Metabolic profiles and protein expression responses of Pacific oyster (Crassostrea gigas) to polystyrene microplastic stress.

The underlying toxicity mechanisms of microplastics on oysters have rarely been explored. To fill this gap, the present study investigated the metabolic profile and protein expression responses of oysters to microplastic stress through metabolomics and biochemical analyses. Oysters were exposed to microplastics for 21 days, and the results indicated that the microplastics induced oxidative stress, with a significant decrease in SOD activity in the 0.1 mg/L exposure group. Metabolomics revealed that exposure to microplastics disturbed many metabolic pathways, such as amino acid metabolism, lipid metabolism, biosynthesis of amino acids, aminoacyl-tRNA biosynthesis, and that different concentrations of microplastics induced diverse metabolomic profiles in oysters. Overall, the current study provides new reference data and insights for assessing food safety and consumer health risks caused by microplastic contamination.

Extraction and characterization of valuable compounds from chicken sternal cartilage: Type II collagen and chondroitin sulfate.

Type II collagen (Col II) and chondroitin sulfate (CS) are the main macromolecules in the extracellular matrix. This study investigated the characteristics of Col II and CS obtained from chicken sternal cartilage (CSC) via enzymatic hydrolysis for various treatment times. For Col II and CS, the highest efficiency of enzymatic hydrolysis was achieved after 24 and 6 h of treatment, respectively. The average molecular weights were α1 chain-130 kDa, ß chain-270 kDa for Col II, and 80.27 kDa for CS. Fourier transform infrared spectroscopy revealed that the Col II samples maintained their triple-helical structure and that the predominant type of CS was chondroitin-4-sulfate. Scanning electron microscopy revealed that the Col II and CS samples possessed fibrillar and clustered structures, respectively. This study suggests that collagen and CS obtained from CSC can be used as promising molecules for application in food and pharmaceutical industries.

Valorization of tomato processing by-products: Predictive modeling and optimization for ultrasound-assisted lycopene extraction.

Lycopene is a carotenoid highly valuable to the food, pharmaceutical, dye, and cosmetic industries, present in ripe tomatoes and other fruits with a distinctive red color. The main source of lycopene is tomato crops. This bioactive component can be successfully isolated from tomato processing waste, commonly called tomato pomace, mostly made from tomato skins, seeds, and some residual tomato tissue. The main investigative focus in this work was the application of green engineering principles in each stage of the optimized ultrasound-assisted extraction (UAE) of enzymatically treated tomato skins to obtain functional extracts rich in lycopene. The experimental plan was designed to determine the influence of studied operating parameters: enzymatic reaction time (60, 120, and 180 min), extraction time (0, 5, 10, 15, 30, 60, and 120 min), and temperature (25, 35 and 45 ℃) on lycopene yield. Process optimization was performed based on the yield of lycopene [1018, 1067, and 1120 mg/kg] achieved at optimal operating conditions. An artificial neural network (ANN) model was developed and trained for predictive modeling of the closed extraction system, with operating parameters used as input neurons and experimentally obtained values for lycopene content defined as the output neural layer. Applied ANN architecture provided a high correlation of experimental output with ANN-generated data (R=0.99914) with a model deviation error for the entire data set of RMSE=5.3 mg/kg. The k-Nearest Neighbor algorithm was introduced to predict lycopene yield using experimental key features: operating temperature, extraction time, and time of enzymatic treatment, split into training and testing sets with an 85/15 ratio. The model interpretation was conducted through the SHAP (SHapley Additive exPlanations) methodology.

Structure, Properties and Degradation of Self-Assembled Fibrinogen Nanofiber Scaffolds.

Self-assembled fibrinogen nanofibers are promising candidates for skin tissue engineering due to their biocompatibility and ability to mimic the native blood clot architecture. Here, we studied the structure-property relationship and degradation of rehydrated fibrinogen nanofibers prepared by salt-induced self-assembly, focusing on the effect of scaffold layering, cross-linking time and freeze-drying. Optimal fiber stability was achieved with cross-linking by formaldehyde (FA) vapor, while treatment with liquid aldehydes, genipin, EDC, and transglutaminase failed to preserve the nanofibrous architecture upon rehydration. Scaffold layering did not significantly influence the mechanical properties but changed the scaffold architecture, with bulk fiber scaffolds being more compact than layered scaffolds. Freeze-drying maintained the mechanical properties and interconnected pore network with average pore diameters around 20 µm, which will enhance the storage stability of self-assembled fibrinogen scaffolds. Varying cross-linking times altered the scaffold mechanics without affecting the swelling behavior, indicating that scaffold hydration can be controlled independently of the mechanical characteristics. Cross-linking times of 240 min increased scaffold stiffness and decreased elongation, while 30 min resulted in mechanical properties similar to native skin. Cross-linking for 120 min was found to reduce scaffold degradation by various enzymes in comparison to 60 min. Overall, after 35 days of incubation, plasmin and a combination of urokinase and plasminogen exhibited the strongest degradative effect, with nanofibers being more susceptible to enzymatic degradation than planar fibrinogen due to their higher specific surface area. Based on these results, self-assembled fibrinogen fiber scaffolds show great potential for future applications in soft tissue engineering that require controlled structure-function relationships and degradation characteristics.

Isothermal retrogradation preparation of type III resistant starch from extruded-debranched starch: Structure and in vitro digestibility.

The extrusion-debranching method is suitable for the industrial production of resistant starch (RS) with high thermal stability. In this study, corn starch treated with extrusion and pullulanase debranching was subjected to different temperatures for different days (1 d, 3 d, and 7 d) and was evaluated by analysing its digestion, crystallization and thermal characteristics. Although the generally accepted optimal retrogradation temperature of starch is 4 °C, it was observed that in vitro digestibility was most reduced by retrogradation at 45 °C, with an RS content of up to 60.19 % on day 7. Retrograding at 45 °C formed more perfect and dense crystals with a mass fractal (Dm) of up to 2.68 and C + V type crystalline pattern. The crystalline pattern of samples stored at 80 °C were A + V and the others were B + V. In addition, samples retrograded at lower temperature showed higher thermal stability. While an increase in storage time at a constant temperature can lead to a reduction in the in vitro digestibility of starch, this effect is not as pronounced as that of temperature.

Pomegranate Peel Extract as 6-Phosphogluconate Dehydrogenase (6PGD) Inhibitor for Treatment of Breast Cancer.

Targeting the enzymes of Pentose Phosphate Pathway (PPP) has been emerged as a novel strategy for treatment of cancer. 6-phosphogluconate dehydrogenase (6PGD) is third enzyme of PPP and converts 6-phosphogluconate (6-PG) into ribulose 5-phosphate (R-5-P) and produces NADPH. The overexpression of 6PGD has been reported in many human cancers especially in breast cancer and is emerged as the potential anti-cancer drug target. The current study is focused to screen an already established library of plant extracts against 6PGD, among which Pomegranate peel extract showed significant 6PGD inhibitory activity with IC50 value = 0.090 µg/mL. Pomegranate peel competitively inhibited NADP+ and 6-phosphogluconate to 6PGD enzyme having Ki constant value = 12.72 ± 5.54 ng/mL. Moreover, anti-breast cancer activity against MCF-7 cells determined Pomegranate peel as the potent inhibitor of cancerous cells with IC50 value = 3.138 µg/mL. Toxicity profiling of pomegranate peel extract (2000mg/kg) did not show any adverse effect on mice. Moreover, Ont the base of literature a library of known compounds of pomegranate was prepared and established and screened against 6PGD for the identification of actual responsible phytochemicals of 6PGD activity by using molecular docking. Computational tools were used to evaluate selected potent hits. Out of 26 compounds, three potent phytochemicals (Procyanidin, Delphinidin and Cyanidin) exhibited the best binding affinities with 6PGD. In addition, these phytochemicals displayed the best favorable hydrogen bonding, binding energy, and protein-ligand interactions as compare to 3PG. Molecular dynamics simulation suggested that these hits form a stable binding complex with the active site of 6PGD. These findings suggest that Pomegranate peel and its secondary metabolites as the potent inhibitors of 6PGD and the best drug candidate for treatment of breast cancer.

Homologous expression of Aspergillus niger α-L-rhamnosidase and its application in enzymatic debittering of Ougan juice.

The α-L-rhamnosidase (rha1) gene was homologously expressed in Aspergillus niger strains CCTCC 206047 and CCTCC 206047ΔpyrG, using hygromycin B and auxotrophic as selection markers. The engineered A. niger strains RHA001-1 and RHA003-1 were screened, yielding α-L-rhamnosidase activities of 20.81 ± 0.56 U/mL and 15.35 ± 0.87 U/mL, respectively. The copy numbers of the rha1 gene in strains RHA001-1 and RHA003-1 were found to be 18 and 14, respectively. Correlation analysis between copy number and enzyme activity in the A. niger strains revealed that α-L-rhamnosidase activity increased with the copy number of the rha1 gene. Recombinant α-L-rhamnosidase was utilized for the enzymatic debittering of Ougan juice, and its process conditions were optimized. Furthermore, the primary bitter substance neohesperidin (2.22 g/L) in Ougan juice was converted into hesperetin 7-O-glucoside (1.47 g/L) and hesperidin (0.143 g/L). This study presents a novel approach for the production of food-grade α-L-rhamnosidase and establishes a technical foundation for its application in the beverage industry.

Enhancing the thermostability of lignin peroxidase: Heme as a keystone cofactor driving stability changes in heme enzymes.

Heme-containing enzymes, critical across life's domains and promising for industrial use, face stability challenges. Despite the demand for robust industrial biocatalysts, the mechanisms underlying the thermal stability of heme enzymes remain poorly understood. Addressing this, our research utilizes a 'keystone cofactor heme-interaction approach' to enhance ligand binding and improve the stability of lignin peroxidase (LiP). We engineered mutants of the white-rot fungus PcLiP (Phanerochaete chrysosporium) to increase thermal stability by 8.66 °C and extend half-life by 29 times without losing catalytic efficiency at 60 °C, where typically, wild-type enzymes degrade. Molecular dynamics simulations reveal that an interlocked cofactor moiety contributes to enhanced structural stability in LiP variants. Additionally, a stability index developed from these simulations accurately predicts stabilizing mutations in other PcLiP isozymes. Using milled wood lignin, these mutants achieved triple the conversion yields at 40 °C compared to the wild type, offering insights for more sustainable white biotechnology through improved enzyme stability.

[Identification and characterization of a novel polyamide hydrolase].

The widespread use of non-naturally degradable plastics is causing increasingly serious harm to the environment. Reducing plastic pollutants has become the core of ecological and environment management. Biological methods such as enzymes demonstrate advantages in depolymerizing plastics with mild reaction conditions and recycling of depolymerization products. However, there are few reports on the biological depolymerization of polyamide plastics. In this study, by using 4-nitropropionanilide as the model substrate, we screened against our plastic depolymerase library and obtained a Meiothermus ruber-derived enzyme (MrABH) that can hydrolyze the polyamide bond. We expressed this enzyme in Escherichia coli and purified the protein by affinity chromatography. Furthermore, we investigated the catalytic properties, enzymatic properties, and catalytic products of this enzyme with polyamide as the substrate. MrABH had good stability at pH 8.0-10.0, with the optimal performance at pH 9.0 and 30 ℃. The catalytic performance of this enzyme for ester bonds and amide bonds was similar. MrABH can catalyze the depolymerization of PA6 and PA66 to produce monomers and oligomers, demonstrating the potential to be used in the depolymerization and recycling of polyamide.

Molecular Networking-Guided Discovery of a New Antitumor Pyranonaphthoquinone from Streptomyces tanashiensis DSM 731: Insights from Single-Molecule Stretching Assays.

Guided by molecular networking based on single-molecule stretching assay, an unprecedented pyranonaphthoquinone, methyl kalafunginate (1) and five known compounds 2-6 were isolated from Streptomyces tanashiensis DSM 731. Compound 1 was characterized through a combination of spectroscopic techniques, including 1D and 2D NMR analysis, ECD calculation, and X-ray crystallography. Interestingly, we discovered that compound 1 was spontaneously converted from kalafungin (4) in methanol solution. All isolated compounds were assessed for their cytotoxic potential against a panel of five human cancer cell lines: A549, HepG2, BxPC-3, SW620, and C4-2B. Compounds 1, 2, 4, and 5 exhibited remarkable cytotoxicity with IC50 values below 2.382 µM, suggesting their potential as promising anticancer agents. These findings highlight the significance of using a combined approach of single-molecule stretching assays and molecular networking for efficiently discovering novel natural products with potential therapeutic applications.

Combining systems and synthetic biology for in vivo enzymology.

Enzymatic parameters are classically determined in vitro, under conditions that are far from those encountered in cells, casting doubt on their physiological relevance. We developed a generic approach combining tools from synthetic and systems biology to measure enzymatic parameters in vivo. In the context of a synthetic carotenoid pathway in Saccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to study in vitro. We designed, built, and analyzed a collection of yeast strains mimicking substantial variations in substrate concentration by strategically manipulating the expression of geranyl-geranyl pyrophosphate (GGPP) synthase. We successfully determined in vivo Michaelis-Menten parameters (KM, Vmax, and kcat) for GGPP-converting phytoene synthase from absolute metabolomics, fluxomics and proteomics data, highlighting differences between in vivo and in vitro parameters. Leveraging the versatility of the same set of strains, we then extracted enzymatic parameters for two of the three phytoene desaturases. Our approach demonstrates the feasibility of assessing enzymatic parameters directly in vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.

[Effects of Film Mulching on Soil Quality, Garlic Yield, and Garlic Quality].

To determine the optimal film management technique for garlic planting, this study aimed to investigate the effects of various film cover methods on soil quality and garlic yield in garlic cropping systems. To achieve these goals, trials with different film cover methods were conducted at the Jiangsu Academy of Agricultural Sciences in Nanjing. To investigate the impact of changes in soil quality and garlic yield, we set up four treatments： no film treatment （CK）, black polyethylene film treatment （HPE）, black poly（butylene- adipate-co-terephthalate） （PBAT） with straw composite film treatment （HSJ）, and white PBAT film treatment （BJ） in a garlic cropping system. Our results indicated that specific mulch coverings had a positive effect on both soil quality and garlic yield. The film cover treatments resulted in significant changes in soil physicochemical properties and bacterial and fungal biomasses and indirectly improved soil quality. Compared to that under the no film treatment, the BJ treatment boosted soil quality by 70%, with the most significant impact, followed by that under the HPE and HSJ treatments, with improvements of 52% and 36%. Random forest modeling indicated that soil organic matter and total nitrogen were the most important factors influencing soil quality. The different film covers significantly increased the diameter of garlic bulbs and single quality. The HSJ treatment exhibited the most significant increase in garlic yield, with 46%, 19%, and 6% improvement compared to that in the CK, HPE, and BJ treatments, respectively. Correlation analysis showed that soil quality under film cover was significantly correlated with the starch content of garlic bulbs, garlic diameter, and single quality. This study highlights that selecting the appropriate mulch film aids in the production of garlic and helps to develop farmland that produces both high-quality and high-yield crops.

Trace Elements in Medicinal Metallomics.

This analytical mini-review focuses on the effects of trace elements, which includes Cu, Mn, Zn, and Se, as well as some rarer microelements, on the regulation of oxidative stress in the body and of certain diseases associated with it. Synergism and competition between certain microelements have been considered a hot topic in the applied molecular pharmacology of these specific bio-effects. Some ideas for further possible directions of research are expressed. Noteworthy, metal coordinating catalytical sites of certain enzymes function as pharmacophore-forming and connecting nanostructures. These sites can be regarded as targets for various effectors, making them pharmacologically significant contributors to biocatalysis.

Registration of activity of a single molecule of horseradish peroxidase using a detector based on a solid-state nanopore.

This work demonstrates the use of a solid-state nanopore detector to monitor the activity of a single molecule of a model enzyme, horseradish peroxidase (HRP). This detector includes a measuring cell, which is divided into cis- and trans- chambers by a silicon nitride chip (SiN structure) with a nanopore of 5 nm in diameter. To entrap a single HRP molecule into the nanopore, an electrode had been placed into the cis-chamber; HRP solution was added into this chamber after application of a negative voltage. The reaction of the HRP substrate, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), oxidation by the enzyme molecule was performed in the presence of hydrogen peroxide. During this reaction, the functioning of a single HRP molecule, entrapped in the nanopore, was monitored by recording the time dependence of the ion current flowing through the nanopore. The approach proposed in our work is applicable for further studies of functioning of various enzymes at the level of single molecules, and this is an important step in the development of single-molecule enzymology.

Ultra-small platinum nano-enzymatic spray with ROS scavenging and anti-inflammatory properties for photoaging treatment.

Photoaging induced by ultraviolet (UV) results in oxidative stress and inflammation. Noble metal nanozymes have strong antioxidant and anti-inflammatory capacity, which are expected to eliminate the excessive reactive oxygen species (ROS) and inflammatory factors in the photoaged skin. Hence, we have synthesized ultrasmall platinum nanoparticles coated with polyvinylpyrrolidone (Pt NPs) with a diameter of nearly 5 nm for photoaging treatment. Thanks to multi-enzymatic capacities (catalase, peroxidase, and superoxide dismutase) of Pt NPs, they can effectively protect fibroblasts from UV-induced ROS attack, relieve fibroblasts from UV-induced cell cycle arrest, downregulate matrix metalloproteinases (MMPs) to regenerate type I collagen, and inhibit M1 macrophage polarization to decrease the expression of inflammatory factors. For photoaged mice treatment, we employ the concept of routine spray skincare and encapsulate Pt NPs solution in a spray bottle. In combination with roller needle, following Pt NPs nano-enzymatic spray given, UV-induced photoaged mice display reduced wrinkle formation in the collagen-depleted dermal tissue of mice and more youthful performance in both appearance and organizational structure. Consequently, multi-enzymatic functions of Pt NPs nano-spray offers a promising avenue for anti-photoaging therapy, providing potential benefits in both preventative and restorative skincare applications.

Analysis of the region-specific characteristics of traditional Korean doenjang produced in Gyeonggi-do.

Doenjang is traditional soybean fermented food in Korea, and traditional doenjang on the market has different qualities like aroma, taste. This study is a preliminary study to investigate the quality characteristics of doenjang produced in each region of Korea. The objective of this study was to analyze the region-specific characteristics of traditional Korean doenjang produced in Gyeonggi-do. Physiochemical characteristics including pH, moisture content, soluble solid content, salinity, color, acid value, titratable acidity, NH2-N, total/reducing sugar, and alcohol contents, and enzymatic activities such as acidic/neutral protease and α/ß-amylase activities, were analyzed. Doenjang produced in Gyeonggi-do was classified into two groups (GDG-A and GDG-B), and the distinction between two groups were the aging period: doenjang samples in GDG-A groups were aged over 3 yrs, while samples in GDG-B groups were aged less than 3 yrs. The results of this study provided the physicochemical characteristics and enzymatic activities of traditional doenjang produced in Gyeonggi-do.

Novel umami-enhancing peptides of beef M. Semimembranosus hydrolysates and interactions with the T1R1/T1R3 taste receptor.

The taste mechanisms of beef umami and umami-enhancing peptides are not well understood. Therefore, novel umami and umami-enhancing peptides from beef M. semimembranosus hydrolysates were explored. Beef hydrolysates treated with Flavourzyme® showed an overall strong umami intensity compared to those treated with Alcalase®, papain, or Protamex®. The peptides were isolated via consecutive separation processes, and 31 potential umami peptides were identified. Molecular docking results showed that WGSEPIRIQ and TERGYSF had considerably low docking energies with the T1R1/T1R3 taste receptor through potential key binding sites for hydrogen bonding, including Ser48, Gly49, and Gln278 in T1R1, and Ser67, Asn68, and Arg247 in the T1R3 subunit. The taste of the identified peptides dissolved in ultrapure water was dominated by sourness. Instead, they demonstrated an umami-enhancing effect in the presence of monosodium glutamate. These results broaden our understanding of the taste mechanisms of beef umami-enhancing peptides and their potential applications as flavoring agents.

Discovery and biosynthesis of non-canonical C16-terpenoids from Pseudomonas.

Biosynthesis of sodorifen with a unique C16-bicyclo[3.2.1]octene framework requires an S-adenosyl methionine-dependent methyltransferase SodC and terpene cyclase SodD. While bioinformatic analyses reveal a wide distribution of the sodCD genes organization in bacteria, their functional diversity remains largely unknown. Herein, two sodorifen-type gene clusters, pcch and pcau, from Pseudomonas sp. are heterologously expressed in Escherichia coli, leading to the discovery of two C16 terpenoids. Enzymatic synthesis of these compounds is achieved using the two (SodCD-like) pathway-specific enzymes. Enzyme assays using different combinations of methyltransferases and terpene synthases across the pcch, pcau, and sod pathways reveal a unifying biosynthetic mechanism: all three SodC-like enzymes methylate farnesyl pyrophosphate (FPP) with subsequent cyclization to a common intermediate, pre-sodorifen pyrophosphate. Structural diversification of this joint precursor solely occurs by the subsequently acting individual terpene synthases. Our findings expand basic biosynthetic understanding and structural diversity of unusual C16-terpenoids.

Inhibition mechanism of buckwheat hulls polyphenols on α-amylase and α-glucosidase using kinetics, spectroscopics and molecular docking approaches.

The work investigated the activity inhibition of phenolic compounds in buckwheat (Fagopyrum esculentum Moench) hulls (BH) on α-amylase and α-glucosidase, and clarified their possible mechanisms based on kinetics, spectroscopics and molecular docking analysis. The total polyphenols (BHP) from BH using an ultrasound-assisted alcohol extraction method was 210.50 mg GAE/g DW. The study identified a total of 33 polyphenolic compounds in the extracts of BH using UPLC-Q-Exactive Orbitrap/MS, revealing that sixteen of these were novel polyphenolic substances not previously documented in this plant. BHP demonstrated significant inhibitory effects on both α-amylase and α-glucosidase enzymes, with IC50 values recorded at 27.16 µg/mL and 7.00 µg/mL, respectively, suggesting noncompetitive and mixed-type inhibition mechanisms. The fluorescence intensity of the enzymes was effectively quenched by BHP through a combination of dynamic and static quenching mechanisms, driven predominantly by hydrophobic interactions. BHP's interaction with the enzymes resulted in conformational changes that reduced their enzymatic activities. Molecular docking further revealed that six polyphenolic components of BHP had a strong affinity for binding within the active sites nestled in the enzymes' hydrophobic cavities, inhibiting their activity and potentially contributing to a reduction in blood glucose levels. The results could provide perspective for using BHP in the functional components of sugar-controlling foods.

Corn-derived Expansin synergistically promotes enzymatic hydrolysis of corn cob.

The enzymatic hydrolysis of lignocellulose is hindered by challenges such as high enzyme usage and associated costs. It is essential to explore effective approaches to improve the efficiency of enzymatic hydrolysis while reducing costs. Expansins are non-enzymatic proteins that can interact with lignocellulose and facilitate the loosening of plant cell walls. Given their natural affinity to plant cell walls, we hypothesized that a corn Expansin could enhance the enzymatic hydrolysis of corn lignocellulose. In this study, we expressed a corn (Zea mays) Expansin, EXPA17, in yeast cells and explored the synergistic effect between EXPA17 and commercial cellulase, and found that EXPA17 exhibited a pronounced synergistic effect on the enzymatic hydrolysis of corn cobs. The addition of 0.015 mg/mL EXPA17 resulted in a 14.00 % increase in glucose yield. Fourier-transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) analysis revealed that EXPA17 proteins disrupted hydrogen bonds in the amorphous regions of corn cobs, leading to a more porous and looser structure, thereby enhancing cellulose accessibility. Our work leveraged the synergistic effect between Expansin and lignocellulose from the same source of corn, providing a novel strategy to improve the efficiency of enzymatic hydrolysis of corn lignocellulose while potentially reducing the associated costs.