Charge neutralization and ß-elimination cleavage mechanism of family 42 L-rhamnose-α-1,4-D-glucuronate lyase revealed using neutron crystallography.

Gum arabic (GA) is widely used as an emulsion stabilizer and edible coating and consists of a complex carbohydrate moiety with a rhamnosyl-glucuronate group capping the non-reducing ends. Enzymes that can specifically cleave the glycosidic chains of GA and modify their properties are valuable for structural analysis and industrial application. Cryogenic X-ray crystal structure of GA-specific L-rhamnose-α-1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1), belonging to the polysaccharide lyase (PL) family 42, has been previously reported. To determine the specific reaction mechanism based on its hydrogen-containing enzyme structure, we performed joint X-ray/neutron crystallography of FoRham1. Large crystals were grown in the presence of L-rhamnose (a reaction product), and neutron and X-ray diffraction datasets were collected at room temperature at 1.80 and 1.25 Å resolutions, respectively. The active site contained L-rhamnose and acetate, the latter being a partial analog of glucuronate. Incomplete H/D exchange between Arg166 and acetate suggested that a strong salt-bridge interaction was maintained. Doubly deuterated His105 and deuterated Tyr150 supported the interaction between Arg166 and the acetate. The unique hydrogen-rich environment functions as a charge neutralizer for glucuronate and stabilizes the oxyanion intermediate. The NE2 atom of His85 was deprotonated and formed a hydrogen bond with the deuterated O1 hydroxy of L-rhamnose, indicating the function of His85 as the base/acid catalyst for bond cleavage via ß-elimination. Asp83 functions as a pivot between the two catalytic histidine residues by bridging them. This His-His-Asp structural motif is conserved in the PL 24, 25, and 42 families.

Multichannel Multijunction Droplet Microfluidic Device to Synthesize Hydrogel Microcapsules with Different Core-Shell Structures and Adjustable Core Positions.

Core-shell hydrogel microcapsules have sparked great interest due to their unique characteristics and prospective applications in the medical, pharmaceutical, and cosmetic fields. However, complex synthetic procedures and expensive costs have limited their practical application. Herein, we designed and prepared several multichannel and multijunctional droplet microfluidic devices based on soft lithography for the effective synthesis of core-shell hydrogel microcapsules for different purposes. Additionally, two different cross-linking processes (ultraviolet (UV) exposure and interfacial polymerization) were used to synthesize different types of core-shell structured hydrogel microcapsules. Hydrogel microcapsules with gelatin methacryloyl (GelMA) as the core and polyacrylamide (PAM) as the thin shell were synthesized using UV cross-linking. Using an interfacial polymerization process, another core-shell structured microcapsule with GelMA as the core and Ca2+ cross-linked alginate with polyethylenimine (PEI) as the shell was constructed, and the core diameter and total droplet diameter were flexibly controlled by carving. Noteworthy, these hydrogel microcapsules exhibit stimuli-responsiveness and controlled release ability. Overall, a novel technique was developed to successfully synthesize various hydrogel microcapsules with core-shell microstructures. The hydrogel microcapsules possess a multilayered structure that facilitates the coassembly of cells and drugs, as well as the layered assembly of multiple drugs, to develop synergistic therapeutic regimens. These adaptable and controllable hydrogel microdroplets shall held great promise for multicell or multidrug administration as well as for high-throughput drug screening.

Transforming the Stability, Encapsulation, and Sustained Release Properties of Calcium Alginate Beads through Gel-Confined Coacervation.

Calcium alginate (Ca2+/alginate) gel beads find use in diverse applications, ranging from drug delivery and tissue engineering to bioprocessing, food formulation, and agriculture. Unless modified, however, these gels have limited stability in alkaline media (including phosphate buffers), and their high solute permeability limits their ability to efficiently encapsulate and slowly release water-soluble small molecules. Here, we show how these limitations can be addressed by mixing the alginate solutions used in the bead preparation with the nontoxic anionic polymer polyphosphate (PP). Upon complexing Ca2+ ions, PP undergoes complex coacervation (i.e., liquid/liquid phase separation into a Ca2+/PP-rich coacervate phase and a dilute supernatant phase). At lower PP concentrations, the Ca2+/PP coacervate appears to simply remain dispersed within the beads. Though its presence makes the beads more stable in alkaline media (phosphate-buffered saline and seawater), it has little impact on the bead stiffness, morphology, and (at least in the absence of substantial payload/coacervate association) encapsulation and release properties. When the PP concentrations exceed a critical value, however, Ca2+/PP coacervation within the gelling Ca2+/alginate beads collapses the resulting beads into more compact, interpenetrating polymer networks. Besides their enhanced stability to alkaline environments, these hybrid beads exhibit irregular morphologies with wrinkled and dimpled surface structures and macroscopic (closed) internal pores, and their collapse into these polymer-rich networks also makes them significantly stiffer than their PP-free counterparts. Crucially, these beads also exhibit a much lower solute permeability, which enables highly efficient encapsulation and multiday release of water-soluble small molecules (with the beads encapsulating >90% of the added model payload and sustaining its release over 3-5 d). Collectively, these findings provide a mild and simple (single-step) pathway to generating ionically cross-linked alginate beads with significantly enhanced stability, encapsulation efficiency, and sustained release.

Self-Assembly Reinforced Alginate Fibers for Enhanced Strength, Toughness, and Bone Regeneration.

Material reinforcement commonly exists in a contradiction between strength and toughness enhancement. Herein, a reinforced strategy through self-assembly is proposed for alginate fibers. Sodium alginate (SA) microstructures with regulated secondary structures are assembled in acidic and ethanol as reinforcing units for alginate fibers. Acidity increases the flexibility of the helix and contributes to enhanced extendibility. Ethanol is responsible for formation of a stiff ß-sheet, which enhances the modulus and strength. The structurally engineered SA assembly exhibits robust mechanical compatibility, and thus reinforced alginate fibers possess an improved tensile strength of 2.1 times, a prolonged elongation of 1.5 times, and an enhanced toughness of 3.0 times compared with SA fibers without reinforcement. The reinforcement through self-assembly provides an understanding of strengthening and toughening mechanism based on secondary structures. Due to a similar modulus with bones, reinforced alginate fibers exhibit good efficacy in accelerating bone regeneration in vivo.

Simultaneous adsorption of chromate and arsenate onto ferrihydrite/alginate composite beads: Competition and mechanism.

Ferrihydrite is an effective adsorbent of chromate and arsenate. In order to gain insight into the application of ferrihydrite in water treatment, macroporous alginate/ferrihydrite beads, synthesized using two different methods (internal and encapsulation processes), were used in this work. The properties of the ferrihydrite were assessed using various techniques, including X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Brunauer-Emmett-Teller (BET) theory, and zetametry. The results showed that the specific surface area of the ferrihydrite was 242 m2/g, and the PZC was pH8. The kinetic and isotherm adsorption properties of the ferrihydrite were evaluated in this study. The results indicate that the pseudo second-order and Freundlich models accurately describe the kinetic and isotherm adsorption properties of chromates and arsenates. For chromate removal, ferrihydrite exhibited a relatively high adsorption capacity (40.7 mgCr/g) compared to other adsorbents. However, the arsenate adsorption capacity of MFHB-SI (140.8 mgAs/g) was shown to be the most optimal. The internal synthesis process was suitable for arsenate retention due to the resulting arsenate precipitation. The competitive adsorption analyses indicated that the presence of chromate does not limit the adsorption of arsenate. However, the presence of arsenate almost completely inhibits the adsorption of chromate when the arsenate concentration is above 50 mg/L, due to the precipitation reaction of arsenate.

Bioremediation of 2,4,6-trichlorophenol by extracellular enzymes of white rot fungi immobilized with sodium alginate/hydroxyapatite/chitosan microspheres.

Hydroxyapatite, a calcium phosphate biomass material known for its excellent biocompatibility, holds promising applications in water, soil, and air treatment. Sodium alginate/hydroxyapatite/chitosan (SA-HA-CS) microspheres were synthesized by cross-linking sodium alginate with calcium chloride. These microspheres were carriers for immobilizing extracellular crude enzymes from white rot fungi through adsorption, facilitating the degradation of 2,4,6-trichlorophenol (2,4,6-TCP) in water and soil. At 50 °C, the immobilized enzyme retained 87.2% of its maximum activity, while the free enzyme activity dropped to 68.86%. Furthermore, the immobilized enzyme maintained 68.09% of its maximum activity at pH 7, surpassing the 51.16% observed for the free enzyme. Under optimal conditions (pH 5, 24 h), the immobilized enzymes demonstrated a remarkable 94.7% removal rate for 160 mg/L 2,4,6-TCP, outperforming the 62.1% achieved by free crude enzymes. The degradation of 2,4,6-TCP by immobilized and free enzymes adhered to quasi-first-order degradation kinetics. Based on LC-MS, the plausible biodegradation mechanism and reaction pathway of 2,4,6-TCP were proposed, with the primary degradation product identified as 1,2,4-trihydroxybenzene. The immobilized enzyme effectively removed 72.9% of 2,4,6-TCP from the soil within 24 h. The degradation efficiency of the immobilized enzyme varied among different soil types, exhibiting a negative correlation with soil organic matter content. These findings offer valuable insights for advancing the application of immobilized extracellular crude enzymes in 2,4,6-TCP remediation.

The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation.

Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

Batch-mode degradation of high-strength phenolic pollutants by Pseudomonas aeruginosa strain STV1713 immobilized on single and hybrid matrices.

Controlled environments are pivotal in all bioconversion processes, influencing the efficacy of biocatalysts. In this study, we designed a batch bioreactor system with a packed immobilization column and a decontamination chamber to enhance phenol and 2,4-dichlorophenol degradation using the hyper-tolerant bacterium Pseudomonas aeruginosa STV1713. When free cells were employed to degrade phenol and 2,4-DCP at a concentration of 1000 mg/L, the cells completely removed the pollutants within 28 h and 66 h, respectively. Simultaneous reductions in chemical oxygen demand and biological oxygen demand were observed (phenol: 30.21 mg/L/h and 16.92 mg/L/h, respectively; 2,4-dichlorophenol: 12.85 mg/L/h and 7.21 mg/L/h, respectively). After assessing the degradation capabilities, the bacterium was immobilized on various matrices (sodium alginate, alginate-chitosan-alginate and polyvinyl alcohol-alginate) to enhance pollutant removal. Hybrid immobilized cells exhibited greater tolerance and degradation capabilities than those immobilized in a single matrix. Among them, polyvinyl alcohol-alginate immobilized cells displayed the highest degradation capacities (up to 2000 mg/L for phenol and 2500 mg/L for 2,4-dichlorophenol). Morphological analysis of the immobilized cells revealed enhanced cell preservation in hybrid matrices. Furthermore, the elucidation of the metabolic pathway through the catechol dioxygenase enzyme assay indicated higher activity of the catechol 1,2-dioxygenase enzyme, suggesting that the bacterium employed an ortho-degradation mechanism for pollutant removal. Additionally, enzyme zymography confirmed the presence of catechol 1,2-dioxygenase, with the molecular weight of the enzyme determined as 245 kDa.

A novel, microfluidic high-throughput single-cell encapsulation of human bone marrow mesenchymal stromal cells.

The efficacy of stem-cell therapy depends on the ability of the transplanted cells to escape early immunological reactions and to be retained at the site of transplantation. The use of tissue engineering scaffolds or injectable biomaterials as carriers has been proposed, but they still present limitations linked to a reliable manufacturing process, surgical practice and clinical outcomes. Alginate microbeads are potential candidates for the encapsulation of mesenchymal stromal cells with the aim of providing a delivery carrier suitable for minimally-invasive and scaffold-free transplantation, tissue-adhesive properties and protection from the immune response. However, the formation of stable microbeads relies on the cross-linking of alginate with divalent calcium ions at concentrations that are toxic for the cells, making control over the beads' size and a single-cell encapsulation unreliable. The present work demonstrates the efficiency of an innovative, high throughput, and reproducible microfluidic system to produce single-cell, calcium-free alginate coatings of human mesenchymal stromal cells. Among the various conditions tested, visible light and confocal microscopy following staining of the cell nuclei by DAPI showed that the microfluidic system yielded an optimal single-cell encapsulation of 2000 cells/min in 2% w/v alginate microcapsules of reproducible morphology and an average size of 28.2 ± 3.7 µm. The adhesive properties of the alginate microcapsules, the viability of the encapsulated cells and their ability to escape the alginate microcapsule were demonstrated by the relatively rapid adherence of the beads onto tissue culture plastic and the cells' ability to gradually disrupt the microcapsule shell after 24 h and proliferate. To mimic the early inflammatory response upon transplantation, the encapsulated cells were exposed to proliferating macrophages at different cell seeding densities for up to 2 days and the protection effect of the microcapsule on the cells assessed by time-lapse microscopy showing a shielding effect for up to 48 h. This work underscores the potential of microfluidic systems to precisely encapsulate cells by good manufacturing practice standards while favouring cell retention on substrates, viability and proliferation upon transplantation.

Development of alginate beads for precise environmental release applications: A design of experiment based approach and analysis.

Controlled release of active ingredients are important for drug delivery and more recently environmental applications including modulated dosing of chemical and biological controls. This study demonstrates the importance of investigating various material science factors that can influence the diffusion rates of alginate beads to improve and tune their performance for marine environmental applications. This investigation aimed to design a rational workflow to aid in leveraging alginate bead use as a carrier matrix for releasing a specific active agent into water. Experiments were conducted to focus on the narrow a large list of relevant material formulation parameters, which included chitosan molecular weight, chitosan concentration, calcium concentration, drop height, and bead size. Once the most relevant material preparation methods were screened, a more robust statistic Design of Experiments approach was performed and results determined the important (and unimportant) factors for increasing dye release kinetics in marine water. The process was further streamlined by narrowing the critical experimental factors to a three-level based on the prior analysis: chitosan MW, chitosan concentration, and bead size. Analysis of the collected data indicated that while chitosan MW had a negligible impact (Fstatistic = 0.22), bead size (Fstatistic = 60.33) significantly influenced the diffusion rates based on surface area. However, chitosan MW had minor effects where lower chitosan MW enabled higher product release rates. This case investigation was a novel application of the design of experiment approach towards environmental applications to understand differences in release rates to marine waters for the first time and the workflow provided also serve as the basis for researchers to optimize other environmental applications requiring optimization when it is unknown how a large number of formulation variables will impact performance in different environmental scenarios.

Assessment of calcium alginate gels as wall materials for encapsulation systems.

BACKGROUND: Calcium alginate gels are widely used to encapsulate active compounds. Some characteristic parameters of these gels are necessary to describe the release of active compounds through mechanistic mathematical models. In this work, transport and kinetics properties of calcium alginate gels were determined through simple experimental techniques. RESULTS: The weight-average molecular weight ( M ¯ w = 192 × 103 Da) and the fraction of residues of α-l-guluronic acid ( F G = 0.356) of sodium alginate were determined by capillary viscometry and 1 H-nuclear magnetic resonance at 25 °C, respectively. Considering the half egg-box model, both values were used to estimate the molecular weight of calcium alginate as M g = 2.02 × 105 Da. An effective diffusion coefficient of water ( D eff , w = 2.256 × 10-9 m2 s-1 ) in calcium alginate was determined using a diffusion cell at 37 °C. Finally, a kinetics constant of depolymerization ( k m = 9.72 × 10-9 m3 mol-1 s-1 ) of calcium alginate was obtained considering dissolution of calcium to a medium under intestinal conditions. CONCLUSION: The experimental techniques used are simple and easily reproducible. The obtained values may be useful in the design, production, and optimization of the alginate-based delivery systems that require specific release kinetics of the encapsulated active compounds. © 2023 Society of Chemical Industry.

The Pseudomonas aeruginosa homeostasis enzyme AlgL clears the periplasmic space of accumulated alginate during polymer biosynthesis.

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of chronic infection in the lungs of individuals with cystic fibrosis. After colonization, P. aeruginosa often undergoes a phenotypic conversion to mucoidy, characterized by overproduction of the alginate exopolysaccharide. This conversion is correlated with poorer patient prognoses. The majority of genes required for alginate synthesis, including the alginate lyase, algL, are located in a single operon. Previous investigations of AlgL have resulted in several divergent hypotheses regarding the protein's role in alginate production. To address these discrepancies, we determined the structure of AlgL and, using multiple sequence alignments, identified key active site residues involved in alginate binding and catalysis. In vitro enzymatic analysis of active site mutants highlights R249 and Y256 as key residues required for alginate lyase activity. In a genetically engineered P. aeruginosa strain where alginate biosynthesis is under arabinose control, we found that AlgL is required for cell viability and maintaining membrane integrity during alginate production. We demonstrate that AlgL functions as a homeostasis enzyme to clear the periplasmic space of accumulated polymer. Constitutive expression of the AlgU/T sigma factor mitigates the effects of an algL deletion during alginate production, suggesting that an AlgU/T-regulated protein or proteins can compensate for an algL deletion. Together, our study demonstrates the role of AlgL in alginate biosynthesis, explains the discrepancies observed previously across other P. aeruginosa ΔalgL genetic backgrounds, and clarifies the existing divergent data regarding the function of AlgL as an alginate degrading enzyme.

Identification of xylan arabinosyl 2-O-xylosyltransferases catalyzing the addition of 2-O-xylosyl residue onto arabinosyl side chains of xylan in grass species.

Grass xylan, the major hemicellulose in both primary and secondary cell walls, is heavily decorated with α-1,3-linked arabinofuranosyl (Araf) residues that may be further substituted at O-2 with xylosyl (Xyl) or Araf residues. Although xylan 3-O-arabinosyltransferases (XATs) catalyzing 3-O-Araf addition onto xylan have been characterized, glycosyltransferases responsible for the transfer of 2-O-Xyl or 2-O-Araf onto 3-O-Araf residues of xylan to produce the Xyl-Araf and Araf-Araf disaccharide side chains remain to be identified. In this report, we showed that a rice GT61 member, named OsXAXT1 (xylan arabinosyl 2-O-xylosyltransferase 1) herein, was able to mediate the addition of Xyl-Araf disaccharide side chains onto xylan when heterologously co-expressed with OsXAT2 in the Arabidopsis gux1/2/3 (glucuronic acid substitution of xylan 1/2/3) triple mutant that lacks any glycosyl substitutions. Recombinant OsXAXT1 protein expressed in human embryonic kidney 293 cells exhibited a xylosyltransferase activity catalyzing the addition of Xyl from UDP-Xyl onto arabinosylated xylooligomers. Consistent with its function as a xylan arabinosyl 2-O-xylosyltransferase, CRISPR-Cas9-mediated mutations of the OsXAXT1 gene in transgenic rice plants resulted in a reduction in the level of Xyl-Araf disaccharide side chains in xylan. Furthermore, we revealed that XAXT1 close homologs from several other grass species, including switchgrass, maize, and Brachypodium, possessed the same functions as OsXAXT1, indicating functional conservation of XAXTs in grass species. Together, our findings establish that grass XAXTs are xylosyltransferases catalyzing Xyl transfer onto O-2 of Araf residues of xylan to form the Xyl-Araf disaccharide side chains, which furthers our understanding of genes involved in xylan biosynthesis.

How to Determine a Suitable Alginate for Biofabrication Approaches using an Extensive Alginate Library?

Alginate-based hydrogels are a promising class of biomaterials due to their usability, biocompatibility, and high water-binding capacity which is the reason for their broad use in biofabrication. One challenge of these biomaterials is, however, the lack of cell adhesion motifs. This drawback can be overcome by oxidizing alginate to alginate dialdehyde (ADA) and by subsequent cross-linking with gelatin (GEL) to fabricate ADA-GEL hydrogels, which offer improved cell-material interactions. The present work investigates four pharmaceutical grade alginates of different algae sources and their respective oxidized forms regarding their molecular weight and M/G ratio using 1H NMR spectroscopy and gel permeation chromatography. In addition, three different methods for determining the degree of oxidation (% DO) of ADA, including iodometric, spectroscopic, and titration methods, are applied and compared. Furthermore, the aforementioned properties are correlated with the resulting viscosity, degradation behavior, and cell-material interactions to predict the material behavior in vitro and thus choose a suitable alginate for an intended application in biofabrication. In the framework of the present work, easy and practicable detection methods for the investigations of alginate-based bioinks were summarized and shown. In this regard, the success of oxidation of alginate was confirmed by the three aforementioned methods and was further proven by solid-state 13C NMR, for the first time in the literature, that only guluronic acid (G) was attacked during the oxidation, leading to the formation of hemiacetals. Furthermore, it was shown that ADA-GEL hydrogels of alginates with longer G-blocks are more suitable for long-term experiments due to their stability over an incubation period of 21 days, while ADA-GEL hydrogels of alginates with longer mannuronic acid (M)-blocks are more suitable for short-term applications such as sacrificial inks due to their extensive swelling and subsequent loss of shape. Finally, it was proven that the M/G ratio did not show any influence on the biocompatibility or printability of the investigated alginate-based hydrogels. The physicochemical findings provide an alginate library for tailored application in biofabrication.

Yeast extracts from different manufacturers and supplementation of amino acids and micro elements reveal a remarkable impact on alginate production by A. vinelandii ATCC9046.

BACKGROUND: In research and production, reproducibility is a key factor, to meet high quality and safety standards and maintain productivity. For microbial fermentations, complex substrates and media components are often used. The complex media components can vary in composition, depending on the lot and manufacturing process. These variations can have an immense impact on the results of biological cultivations. The aim of this work was to investigate and characterize the influence of the complex media component yeast extract on cultivations of Azotobacter vinelandii under microaerobic conditions. Under these conditions, the organism produces the biopolymer alginate. The focus of the investigation was on the respiration activity, cell growth and alginate production. RESULTS: Yeast extracts from 6 different manufacturers and 2 different lots from one manufacturer were evaluated. Significant differences on respiratory activity, growth and production were observed. Concentration variations of three different yeast extracts showed that the performance of poorly performing yeast extracts can be improved by simply increasing their concentration. On the other hand, the results with well-performing yeast extracts seem to reach a saturation, when their concentration is increased. Cultivations with poorly performing yeast extract were supplemented with grouped amino acids, single amino acids and micro elements. Beneficial results were obtained with the supplementation of copper sulphate, cysteine or a combination of both. Furthermore, a correlation between the accumulated oxygen transfer and the final viscosity (as a key performance indicator), was established. CONCLUSION: The choice of yeast extract is crucial for A. vinelandii cultivations, to maintain reproducibility and comparability between cultivations. The proper use of specific yeast extracts allows the cultivation results to be specifically optimised. In addition, supplements can be applied to modify and improve the properties of the alginate. The results only scratch the surface of the underlying mechanisms, as they are not providing explanations on a molecular level. However, the findings show the potential of optimising media containing yeast extract for alginate production with A. vinelandii, as well as the potential of targeted supplementation of the media.

Rotenone encapsulated in pH-responsive alginate-based microspheres reduces toxicity to zebrafish.

Rotenone is a botanical pesticide and has long been used for control of insect pests and also as a natural piscicide for management of fish populations in many countries. Field application for pest control, however, often encounters the movement of rotenone into surface water due to spray drift or surface runoff after rainfall, which could potentially result in water pollution and unexpected death of fishes. To minimize its effect on freshwater and the problem of fish dying, one solution was to encapsulate rotenone in specific microspheres, limiting its release and reducing its toxicity since rotenone can be quickly degraded under sunlight. In this study, pH-responsive alginate-based microspheres were synthesized to encapsulating rotenone, which were designated as rotenone beads. The rotenone beads, along with alginate beads (devoid of rotenone) were characterized and evaluated for their responses to pH and effects on zebrafish. Results showed that the microspheres had high loading efficiency (4.41%, w/w) for rotenone, and rotenone beads well responded to solution pH levels. The cumulative release rates of rotenone from the beads were 27.91%, 42.72%, and 90.24% at pH 5.5, 7.0, and 9.0, respectively. Under acidic conditions, the rotenone release rate was lower due to hydrogen bonding. On the contrary, rotenone became more quickly released at the high pH due to intermolecular repulsion. The toxicity of rotenone beads to zebrafish and fish embryos at a pH of 5.5 was reduced by 2- and 4-fold than chemical rotenone. Since pH levels in most freshwater lakes, ponds, and streams vary from 6 to 8, rotenone release from the beads in such freshwater could be limited. Thus, the synthesized rotenone beads could be relatively safely used for pest control with limited effects on freshwater fishers.

Ions-Induced Alginate Gelation According to Elemental Analysis and a Combinatorial Approach.

This study considers the potential of elemental analysis of polysaccharide ionotropic gels in elucidating the junction zones for different divalent cations. The developed algorithm ensures the correct separation of contributions from physically adsorbed and structure-forming ionic compounds, with the obtained results scaled to alginate C12 block. Possible versions of chain association into dimers and their subsequent integration into flat junction zones were analyzed within the framework of the "egg-box" model. The application of combinatorial analysis made it possible to derive theoretical relations to find the probability of various types of egg-box cell occurrences for alginate chains with arbitrary monomeric units ratio µ = M/G, which makes it possible to compare experimental data for alginates of different origins. Based on literature data and obtained chemical formulas, the possible correspondence of concrete biopolymer cells to those most preferable for filling by alkaline earth cations was established. The identified features of elemental composition suggest the formation of composite hydrated complexes with the participation of transition metal cations. The possibility of quantitatively assessing ordered secondary structures formed due to the physical sorption of ions and molecules from environment, correlating with the sorption capabilities of Me2+ alginate, was established.

Development and characterization of letrozole-encapsulated polymeric beads for sustained release.

The study aimed to prepare and characterize biodegradable sustained-release beads of letrozole (LTZ) for treating cancerous disease. The ionotropic gelation method was used for the preparation and calcium chloride (CaCl2) was used as a gelating agent, while chitosan (CTS) and sodium alginate (NaAlg) as biodegradable polymeric matrices in the blend hydrogel beads. The beads were characterized for their size, surface morphology, drug entrapment efficiency, drug-polymer interaction and crystallinity using different analytic techniques, including optical microscopy, Scanning Electron Microscopy (SEM), UV-spectroscopy, Fourier-transform Infrared Spectroscopy (FTIR), Thermo gravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC) and X-ray Diffraction Analysis (XRD) respectively. In vitro swelling studies were also applied to observe the response of these polymeric networks against different pH (at 1.2, 6.8 and 7.4 pH). The results from TGA and DSC exhibited that the components in the formulation possess better thermal stability. The XRD of polymeric networks displays a minor crystalline and significant amorphous nature. The SEM micrographs revealed that polymeric networks have uneven surfaces and grooves. Better swelling and in vitro outcomes were achieved at a high pH (6.8,7.4), which endorsed the pH-responsive characteristics of the prepared beads. Hence, beads based on chitosan and sodium alginate were successfully synthesized and can be used for the controlled release of letrozole.

QM/MM Study of the Reaction Mechanism of Thermophilic Glucuronoyl Esterase for Biomass Treatment.

Hydrolysis of lignocellulosic biomass, composed of a lignin-carbohydrate-complex (LCC) matrix, is critical for producing bioethanol from glucose. However, current methods for LCC processing require costly and polluting processes. The fungal Thermothelomyces thermophila glucuronoyl esterase (TtGE) is a promising thermophilic enzyme that hydrolyses LCC ester bonds. This study describes the TtGE catalytic mechanism using QM/MM methods. Two nearly-degenerate rate-determining transition states were found, with barriers of 16 and 17 kcal ⋅ mol-1 , both with a zwitterionic nature that results from a proton interplay from His346 to either the Ser213-hydroxyl or the lignin leaving group and the rehybridisation of the ester moiety of the substrate to an alkoxide. An oxyanion hole, characteristic of esterases, was provided by the conserved Arg214 through its backbone and sidechain. Our work further suggests that a mutation of Glu267 to a non-negative residue will decrease the energetic barrier in ca. -5 kcal ⋅ mol-1 , improving the catalytic rate of TtGE.

Structural basis of catalysis and substrate recognition by the NAD(H)-dependent α-d-glucuronidase from the glycoside hydrolase family 4.

Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and ß-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product d-glucuronic acid, at 2.15, 1.97 and 1.85 Šresolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure-function relationships within the unique GH4 family.