraction in the debranched starch analyzed by HPSEC. Characterizing high-amylose durum wheat starch provides information to determine the structure-function relationship and design strategies to produce crosses with structural characteristics of AM and AP for specific applications.

Measurement of α-synuclein as protein cargo in plasma extracellular vesicles

Extracellular vesicles (EVs) are released by all cells and hold great promise as a class of biomarkers. This promise has led to increased interest in measuring EV proteins from both total EVs as well as brain-derived EVs in plasma. However, measuring cargo proteins in EVs has been challenging because EVs are present at low levels, and EV isolation methods are imperfect at separating EVs from free proteins. Thus, knowing whether a protein measured after EV isolation is truly inside EVs is difficult. In this study, we developed methods to measure whether a protein is inside EVs and quantify the ratio of a protein in EVs relative to total plasma. To achieve this, we combined a high-yield size-exclusion chromatography protocol with an optimized protease protection assay and Single Molecule Array (Simoa) digital enzyme-linked immunoassays (ELISAs) for ultrasensitive measurement of proteins inside EVs. We applied these methods to analyze α-synuclein and confirmed that a small fraction of the total plasma α-synuclein is inside EVs. Additionally, we developed a highly sensitive Simoa assay for phosphorylated α-synuclein (phosphorylated at the Ser129 residue). We found enrichment in the phosphorylated α-synuclein to total α-synuclein ratio inside EVs relative to outside EVs. Finally, we applied the methods we developed to measure total and phosphorylated α-synuclein inside EVs from Parkinson's disease and Lewy body dementia patient samples. This work provides a framework for determining the levels of proteins in EVs and represents an important step in the development of EV diagnostics for diseases of the brain, as well as other organs.

Development of imaged capillary isoelectric focusing as a platform mispairing byproduct testing method for asymmetric WuXiBody-based bispecific antibodies

This study explores the application of imaged capillary isoelectric focusing (icIEF) to distinguish and quantify mispairing byproducts in asymmetric WuXiBody-based bispecific antibodies (AsWXbsAbs). Bispecific antibody (BsAb), developed using Knobs-into-Holes (KiH) technology, often result in byproducts such as knob-knob (KK) and hole-hole (HH) homodimers, which share similar sizes with the target BsAb, complicating their separation by traditional methods like size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Our approach leverages the unique pI differences introduced by substituting the CH1/CL domain with the T cell receptor (TCR) constant domain in AsWXbsAbs. This modification enables icIEF to effectively differentiate between the KK and HH homodimers and the target BsAb. Through the construction and expression of heavy and light chain variants, we validated that the experimental pI values aligned with theoretical predictions, confirming icIEF’s capability in distinguishing these entities. Enrichment of in-process K-related and H-related species was achieved, allowing for high-purity samples necessary for icIEF method development. The method was qualified and showed good specificity and linearity, with a quantitation limit of 4 % for K-related species (R2 = 0.9919) and 1 % for H-related species (R2 = 0.9805). This method was used effectively as an in-process test and release assay, proving its simple and quick utility in multiple AsWXbsAbs projects.

2D Membranes Interlayered with Bimetallic Metal-Organic Frameworks for Lithium Separation from Brines.

Efficient lithium extraction from salt lakes is essential for a sustainable resource supply. This study tackles the challenge of separating Li+ from Mg2+ in complex brines by innovatively integrating two-dimensional (2D) graphene oxide (GO) with bimetallic metal-organic frameworks (MOFs). Zn2+ and Co2+ ions are confined within GO interlayers through an in situ synthesis, forming a 2D Zn-Co MOFs/GO membrane (Zn-Co-GOM). This design exploits the unique advantages of bimetallic MOFs, including enhanced structural stability and superior ion separation capabilities due to the synergistic effects of Zn and Co. The Zn-Co-GOM demonstrates an impressive separation factor of 191 for Li+ over Mg2+, significantly surpassing traditional membranes. This exceptional selectivity is achieved through a combination of size exclusion effects and ion transport energy barriers. Our approach not only enhances the practical application of membrane technology for lithium extraction from salt lakes but also provides valuable insights into the underlying separation mechanisms.

Biochemical characterization of a recombinant laccase from Halalkalibacterium halodurans C-125 and its application in the biotransformation of organic compounds

ObjectivesThis study aimed to produce an engineered recombinant laccase from extremophilic Halalkalibacterium halodurans C-125 (Lac-HhC-125) with higher protein yield, into a more active conformation and with properties that meet the fundamental needs of biotechnological application.ResultsThe rLac-HhC125 was partially purified by size exclusion chromatography and concentrated by ultrafiltration (10 kDa) with a yield of 57.6%. Oxidation reactions showed that adding 2 mM CuSO4 to the assay solution led to activating the laccase. To increase its initial activity, the rLac-HhC125 was treated at 50 °C for 20 min before the assays, improving its performance by fourfold using the syringaldazine as a substrate. When treated with EDTA, methanol, ethanol, and DMSO, the rLac-HhC125 maintained more than 80% of its original activity. Interestingly, the acetonitrile induced a twofold activity of the rLac-HhC125. The putative rLac-HhC125 demonstrated a capability of efficient transformation of different organic compounds at pH 6, known as dye precursors, into coloured molecules.ConclusionThe rLac-HhC125 was active at high temperatures and alkaline pH, exhibited tolerance to organic solvents, and efficiently transformed different hydroxy derivatives into coloured compounds, which indicates that it can be used in various biotechnological processes.