Cryogel with Modular and Clickable Building Blocks: Toward the Ultimate Ideal Macroporous Medium for Bacterial Separation.

The lack of practical platforms for bacterial separation remains a hindrance to the detection of bacteria in complex samples. Herein, a composite cryogel was synthesized by using clickable building blocks and boronic acid for bacterial separation. Macroporous cryogels were synthesized by cryo-gelation polymerization using 2-hydroxyethyl methacrylate and allyl glycidyl ether. The interconnected macroporous architecture enabled high interfering substance tolerance. Nanohybrid nanoparticles were prepared via surface-initiated atom transfer radical polymerization and immobilized onto cryogel by click reaction. Alkyne-tagged boronic acid was conjugated to the composite for specific bacteria binding. The physical and chemical characteristics of the composite cryogel were analyzed systematically. Benefitting from the synergistic, multiple binding sites provided by the silica-assisted polymer, the composite cryogel exhibited excellent affinity toward S. aureus and Salmonella spp. with capacities of 91.6 × 107 CFU/g and 241.3 × 107 CFU/g in 0.01 M PBS (pH 8.0), respectively. Bacterial binding can be tuned by variations in pH and temperature and the addition of monosaccharides. The composite was employed to separate S. aureus and Salmonella spp. from spiked tap water, 40% cow milk, and sea cucumber enzymatic hydrolysate, which resulted in high bacteria separation and demonstrated remarkable potential in bacteria separation from food samples.

Characterization of a λ-Carrageenase Mutant with the Generation of Long-Chain λ-Neocarrageenan Oligosaccharides.

λ-carrageenan oligosaccharides can be widely applied in the food, pharmaceutical, medicine and cosmetic industries due to their abundant bioactivities, and they are important products for the high-value utilization of λ-carrageenan. However, oligosaccharides with different degrees of polymerization have different properties, and the final products of λ-carrageenase reported so far are mainly λ-neocarrabiose, λ-neocarratetraose and λ-neocarrahexaose without longer-chain oligosaccharides. Further research is consequently required. Herein, a mutant λ-carrageenase was constructed by deleting the pyrroloquinoline quinone-like domain of OUC-CglA derived from Maribacter vaceletii. Interestingly, it was discovered that the majority of final products of the mutant OUC-CglA-DPQQ were long-chain oligosaccharides with a polymerization degree of 10-20, which underwent significant changes compared to that of OUC-CglA. Additionally, without the pyrroloquinoline quinone-like domain, fewer inclusion bodies were produced throughout the expression process, and the yield of the λ-carrageenase increased about five-fold. However, compared to its parental enzyme, significant changes were made to its enzymatic properties. Its optimal temperature and pH were 15 °C and pH 7.0, and its specific activity was 51.59 U/mg. The stability of the enzyme decreased. Thus, it was found that the deleting domain was related to the formation of inclusion bodies, the stability of the enzyme, the activity of the enzyme and the composition of the products.

In Situ Preparation of Star-Shaped Protein-"Smart" Polymer Conjugates with pH and Thermo-Dual Responsibility for Bacterial Separation.

To achieve effective separation and enrichment of bacteria, a novel synthetic scheme was developed to synthesize star-style boronate-functionalized copolymers with excellent hydrophilicity and temperature and pH responsiveness. A hydrophilic copolymer brush was synthesized by combining surface-initiated atom-transfer radical polymerization with amide reaction using bovine serum albumin as the core. The copolymer brush was further modified by introducing and immobilizing fluorophenylboronic acids through an amide reaction, resulting in the formation of boronate affinity material BSA@poly(NIPAm-co-AGE)@DFFPBA. The morphology and organic content of BSA@poly(NIPAm-co-AGE)@DFFPBA were systematically characterized. The BSA-derived composites demonstrated a strong binding capacity to both Gram-positive and Gram-negative bacteria. The binding capabilities of the affinity composite to Staphylococcus aureus and Salmonella spp. were 195.8 × 1010 CFU/g and 79.2 × 1010 CFU/g, respectively, which indicates that the novel composite exhibits a high binding capability to bacteria and shows a particularly more significant binding capacity toward Gram-positive bacteria. The bacterial binding of BSA@poly(NIPAm-co-AGE)@DFFPBA can be effectively altered by adjusting the pH and temperature. This study demonstrated that the star-shaped affinity composite had the potential to serve as an affinity material for the rapid separation and enrichment of bacteria in complex samples.

Catalytic properties characterization and degradation mode elucidation of a polyG-specific alginate lyase OUC-FaAly7.

Polymerized guluronates (polyG)-specific alginate lyase with lower polymerized mannuronates (polyM)-degrading activity, superior stability, and clear action mode is a powerful biotechnology tool for the preparation of AOSs rich in M blocks. In this study, we expressed and characterized a polyG-specific alginate lyase OUC-FaAly7 from Formosa agariphila KMM3901. OUC-FaAly7 belonging to polysaccharide lyase (PL) family 7 had highest activity (2743.7 ± 20.3 U/µmol) at 45 °C and pH 6.0. Surprisingly, its specific activity against polyG reached 8560.2 ± 76.7 U/µmol, whereas its polyM-degrading activity was nearly 0 within 10 min reaction. Suggesting that OUC-FaAly7 was a strict polyG-specific alginate lyase. Importantly, OUC-FaAly7 showed a wide range of temperature adaptations and remarkable temperature and pH stability. Its relative activity between 20 °C and 45 °C reached >90 % of the maximum activity. The minimum identifiable substrate of OUC-FaAly7 was guluronate tetrasaccharide (G4). Action process and mode showed that it was a novel alginate lyase digesting guluronate hexaose (G6), guluronate heptaose (G7), and polymerized guluronates, with the preferential generation of unsaturated guluronate pentasaccharide (UG5), although which could be further degraded into unsaturated guluronate disaccharide (UG3) and trisaccharide (UG2). This study contributes to illustrating the catalytic properties, substrate recognition, and action mode of novel polyG-specific alginate lyases.

Marine phospholipid nanoliposomes: A promising therapeutic approach for inflammatory bowel disease: Preparation, safety, and efficacy evaluation.

Promising findings have been emerged from studies utilizing n3 polyunsaturated fatty acids (PUFA) supplementation in animal models of inflammatory bowel disease (IBD). Introduction of marine phospholipids which combine n3 PUFA with phosphatidylcholine in a nanoliposome formulation offers enhanced pharmacological efficacy due to physical stability, improved bioavailability, and specific targeting to inflamed colitis tissues. In the present study, a marine phospholipid-based nanoliposome formulation was developed and optimized, resulting in nanovesicles of approximately 107.7 ± 1.3 nm in size, 0.18 ± 0.01 PDI, and - 32.03 ± 3.16 mV ZP. The nanoliposomes exhibited spherical vesicles with stable properties upon incubation at SGF as shown by the TEM, DLS, and turbidity measurements over 3 h. MPL nanoliposomes were cytocompatible until the concentration of 500 µg/mL as per MTT assay and taken by macrophages through macropinocytosis and caveolae pathways, and demonstrated significant inhibitory activity against reactive oxygen species (ROS) in LPS-stimulated macrophages. They were also shown to be blood-compatible and safe for administration in healthy mice. In a colitis mouse model, the nanoliposomes displayed preferential distribution in the inflamed gut, delaying the onset of colitis when administered prophylactically. These findings highlight the potential of marine phospholipid nanoliposomes as a promising therapeutic approach for managing inflammatory bowel disease.

High-level and reusable preparation of sulforaphane by yeast cells expressing myrosinase.

Myrosinase is a key tool for the fast and efficient preparation of sulforaphane which is one of the prominent natural ingredients found in brassicaceous vegetables. Here, the glucoraphanin-hydrolyzing activity of a Yarrowia lipolytica 20-8 harboring myrosinase reached 73.28 U/g dry cell weight, indicating that it had a potential application in sulforaphane preparation from glucoraphanin. An efficient and reusable process for sulforaphane preparation via myrosinase produced by Y. lipolytica 20-8 was constructed. In detail, as high as 10.32 mg sulforaphane could be produced from 1 g broccoli seed under the reaction of 40 U yeast whole-cell catalyst within 15 min with the conversion efficiency of 99.86%. Moreover, when the yeast whole-cell catalyst was reused 7 and 10 times, as high as 92.53% and 87.56% of sulforaphene yield of the initial level could be retained, respectively. Therefore, this yeast whole-cell is a potent biocatalyst for the efficient and reusable preparation of sulforaphane.

Distinct roles of carbohydrate-binding modules in multidomain ß-1,3-1,4-glucanase on polysaccharide degradation.

Lam16A is a novel GH16 ß-1,3-1,4-lichenase isolated from the genus Caldicellulosiruptor which can utilize untreated carbohydrate components of plant cell walls. Its catalytic module has been characterized that the six carbohydrate-binding modules (CBMs) were queued in the C-terminus, but their roles were still unclear. Here, full-length and CBM-truncated mutants of Lam16A were purified and characterized through heterologous expression in Escherichia coli. The profiles of these proteins, including the enzyme activity, degrading efficiency, substrate-binding affinity, and thermostability, were explored. Full-length Lam16A with six CBMs showed excellent thermostability and the highest activity against barley ß-glucan and laminarin with optimum pH of 6.5. The CBMs stimulated degrading ability of the catalytic module, especially against ß-1,3(4)-glucan-based polysaccharides. The released products from ß-1,3-1,4-glucan by Lam16A or its truncated mutants revealed an endo-type glycoside hydrolase. Lam16As exhibited strong binding affinities to the insoluble polysaccharides, especially Lam16A-1CBM. The degradation of yeast cell walls by Lam16A enzyme solution relative to the control reduced the absorbance values at OD800 by ~ 85% ± 1.2, enabling the release of up to ~ 0.057 ± 0.0039 µg/mL of the cytoplasmic protein into the supernatant, lowering the viability of the cells by ~ 70.3% ± 6.9, thus causing significant damage in the cell wall structure. Taken together, CBMs could influence the substrate specificity, thermal stability, and binding affinity of ß-1,3-1,4-glucanase. These results demonstrate the great potential of these enzymes to promote the bioavailability of ß-1,3-glucan oligosaccharides for health benefits. KEY POINTS: • Carbohydrate-binding modules strongly influenced the enzyme activity and binding affinity, and further impacted glycoside hydrolase activity. • Lam16A enzymes have sufficient ability to hydrolyze ß-1,3-1,4-glucan-based polysaccharides. • Lam16As provide a powerful tool to promote the bioavailability of ß-1,3-glucan oligosaccharides.

Statistical optimization of bioethanol production from giant reed hydrolysate by Candida tropicalis using Taguchi design.

The economic production of bioethanol as a sustainable liquid fuel is particularly needed and attractive. Giant reed as a low-cost and renewable biomass can be utilized as a sustainable feedstock for bioethanol development. The current research focuses on optimizing the fermentation parameters to increase ethanol concentration while lowering production costs. In this work, the giant reed was hydrolyzed thermochemically using HCl; cellulose and hemicellulose fractions were maximally converted at optimized hydrolysis conditions (5% HCl, 30 min, and 120 °C), resulting in a high sugar concentration (≈ 55 g/L), which were fermented by Candida tropicalis Y-26 for bioethanol production (≈ 15 g/L). Taguchi design was used to optimize the fermentation parameters (temperatures, pH, incubation period, and nitrogen sources). Under optimum fermentation conditions (25 °C; 24 h.; pH 5.5; and ammonium nitrate as a nitrogen source), the ethanol concentration at flask level accomplished ≈ 21 g/L, while its scale-up to bioreactor level contributed ≈ 25 g/L (equivalent to 250 kg ethanol/ton biomass) with ≈ 67% increase than the fermentation under unoptimized conditions. Overall, these findings proved that optimizing the fermentation parameters by Taguchi design and scaling up at a bioreactor could improve bioethanol production from giant reed biomass.

Multifunctional nanoparticles based on marine polysaccharides for apremilast delivery to inflammatory macrophages: Preparation, targeting ability, and uptake mechanism.

Hydrophobic drug encapsulation inside targeted nanoparticles can enhance accumulation in inflamed sites, limit toxicity to healthy tissue, and improve pharmacokinetics compared to free drug dosing. This study reports a functionalized marine polysaccharide nanoparticle with a controlled release, targeting abilities, and in-situ imaging properties. Carbon dots functionalized Enteromorpha polysaccharide/Mannose/Methionine functionalized Chitosan (CDs.EP/Man/Meth.Cs) NPs could deliver apremilast to inflammatory macrophages and Caco-2 intestinal cells as an in vitro model for application in oral drug delivery to cure IBD. The nanoparticles were simply a polyelectrolyte complex between cationic functionalized chitosan and anionic polysaccharide of Enteromorpha prolifera. Functionalized polysaccharides and the prepared NPs were well characterized. The functionalized nanoparticles could overcome the limitation of poor drug bioavailability and showed a high loading capacity of (45 %) with a controlled release of about (74.5 %). Confocal laser scanning imaging showed higher cellular uptake of the modified nanoparticles than that of the unmodified nanoparticles in LPS-activated RAW 264.7 macrophages and Caco-2 cells. The effect of functionalization on the cellular uptake targetability was assessed using spectrofluorometric measurements after mannose competition. Anti-inflammatory activity of apremilast-loaded NPs is more elevated than the free drug. These results suggest the feasibility of using functionalized EP/Cs nanoparticles in IBD oral drug delivery.

Biochemical Characterization of a Cold-Adapted λ-Carrageenase OUC-CglA from Maribacter vaceletii: An Efficient Tool for λ-Carrageenan Degradation.

λ-Carrageenase with high activity is an effective and environmentally friendly tool enzyme for the preparation of λ-carrageenan oligosaccharides with various biological activities. Herein, a novel GH150 (glycoside hydrolases family 150) λ-carrageenase OUC-CglA from Maribacter vaceletii was heterologously expressed, purified, and characterized. The recombinant OUC-CglA performs strict selectivity toward λ-carrageenan with a specific activity of 418.7 U/mg under its optimal reaction conditions of 20 °C and pH 7.0. Additionally, OUC-CglA is a typical cold-adapted λ-carrageenase because it unfolds 90% and 63% of its maximum activity at 15 and 10 °C, respectively. The hydrolysis process suggests that OUC-CglA is an endotype λ-carrageenase with the final products consisting of λ-neocarrabiose, λ-neocarratetraose, λ-neocarrahexaose, and other long-chain λ-neocarrageenan oligosaccharides. As a result, high activity, cold-adaptation, and principal products of OUC-CglA make it a potential biocatalyst for the effective preparation of λ-carrageenan oligosaccharides.

Expression and Biochemical Characterization of a Novel Fucoidanase from Flavobacteriumalgicola with the Principal Product of Fucoidan-Derived Disaccharide.

Fucoidan is one of the main polysaccharides of brown algae and echinoderm, which has nutritional and pharmacological functions. Due to the low molecular weight and exposure of more sulfate groups, oligo-fucoidan or fucoidan oligosaccharides have potential for broader applications. In this research, a novel endo-α-1,4-L-fucoidanase OUC-FaFcn1 which can degrade fucoidan into oligo-fucoidan was discovered from the fucoidan-digesting strain Flavobacterium algicola 12,076. OUC-FaFcn1 belongs to glycoside hydrolases (GH) family 107 and shows highest activity at 40 °C and pH 9.0. It can degrade the α-1,4 glycosidic bond, instead of α-1,3 glycosidic bond, of the fucoidan with a random tangent way to generate the principal product of disaccharide, which accounts for 49.4% of the total products. Therefore, OUC-FaFcn1 is a promising bio-catalyst for the preparation of fucoidan-derived disaccharide. These results further enrich the resource library of fucoidanase and provide the basis for the directional preparation of fucoidan-derived oligosaccharide with specific polymerization.

Biochemical Characterization of a Novel Myrosinase Rmyr from Rahnella inusitata for High-Level Preparation of Sulforaphene and Sulforaphane.

Myrosinase is a biotechnological tool for the preparation of sulforaphane and sulforaphene with a variety of excellent biological activities. In this study, a gene encoding the novel glycoside hydrolase family 3 (GH3) myrosinase Rmyr from Rahnella inusitata was heterologously expressed in Escherichia coli BL21 (DE3). The purified Rmyr shows the highest activity at 40 °C and pH 7.0; meanwhile, its half-life at 30 °C reaches 12 days, indicating its excellent stability. Its sinigrin-, glucoraphenin-, and glucoraphanin-hydrolyzing activities were 12.73, 4.81, and 6.99 U/mg, respectively. Rmyr could efficiently degrade the radish seed-derived glucoraphenin and the broccoli seed-derived glucoraphanin into sulforaphene and sulforaphane within 10 min with the highest yields of 5.07 mg/g radish seeds and 9.56 mg/g broccoli seeds, respectively. The highest conversion efficiencies of sulforaphane from glucoraphanin and sulforaphene from glucoraphenin reached up to 92.48 and 97.84%, respectively. Therefore, Rmyr is a promising and potent biocatalyst for efficient and large-scale preparation of sulforaphane and sulforaphene.

Preparation, urease inhibition mechanisms, and anti-Helicobacter pylori activities of hesperetin-7-rhamnoglucoside.

This work investigated the effects of the bioflavonoid hesperetin-7-rhamnoglucoside isolated from Citrus uranium fruit peel on Helicobacter pylori (H. pylori). Separation and purity, crystalline state, and urease inhibition assays were carried out. Then, molecular docking and molecular dynamics (MD) simulations were conducted with urease as the target protein. Hesp was isolated from citrus peel with a purity of 95.14 µg mg-1 of dry raw material. X-ray diffraction analysis, hydrogen-1 nuclear magnetic resonance, Fourier transform infrared spectroscopy, and differential scanning calorimetry revealed that pure Hesp had the same crystallinity rating as the Hesp standard. The kinetic inhibition study demonstrated that Hesp inhibited H. pylori urease in a competitive and concentration-dependent manner with jack bean urease. In addition, bioimaging studies with laser scanning confocal microscopy and scanning electron microscopy illustrated that Hesp interacted with bacterial cells and induced membrane disruption by creating holes in the outer membranes of the bacterial cells, resulting in the leakage of amino acids. Importantly, molecular docking and 20 ns MD simulations revealed that Hesp inhibited the target protein through slow-binding inhibition and hydrogen bond interactions with active site residues, namely, Gly11 (O⋯H distance = 2.2 Å), Gly13 (O⋯H distance = 2.4 Å), Ser12 (O⋯H distance = 3.3 Å), Lys14 (O⋯H distance = 3.3 Å), and Arg179 (O⋯H distance = 2.7 Å). This work presents novel anti- H. pylori agents from natural sources.

Exploration of Two Pectate Lyases from Caldicellulosiruptor bescii Reveals that the CBM66 Module Has a Crucial Role in Pectic Biomass Degradation.

Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using selected microorganisms that encode pectinolytic enzymes. Pectate lyases that cleave the α-1,4-galacturonosidic linkage of pectin are widely used in industries such as papermaking and fruit softening. However, there are few reports on pectate lyases with good thermostability. Here, two pectate lyases (CbPL3 and CbPL9) from a hyperthermophilic bacterium, Caldicellulosiruptor bescii, belonging to family 3 and family 9 polysaccharide lyases, respectively, were investigated. The biochemical properties of the two CbPLs were shown to be similar under optimized conditions of 80°C to 85°C and pH 8 to 9. However, the degradation products from pectin and polygalacturonic acids (pGAs) were different. A family 66 carbohydrate-binding module (CbCBM66) located in the N terminus of the two CbPLs shares 100% amino acid identity. A CbCBM66-truncated mutant of CbPL9 showed lower activities than the wild type, whereas CbPL3 with a CbCBM66 knockout portion was reported to have enhanced activities, thereby revealing the different effect of CbCBM66. Prediction by the I-TASSER server revealed that CbCBM66 is structurally close to BsCBM66 from Bacillus subtilis; however, the COFACTOR and COACH programs indicated that the substrate-binding sites between CbCBM66 and BsCBM66 are different. Furthermore, a substrate-binding assay indicated that the catalytic domains in the two CbPLs had strong affinities for pectate-related substrates, but CbCBM66 showed a weak interaction with a number of lignocellulosic carbohydrates. Finally, scanning electron microscopy (SEM) analysis and a total reducing sugar assay showed that the two enzymes could improve the saccharification of switchgrass. The two CbPLs are impressive sources for the degradation of plant biomass.IMPORTANCE Thermophilic proteins could be implemented in diverse industrial applications. We sought to characterize two pectate lyases, CbPL3 and CbPL9, from a thermophilic bacterium, Caldicellulosiruptor bescii The two enzymes share a high optimum temperature, a low optimum pH, and good thermostability at the evaluated temperature. A family 66 carbohydrate-binding module (CbCBM66) was identified in the two CbPLs, sharing 100% amino acid identity. The deletion of CbCBM66 dramatically decreased the activity of CbPL9 but increased the activity and thermostability of CbPL3, suggesting different roles of CbCBM66 in the two enzymes. Moreover, the degradation products of the two CbPLs were different. These results revealed that these enzymes could represent potential pectate lyases for applications in the paper and textile industries.

Combinations of alkaline hydrogen peroxide and lithium chloride/N,N-dimethylacetamide pretreatments of corn stalk for improved biomethanation.

The purpose of this study was to improve methane generation from corn stalk (CS) through alkaline hydrogen peroxide and lithium chloride/N,N-dimethylacetamide (AHP-LiCl/DMAc) pretreatment. Changes in the structures of treated and untreated CSs were investigated, and biomass components, including cellulose, hemicellulose and lignin, were analysed. Our findings revealed that AHP-LiCl/DMAc pretreatment improved accumulative methane yield by forceful delignification and effectively destroyed the structure of cellulose. The AHP-LiCl/DMAc-treated group had a maximum methane yield of 318.6 ± 17.85 mL/g volatile solid, which was 40.08% and 10.10% higher than the maximum methane yields of the untreated and AHP-treated group, respectively. This result showed enhanced cellulose dissolution by the ionic solvent of LiCl/DMAc and improved enzymatic saccharification in fermentative bacteria without structural modifications. The AHP-LiCl/DMAc treated group had higher glucose level, acetate followed by biomethanation process. Furthermore, the decrease in crystallinity indexes for AHP-LiCl/DMAc treated group was reported. Overall, this investigation proved a promising pretreatment approach for enhancing the degradation of CS into reducing sugars and improving methane generation.

Structure analysis of yeast glutaredoxin Grx6 protein produced in Escherichia coli.

BACKGROUND: Grx6 is a yeast Golgi/endoplasmic reticulum protein involved in iron-sulfur binding that belongs to monothiol glutaredoxin-protein family. Grx6 has been biochemically characterized previously. Grx6 contains a conserved cysteine residue (Cys-136). Depending on the active-site sequences, Grxs can be classified to classic dithiol Grxs with a CXXC motif known as classes II and monothiol Grxs with a CXXS motif known as classes I, and Grx6 belongs to the class I with a CSYS motif. RESULTS: Our results showed how the loop between the N-terminal and C-terminal can affect the stability. When Grx6 was incubated with FeSO4·7H2O and (NH4)2Fe(SO4)2·6H2O, a disulfide bond was formed between the cysteine 136 and glutathione, and the concentration of dimer and tetramer was increased. The results presented various levels of stability of Grx6 with mutant and deleted amino acids. We also highlighted the difference between the monomer and dimer forms of the Grx6, in addition to comparison of the Fe-S cluster positions among holo forms of poplar Grx-C1, human Grx2 and Saccharomyces cerevisiae Grx6. CONCLUSIONS: In this paper, we used a combination of spectroscopic and proteomic techniques to analyse the sequence and to determine the affected mutations and deletions in the stability of Grx6. Our results have increased the knowledge about the differences between monomer and dimer structures in cellular processes and proteins whose roles and functions depend on YCA1 in yeast.

Response Surface Optimization of Bioethanol Production from Sugarcane Molasses by Pichia veronae Strain HSC-22.

Pichia veronae strain HSC-22 (accession number KP012558) showed a good tolerance to relatively high temperature, ethanol and sugar concentrations. Response surface optimization based on central composite design of experiments predicted the optimal values of the influencing parameters that affect the production of bioethanol from sugarcane molasses to be as follows: initial pH 5, 25% (w : v) initial molasses concentration, 35°C, 116 rpm, and 60 h. Under these optimum operating conditions the maximum bioethanol production on a batch fermenter scale was recorded as 32.32 g/L with 44% bioethanol yield.