Search

Objective: The goal of this study is the detection of the gen that responsible for production the vaginolysin toxin, discovered the optimal condition for produce vaginolysin, purification and characterization of the toxin and show the cytotoxic effect of vaginolysin on VK2 cell and HeLa cell. Methods: Among 592 specimens isolated from pregnant women suffering from vaginal inflammation and threatened with abortion (these women is suffering from strong pain in the cervical region), only 62 of isolates identified as G vaginalis. From Baghdad teaching hospital. detection of the gen that responsible for production the vaginolysin toxin. Study of the optimal condition to produce vaginolysin. The purification of the toxin by using ammonium sulfate, ion exchange chromatography and gel filtration, toxin characterization of pH and temperature activity and stability, the study of cytotoxic effect of vaginolysin on VK2 and HeLa cells. Result: The optimal condition to produce the toxin is pH level in 4, best temperature is 37 °C, the best media is TSB broth, and the best hours is 24 hr. The best range for precipitation saturation ratio 50%. Ion exchanges have specific activity 4 mg. gel filtrations show an increase in the specific activity of purified toxin (10 U/mg). The characterization done by pH and temperature, Vaginolysin was active in 0.5 U/mL at pH 7, and stable in pH 7 activity, the best temperature for vaginolysin activity is 0.5 U/mL at 37°C and maximum stability was recorded at 37°C. The cytotoxic effect of the toxin on VK2 cell observed the viability ratio 69.98%, at higher toxin concentration, while the result for HeLa cell viability ratio 38.77%, at higher toxin concentration. Conclusion: Vaginolysin cause lysis of vaginal cell and cervical cell may lead to abortion, the killing ratio increase by increasing the concentration of the toxin and the effect of the toxin on HeLa cell is more than on VK2 cell. [ABSTRACT FROM AUTHOR]

Introduction: Rana dybowskii Guenther (RDG), as a traditional Chinese medicine, has been shown to have antioxidant effects. However, studies on the anti-aging effect of RDG are still limited. Methods: In this study, we prepared polysaccharides from the skin of RDG (RDGP) by hot water extraction, alcohol precipitation, ion-exchange chromatography and gel chromatography. The proteins were removed using the Sevage method in combination with an enzymatic method. The structural features were analyzed using high-performance gel permeation chromatography, γ-elimination reaction and Fourier transform infrared spectra. The anti-aging effect of RDGP was investigated by using D-Gal to establish an aging model in mice, and pathological changes in the hippocampus were observed under a microscope. Results: We obtained the crude polysaccharide DGP from the skin of RDG, with a yield of 61.8%. The free protein was then removed by the Sevage method to obtain DGPI and deproteinated by enzymatic hydrolysis combined with the Sevage method to further remove the bound protein to obtain the high-purity polysaccharide DGPII. Then, DGPIa (1.03 × 105 Da) and DGPIIa (8.42 × 104 Da) were obtained by gel chromatography, monosaccharide composition analysis showed that they were composed of Man, GlcA, GalNAc, Glc, Gal, Fuc with molar ratios of 1: 4.22: 1.55: 0.18: 8.05: 0.83 and 0.74: 1.78: 1: 0.28: 5.37: 0.36, respectively. The results of the γ-elimination reaction indicated the presence of O-glycopeptide bonds in DGPIa. The Morris water maze test indicated that mice treated with DGPIIa exhibited a significantly shorter escape latency and increased time spent in the target quadrant as well as an increase in the number of times they traversed the platform. Pathologic damage to the hippocampus was alleviated in brain tissue stained with hematoxylin-eosin. In addition, DGPIIa enhanced the activities of SOD, CAT, and GSH-Px and inhibited the level of MDA in the serum and brain tissues of aging mice. Discussion: These results suggest that RDGP has potential as a natural antioxidant and provide useful scientific information for anti-aging research. [ABSTRACT FROM AUTHOR]

A thermostable l-asparaginase was produced from Bacillus licheniformis UDS-5 (GenBank accession number, OP117154). The production conditions were optimized by the Plackett Burman method, followed by the Box Behnken method, where the enzyme production was enhanced up to fourfold. It secreted l-asparaginase optimally in the medium, pH 7, containing 0.5% (w/v) peptone, 1% (w/v) sodium chloride, 0.15% (w/v) beef extract, 0.15% (w/v) yeast extract, 3% (w/v) l-asparagine at 50 °C for 96 h. The enzyme, with a molecular weight of 85 kDa, was purified by ion exchange chromatography and size exclusion chromatography with better purification fold and percent yield. It displayed optimal catalysis at 70 °C in 20 mM Tris–Cl buffer, pH 8. The purified enzyme also exhibited significant salt tolerance too, making it a suitable candidate for the food application. The l-asparaginase was employed at different doses to evaluate its ability to mitigate acrylamide, while preparing French fries without any prior treatment. The salient attributes of B. licheniformis UDS-5 l-asparaginase, such as greater thermal stability, salt stability and acrylamide reduction in starchy foods, highlights its possible application in the food industry. [ABSTRACT FROM AUTHOR]

Polyphenol oxidase (PPO) is an important metalloenzyme in the tea plant (Camellia sinensis). However, there has recently been a lack of comprehensive reviews on Camellia sinensis PPO. In this study, the methods for extracting PPO from Camellia sinensis, including acetone extraction, buffer extraction, and surfactant extraction, are compared in detail. The main purification methods for Camellia sinensis PPO, such as ammonium sulfate precipitation, three-phase partitioning, dialysis, ultrafiltration, ion exchange chromatography, gel filtration chromatography, and affinity chromatography, are summarized. PPOs from different sources of tea plants are characterized and systematically compared in terms of optimal pH, optimal temperature, molecular weight, substrate specificity, and activators and inhibitors. In addition, the applications of PPO in tea processing and the in vitro synthesis of theaflavins are outlined. In this review, detailed research regarding the extraction, purification, properties, and application of Camellia sinensis PPO is summarized to provide a reference for further research on PPO. [ABSTRACT FROM AUTHOR]

In recent years, the biological activity of plant polysaccharides has received increasing attention. Polysaccharides, as one of the main components of lilies, have pharmacological effects in regulating immunity, anti-tumor, antioxidant, antibacterial, and hypoglycemic effects. To systematically analyze the structural characteristics of the polysaccharide, the polysaccharide LP-1 was prepared from Lilium lancifolium bulbs by water extraction and ethanol precipitation, ion exchange chromatography and gel filtration chromatography. Structural characterizations show that the weight-average relative molecular weight of LP-1 is 5.3 kDa. LP-1 consists of mannose and glucose at a molar ratio of 1.4:1. Its primary structure is Glcp-α-D-(1 → {4)-β-D-Glcp-(1 → 4)-β-D-2-O-acetyl-Manp-(1 → [4)-β-D-Glcp-(1 → 4)-β-D-Glcp-(1 → 4)-β-D-Manp-(1 → 4)-β-D-Manp-(1 → 4)-β-D-Manp-(1 → 4)-β-D-Manp-(1]2}2 → 4)-α-D-Glcp. Acetylation occurs at the O-2 site of mannose. Acetyl substitution degree is 8.21%. The results of structure elucidation showed that LP-1 was a low-molecular weight linear O-acetylated glucomannan. The results of oxidative stress experiments indicate that LP-1 exerts antioxidant effects on HUVEC by activating the Nrf2/HO-1 signaling pathway, thereby improving endothelial cell damage induced by H2O2, and has the potential to be developed as an antioxidant drug. [ABSTRACT FROM AUTHOR]

Background: Large-scale vaccine production requires downstream processing that focuses on robustness, efficiency, and cost-effectiveness. Methods: To assess the robustness of the current vaccine production process, three batches of COVID-19 Omicron BA.1 strain hydrolytic concentrated solutions were selected. Four gel filtration chromatography media (Chromstar 6FF, Singarose FF, Bestarose 6B, and Focurose 6FF) and four ion exchange chromatography media (Maxtar Q, Q Singarose, Diamond Q, and Q Focurose) were used to evaluate their impact on vaccine purification. The quality of the vaccine was assessed by analyzing total protein content, antigen content, residual Vero cell DNA, residual Vero cell protein, and residual bovine serum albumin (BSA). Antigen recovery rate and specific activity were also calculated. Statistical analysis was conducted to evaluate process robustness and the purification effects of the chromatography media. Results: The statistical analysis revealed no significant differences in antigen recovery (p = 0.10), Vero HCP residue (p = 0.59), Vero DNA residue (p = 0.28), and BSA residue (p = 0.97) among the three batches of hydrolytic concentrated solutions processed according to the current method. However, a significant difference (p < 0.001) was observed in antigen content. Conclusions: The study demonstrated the remarkable robustness of the current downstream process for producing WIBP-CorV vaccines. This process can adapt to different batches of hydrolytic concentrated solutions and various chromatography media. The research is crucial for the production of inactivated SARS-CoV-2 vaccines and provides a potential template for purifying other viruses. [ABSTRACT FROM AUTHOR]

A polysaccharide was extracted from the fresh ginger by water extraction and alcohol precipitation. A homogeneous polysaccharide was obtained by Sevage deproteinization, dialysis, DEAE-52 ion exchange chromatography and Sephadex G-100 gel column chromatography. The molecular weight of the ginger polysaccharide was determined by high performance gel permeation chromatography. The monosaccharide composition, scanning electron microscope and other physicochemical parameters of the ginger polysaccharide were further determined. The spectral properties of the ginger polysaccharide were investigated by ultraviolet and infrared spectroscopy. On this basis, methylation analysis, 1D and 2D NMR analysis were used to investigate its possible chemical structure. The results showed that the ginger polysaccharide group was divided into neutral polysaccharide components with high homogeneity, Mw = 35.52 kDa, Mn = 216.56 kDa and the sugar content was 93.5%. The ginger polysaccharide is mainly composed of Fuc, Ara, Rha, Gal, Glc, Xyl, Man, Gal-UA and Glc-UA, the amount of substance ratio was 1.01:1.96:9.54:14.82:64.20:3.84:2.07:1.41:1.16. To study the structure–activity relationship of ginger polysaccharide, acetylated ginger polysaccharide and phosphate ginger polysaccharide were prepared by chemical methods in this study. The results of antioxidant activity in vitro showed that the ginger polysaccharide and its derivatives had good biological activity in freeing ABTS + radical scavenging, freeing hydroxyl radical scavenging, freeing DPPH radical scavenging and reducing ability. Interestingly, the results showed that the ginger polysaccharide had a certain inhibitory effect on the growth of E. coli in vitro. In general, this study provides a certain experimental basis and reference for the further research on the activity of ginger polysaccharide and its derivatives. [ABSTRACT FROM AUTHOR]

Proteases have various applications in the food, pharmaceutical, medicine, pathogenicity of some pathogenic bacteria, and detergent sectors as well as meeting the needs of approximately 60% of the global enzyme industry, whereas they catalyze the breakdown of protein molecules into peptides and amino acids. Production and purification of protease enzyme by the isolate Escherichia coli AJ55 was scrutinized in the present study. Cultivation optimum conditions, were various complex medium, carbon source, nitrogen source, temperature, pH of the medium, and time of incubation were optimized to enhance the total protease production in shake flask culture of E.coli AJ55. The nutrient broth supplemented with 2% glucose and 2% yeast extract, with a pH of 7.0 and incubated at 37°C for 24 hours, better conditioned for producing the maximum production of protease. Escherichia coli AJ55 proteolytic enzyme was separated and purified using ion-exchange chromatography on a DEAE-Cellulose column and Sephadex G-150 gel after being precipitated with 0-70% saturated ammonium sulfate. Protease that had been partially purified had a yield of 34%, a purification fold of 13.4, an activity of 12.16 U/ml, a protein concentration of 0.005 mg/ml, and a specific activity of 2432 U/mg. By using gel filtration chromatography on a Sephadex G-150 gel, partially purified protease was examined for its ability to cleave the mucin protein. The findings of mucin biodegradation showed that the five fractions of the small peptides were produced after treatment of mucin with partially purified protease. [ABSTRACT FROM AUTHOR]

Copyright of Modern Food Science & Technology is the property of Editorial Office of Modern Food Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Small-aroma chicken with a high protein content, is widely distributed in the mountainous area of Yunnan-Guizhou Plateau, China. In this work, three novel ACE-inhibitory peptides, Leu-Thr-Glu-Lys-Val-Val-Phe (LTQKVVF), Leu-Asp-Asp-His-Phe-Leu (LDDHFL), and Val-Pro-Gly-Pro-Glu-Pro-Lys-Pro (VPGPEPKP), were prepared from the Small-Aroma Chicken breast. The purification and identification protocol employed comprised ion-exchange chromatography, gel filtration chromatography, reverse phase-high performance liquid chromatography (RP-HPLC), and nano liquid chromatography-electrospray ionization–tandem mass (Nano-LC-ESI–MS/MS). Compared with VPGPEPKP and VPGPEPKP, LTQKVVF displayed noticeable ACE inhibitory activity, with IC 50 values of 189.34 μM. What's more, the Lineweaver-Burk plots revealed peptides LTQKVVF and LDDHFL acted as the mixed-competitive inhibitor while VPGPEPKP inhibited ACE in a manner similar to uncompetitive inhibition. The binding free energies of LTQKVVF, LDDHFL, and VPGPEPKP for ACE were −8.1 kcal/mol, −8.6 kcal/mol, and −9.6 kcal/mol, respectively. The strong inhibition of ACE by VPGPEPKP may be attributed to the interaction of hydrogen bonds, alkyl, and pi-alkyl, which formed by crucial residues of ACE. [Display omitted] • Three novel peptides were characterized from small-aroma chicken. • The peptides exhibited inhibition activity on angiotensin converting enzyme (ACE). • Lineweaver Burk plots was used to identify the inhibition mode of peptides. • The interactions between peptides and ACE were simulated by molecular docking. [ABSTRACT FROM AUTHOR]

To identify structures and differences in physiological activity of polysaccharides from different Hovenia dulcis tissues, three populations of H. dulcis polysaccharides (HDP), including whole fruit polysaccharides (HDPW), seed polysaccharides (HDPS) and peduncle polysaccharides (HDPP), were prepared using water extraction and alcohol precipitation. Their structures were analyzed using UV-Vis spectroscopy, high-performance size exclusion chromatography, ion exchange chromatography, Fourier transform infrared spectrometry, and scanning electron microscopy. All three HDP preparations comprised the acidic polysaccharides fucose, rhamnose, arabinose, galactose, glucose, mannose, galacturonic acid, and glucuronic acid in different molar ratios. HDPW, HDPS, and HDPP each eluted in size exclusion chromatography with three chromatographic peaks, and the low and medium molecular weight HDPS peaks (45.72x104~165.75x104 u) displayed higher molecular weights than those of HDPW (0.34x104~12.53x104 u) and HDPP (0.51x104~21.26x104 u). Infrared spectra showed that HDPW and HDPP contained α-glycosidic bonds, while HDPS contained β-glycosidic linked polysaccharides. The respective median inhibitory concentrations of HDP for DPPH free radicals, ABTS free radicals, and α-glucosidase were 0.015~0.043, 0.47~1.21, and 0.13~9.99 mg/mL. When the mass/volume concentrations of HDPW and HDPP were 0.1 mg/mL or above, the glucose consumption rate of insulin-resistant HepG2 cells (IR-HepG2) was significantly increased, with the highest increase compared with the model group being 7.66%. Compared with HDPS, HDPW was similar to HDPP in structure, and both showed better antioxidant and α-glucosidase inhibitory activity and stronger IR-HepG2 glucose uptake promotion ability. This study provides a theoretical basis and data supporting the comprehensive utilization of H. dulcis. [ABSTRACT FROM AUTHOR]

In this article, we review stationary phase‐assisted sample prefractionation in proteomic analysis in 2019–2022 period. Applications are grouped according to the mode of retention used in the prefractionation step prior to the final analysis by low‐pH reversed‐phase liquid chromatography‐tandem mass spectrometry. Online or offline instrumental configurations are also commented on, with a highlight on novel online platforms. From the articles reviewed in this period, the order of popularity of chromatographic modes for sample fractionation is affinity chromatography > size exclusion chromatography > hydrophilic interaction chromatography > high‐pH reversed‐phase liquid chromatography > ion exchange chromatography. [ABSTRACT FROM AUTHOR]

Phosphatidylserine (PS) has significant biological and nutritional effects and finds wide applications in the food, pharmaceutical, and chemical industries. To produce high-value PS efficiently, phospholipase D (PLD)-induced transphosphatidylation of low-value phosphatidylcholine (PC) with L-serine has been explored. In this research, we purified recombinant PLD from Streptomyces antibioticus SK-3 using ion exchange chromatography and gel filtration chromatography. Subsequently, we thoroughly characterized the purified enzyme and optimized the transphosphatidylation conditions to identify the most favorable settings for synthesizing PS in a biphasic system. The purified recombinant PLD displayed a robust transphosphatidylation function, facilitating efficient catalysis in the synthesis of PS. Under the optimal conditions (butyl acetate/enzyme solution 1:1, L-serine 160 mg/mL, soybean lecithin 2 mg/mL, and MgCl2 15 mM, at 50 °C for 2.5 h with shaking), we achieved a conversion rate of 91.35% and a productivity of 0.73 g/L/h. These results demonstrate the applicability of the process optimization strategy for using the candidate enzyme in the efficient synthesis of PS. Overall, this study presents a novel and scalable approach for the efficient large-scale synthesis of PS. [ABSTRACT FROM AUTHOR]

Food-derived antioxidant peptides can be explored as natural antioxidants due to their potential health benefits. In this study, antioxidant peptides were isolated and purified from pea protein hydrolysates (PPH). The DPPH and ABTS radical scavenging activities were used as indexes to purify the antioxidant peptides by a series of purification steps including ultrafiltration, ion exchange chromatography, G25 gel filtration chromatography, and reversed-phase chromatography. Three novel antioxidant peptides YLVN, EEHLCFR and TFY were identified, which all exhibited strong antioxidant activity in vitro. EEHLCFR showed stronger DPPH scavenging activity with an IC50 value of 0.027 mg/mL. YLVN showed stronger ABTS scavenging activity with an IC50 value of 0.002 mg/mL and higher ORAC values of 1.120 ± 0.231 μmol TE/μmol, which is even better than that of GSH. Three novel antioxidant peptides significantly elevated LO2 cells viability even at the concentration of 0.025 mg/mL, and cell viability enhanced to 53.42 ± 1.19%, 55.78 ± 1.03%, and 51.09 ± 1.06% respectively, compared to that of H2O2 injury group (48.35 ± 0.96%), and prevented the accumulation of ROS by enhancing the activities of antioxidant enzymes in H2O2-induced oxidative stress LO2 cells. The molecular docking results showed that the potential molecular mechanism of the three novel antioxidant peptides may be in high correlation with the activation of the Keap1-Nrf2 pathway by occupying the Keap1-Nrf2 binding site. These results demonstrate that the three novel antioxidant peptides are potential natural antioxidants that can be devoted to medicine or functional food ingredients. [ABSTRACT FROM AUTHOR]

A biocontrol Bacillus velezensis strain, NT35, was isolated from the rhizosphere soil of ginseng, and its sterile filtrate was obtained through a 0.22 μm filter which had a significant inhibitory effect against Ilyonectria robusta, which causes rusty root rot in Panax ginseng. In order to obtain the best sterile filtrate, the medium fermentation conditions of the strain NT35 were optimized using response surface methodology (RSM), and the best composition was obtained. Therefore, the fermentation medium was composed of yeast extract powder 2.5%, cornmeal 1.5%, K2HPO4 1.5%, and (NH4)2SO4 2.5%. The optimal inoculum amount was 6%, at an initial pH value of 7.0 and culturing at 34 °C at 180 rpm. The antifungal protein 1-4-2F was obtained through precipitation via 30% (NH4)2SO4 precipitation, desalting by Sephadex G-25, ion-exchange chromatography, and gel filtration chromatography. Tricine-SDS-PAGE showed that the purified protein had a relative molecular weight of approximately 6.5 kDa. The protein 1-4-2F was relatively stable and had better antifungal activity at pH 4–10 and 20–100 °C under ultraviolet irradiation of 30 W. The amino acid sequence of protein 1-4-2F was obtained using mass spectrometry, and had 100% similarity to a hypothetical protein from B. velezensis YAU B9601-Y2 (Accession No: AFJ62117). Its molecular weight was 10.176 kDa, the isoelectric point was 9.08, and its sequence coverage reached 49%. The EC50 value of the protein 1-4-2F against I. robusta was 1.519 μg·mL−1. The mycelia morphology of I. robusta changed significantly after treatment with antifungal protein under microscopic observation; the branches of the mycelia increased, distorted, partially swelled into a spherical or elliptical shape, and even ruptured; and the cells became vacuoles. [ABSTRACT FROM AUTHOR]

In the present study, Phenylalanine ammonia lyase (PAL) activity was detected in different parts of grape plant (leaves, pulp, whole seeds and defatted seeds) extracts. The results revealed that the whole seed possess highest PAL activity (69.44 U), followed by defatted seed (44.22 U), whereas extracts of leaves and pulp gave the lowest activity values (22.77 and 5.11 U, respectively). The optimum conditions for crude PAL extraction achieved using 50mM potassium phosphate buffer (pH 7) at mixing ratios of (1:7) for 1 h. extraction time. PAL was shown to precipitate out at the saturation range between 20-90%. For further purification, gel filtration chromatography using Sephadex G-200 column (1.5×70) and ion exchange chromatography using DEAE- Cellulose (DE 52) column (3.2× 10) were applied. The specific activity, number of folds and yield of purified PAL was 1157 U/mg, 19.94 and 15.32 % respectively. [ABSTRACT FROM AUTHOR]

The preparation of mock-ups in heritage science studies represents a valid alternative for investigation purposes, avoiding extensive sampling of cultural heritage objects. This work presents for the first time the successful preparation of three dimensional (3D) mock-ups made of celluloid, considering a combination of historical industrial production strategies and small-scale lab facilities. Prefabricated transparent celluloid sheets were acquired and then shaped through compression molding for creating mock-ups with 3D geometries. These reflected common and representative shapes encountered in the collection of the Deutsches Museum. Visual inspection of the mock-ups allowed determining the best compression molding conditions. Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) confirmed the absence of molecular heterogeneity due to the processing method. Artificial aging of the mock-ups was conducted to reach degradation states comparable with naturally aged objects. ATR-FTIR investigation offered first insights into the induced artificial degradation. Ion chromatography (IC) and gel permeation chromatography (GPC) analyses allowed to assess the extent of the artificial aging of the celluloid mock-ups and confirmed the occurrence of loss of camphor, denitration, and main chain polymer scission, the latter being the predominant decay path. The comparison with historical objects highlighted that the mock-ups are representative of moderately aged artifacts. As such, this study paves the way for implementing moderately aged celluloid 3D mock-ups in heritage science research, enabling in-depth testing for the scope of conservation. [ABSTRACT FROM AUTHOR]

This study was performed to investigate the fibrinolytic enzyme-producing potentials of locally isolated soil bacteria and to purify and characterize the fibrinolytic enzyme of the most potent bacterial isolate. Among 40 isolates, the isolate V4 was found to have the highest potential to produce the fibrinolytic enzyme. According to 16 S rRNA sequence analysis, the isolate V4 was identified as Bacillus atrophaeus (GenBank number: OL662991). The optimal parameters for fibrinolytic enzyme production from B. atrophaeus were determined as a skim milk powder concentration of 15 g/L, an initial pH of 7.0, a temperature of 35 °C and an incubation time of 72 h. The molecular weight of the purified enzyme was calculated as 36 kDa. After ammonium sulphate precipitation, ion-exchange chromatography and gel filtration chromotography-based processes, the specific activity of the fibrinolytic enzyme was determined as 6414.7 U/mg. The purified enzyme showed the maximum activity at pH 7.0 and 35 °C. The enzyme was inhibited by PMSF and EDTA. The substrate specifity of the enzyme was in the following order; fibrin (62.3 U/mg) > fibrinogen (46.2 U/mg) > casein (42.1 U/mg) > serum albumin (6.8 U/mg). This is the first report on the fibrinolytic enzyme production potential of the species B. atrophaeus. [ABSTRACT FROM AUTHOR]

Background: a-Amylase enzyme is digestive enzyme that breaks down complex carbohydrates like starch into simpler sugars; it is being studied as a potential target for anti-cancer therapies. Objective: The current study aimed to evaluate the general characterization, and inhibition of a-Amylase enzyme, which had been purified from the sera of patients diagnosed with Breast Carcinoma. Materials and Methods: A total of 100 blood serum samples were collected from patients with Breast cancer from Baghdad National Hope Hospital in Baghdad. a-Amylase enzyme was purified from serum clinical isolate in three stages; precipitation with 65% saturated ammonium sulphate, ion exchange chromatography utilizing a DEAEcellulose column, and gel filtration chromatography with a sephadex G-200 column. Changing conditions in pH, temperature, inhibitor concentration, and kinetics all affect the activity of the a-Amylase enzyme. Results: a-Amylase inhibition assay showed that Vitis vinifera L. was the most potent inhibitor (71.8%). During the kinetic investigation of the enzyme, it was discovered that the inhibitory mechanism that the extracts use is noncompetitive. Vmax was 0.023, and 0.012 mmol min -l at 75 µg/ml of Vitis vinifera L., Verbena Officinalis L.extracts, respectively. Conclusions: High specific activity for a-Amylase purified from sera of Iraqi breast cancer patients was obtained after three purification steps. Ethanolic plant extracts showed inhibitory effect on a-Amylase while Vitis vinifera L. was the most potent inhibitor. [ABSTRACT FROM AUTHOR]

This study was designed to isolate an anti-inflammatory activity oligopeptide from goose blood (GBP) for ameliorating LPS-mediated inflammation response and oxidative stress in RAW264.7 macrophages. In this study, GBP was isolated by tangential flow ultrafiltration system (TFUS) combined with size exclusion chromatography (SEC), ion exchange chromatography (IEC), and reversed-phase liquid chromatography (RP-LC), and then identified by liquid chromatography mass spectrometry (LC–MS/MS). The experiment results indicated that the amino acid sequence of oligopeptide with the best anti-inflammatory activity was IIe-Val-Tyr-Pro-Trp-Thr-Gln-Arg (IVYPWTQR), which had a molecular weight of 1062.5720 Da, and was derived from haemoglobin subunit beta OS in goose blood. In addition, IVYPWTQR was confirmed to have satisfactory stability and maintained high anti-inflammatory activity in a simulated gastrointestinal digestion. The mechanism by which the IVYPWTQR protected against LPS-mediated inflammation response was attributed to downregulating the TLR4/NF-kB/iNOS pathway. Moreover, IVYPWTQR ameliorated oxidative stress damage in inflammatory state was attributed to activating antioxidant defence system, which was regulated by Keap-1/NRF2/HO-1 signalling pathway for decreasing the accumulation of reactive oxide species (ROS). In summary, these results indicated GBP could serve as a potential functional factor for prevention and improvement of inflammation mediated by LPS and provided an affordable dietary intervention strategy to prevent inflammation. [ABSTRACT FROM AUTHOR]

Rationale: Reversed‐phase, high‐performance liquid chromatography (RP‐HPLC) and high‐performance size exclusion chromatography (HPSEC) methods were developed to effectively separate unknown impurities and polymerized impurities in cefamandole nafate. The liquid chromatography‐tandem ion trap/time‐of‐flight mass spectrometry (LC‐IT‐TOF‐MS) was applied to characterize the structures of the impurities. Ultraviolet (UV) spectrum characteristics and mass spectrum characteristics of △3‐isomer and 7‐epimer in cefamandole nafate were studied to distinguish the isomers. Methods: RPLC‐IT‐TOF‐MS was used to characterize the structures of unknown impurities and polymerized impurities eluted from the C18 column. On this basis, the two‐dimensional (2D) HPSEC‐IT‐TOF‐MS was used to confirm the structures of polymerized impurities eluted from the TSK‐gel G2000SWxl column. Complete fragmentation patterns of impurities were studied and used to obtain information about the structures of the impurities. Results: The structures of 19 unknown impurities in cefamandole nafate were elucidated based on the high‐resolution MSn data with both positive and negative modes, assisted by the UV spectra and stress testing, of which 2 impurities were polymerized impurities. Cefamandole nafate produced a series of degradation impurities, and another principal component cefamandole acid also produced a series of similar degradation impurities. The disciplines between mass fragmentation pattern/UV spectrum and structure for △3‐isomer and 7‐epimer were presented to distinguish their structures. Conclusions: The results of this study provided a scientific basis for the improvement of official monographs in pharmacopoeias to effectively control the impurities and ensure drug safety for the public. This study also revealed the formation mechanisms of degradation impurities in cefamandole nafate, which may guide industry to improve the manufacturing process and storage conditions to reduce the content of impurities in products. [ABSTRACT FROM AUTHOR]

The aim of this study was to isolate and identify antioxidative peptide from goose liver hydrolysate (GLHP) for ameliorating oxidative stress damage by alcohol in HHL-5 hepatocytes. In this research, the target antioxidative peptides in GLHP were separated, purified, and identified via a tangential flow ultrafiltration system combined with size exclusion chromatography (SEC), ion exchange chromatography (IEC), reversed-phase liquid chromatography (RP-LC), and LC-MS/MS. The results suggested that the amino acid sequence of the target antioxidative peptide for ameliorating alcohol-mediated oxidative stress damage in HHL-5 hepatocytes was Leu-Pro-Leu-Pro-Phe-Pro (LPLPFP), which had a molecular weight of 683.41 Da, and was derived from NADH-ubiquinone oxidoreductase chain 1 in goose liver. In addition, LPLPFP was confirmed to have a satisfactory stability and maintained high hepatic protective activity in a simulated gastrointestinal digestion. Moreover, the mechanism of LPLPFP prevented against oxidative stress damage in HHL-5 hepatocytes was attributed to inhibiting the production of reactive oxide species (ROS) by upregulating genes expression in the Ahr-NQO1 signal pathway. In conclusion, these results indicated that dietary GLHP supplementation could ameliorate alcohol-mediated oxidative stress damage and provide an affordable dietary intervention strategy to prevent alcohol-mediated hepatocyte damage. [ABSTRACT FROM AUTHOR]

Herein, polyphenol oxidase (PPO) was purified from Coleus forskohlii via a three-step process involving precipitation by (NH4)2SO4, ion exchange chromatography and gel filtration. PPO was purified 15-fold with a total yield of 31% and a specific activity of 3168 U/mg, and the observed molecular weight was 42 kDa. To improve the reusability and stability of the purified enzyme, calcium alginate incorporated with zinc oxide nanoparticles (Ca-ALG-ZnO NPs) was successfully prepared and characterized through scanning electron microscopy and Fourier transform infrared spectroscopy and utilized as a support for PPO immobilization. The immobilization yield and enzyme activity reached 83% and 3950 U/g support, respectively. The results showed that immobilized PPO could be reused ten times while retaining 69% of its original activity. The purified and immobilized PPO exhibited an optimal pH of 7.0 and 7.5 and an optimal temperature of 40 °C and 50 °C, respectively. The purified and immobilized PPO showed Vmax values of 255.75 and 251.89 Units/ml, and Km values of 4.99 and 3.12 mM, respectively. As a result, this work provides an opportunity to understand and manage the influence of the coleus forskohlii PPO enzyme in key immobilization, industrial. Additionally, the results indicated that the Ca-ALG-ZnO NPs used as supports had a significant effect on the enzyme activity and stability of immobilized PPO. [ABSTRACT FROM AUTHOR]

In the past few years, the production of shrimp shell waste from the seafood processing industries has confronted a significant surge. Furthermore, insignificant dumping of waste has dangerous effects on both nature and human well-being. This marine waste contains a huge quantity of chitin which has several applications in different fields. The chitinase enzyme can achieve degradation of chitin, and the chitin itself can be used as the substrate as well for production of chitinase. In the current study, the chitinase enzyme was produced by Thermomyces lanuginosus. The extracellular chitinase was purified from crude extract using ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography and Sephadex G-100 gel filtration chromatography. The stability and activity of chitinase with different pH, temperature, different times for a reaction, in the presence of different metal ions, and different concentration of enzyme and substrate were analyzed. The chitinase activity was found to be highest at pH 6.5, 50 °C, and 60 min after the reaction began. and the chitinase showed the highest activity and stability in the presence of β-mercaptoethanol (ME). The SDS-PAGE of denatured purified chitinase showed a protein band of 18 kDa. The characterization study concludes that Cu2+, Hg2+, and EDTA have an inhibitory effect on chitinase activity, whereas β-ME acts as an activator for chitinase activity. The utilization of chitin to produce chitinase and the degradation of chitin using that chitinase enzyme would be an opportunity for bioremediation of shrimp shell waste. [ABSTRACT FROM AUTHOR]

α-galactosidase (EC 3.2.1.22) are glycosidases that catalyze the hydrolysis of α-1,6-linked D-galactosyl residues of different substrates, which has been widely applied in the food industry. Oudemansiella radicata is a kind of precious edible medicinal mushroom, which is a healthy, green, and safe food-derived enzyme source. In this study, a novel acidic α-galactosidase was purified from the dry fruiting bodies of O. radicata by ion-exchange chromatography and gel filtration, and designated as ORG (O. radicata α-galactosidase). ORG was further immobilized to obtain iORG by the sodium alginate–chitosan co-immobilization method. Then, the characterization of free and immobilized enzymes and their potential application in the removal of the RFOs from soymilk were investigated. The results showed that ORG might be a 74 kDa heterodimer, and it exhibited maximum activity at 50 °C and pH 3.0, whereas iORG showed maximum activity at 50 °C and pH 5.5. In addition, iORG exhibited higher thermal stability, pH stability, storage stability, and a better degradation effect on raffinose family oligosaccharides (RFOs) in soymilk than ORG, and iORG completely hydrolyzed RFOs in soymilk at 50 °C within 3 h. Therefore, iORG might be a promising candidate in the food industry due to its excellent stability, high removal efficiency of RFOs from soymilk, and great reusability. [ABSTRACT FROM AUTHOR]

A glycoprotein (MGP2) from mountain-cultivated ginseng (MCG) was purified by Tris-HCl extraction followed by DEAE-52 ion exchange chromatography and Sephadex G-100 gel filtration chromatography. The approximate molecular weight (27.0 kDa) and monomeric nature were determined by reduced and non-reduced SDS-PAGE. The structure of MGP2 was characterized by a practical and reliable "protein-polysaccharide analyzed by spectroscopy combined with chemical analysis" strategy. The results showed that MGP2 belonged to Arabinogalactan proteins (AGPs) which contained high amount of Glc (35.1 %). The hemagglutination test concluded that MGP2 was not a lectin. In addition, the MGP2 exhibited antioxidant activity by scavenging radical capacity tests and the ability to protect human erythrocytes and RAW264.7 cells from oxidative damage induced by AAPH. Therefore, these results suggested that glycoprotein MGP2 could be used as a natural antioxidant in drug and food industry. • A glycoprotein (MGP2) was purified from mountain-cultivated ginseng (MCG). • "Protein-polysaccharide analyzed by spectroscopy combined with chemical analysis" strategy. • MGP2 was an arabinogalactan proteins (AGPs). • MGP2 exhibited obvious antioxidant activity. [ABSTRACT FROM AUTHOR]

A component of licorice polysaccharide (GPS-1) was extracted from licorice, its primary structure was identified and characterized for the first time, and its immunomodulatory activity was studied. Crude licorice polysaccharide was isolated and purified by DEAE sepharose FF ion-exchange column chromatography and Chromdex 200 PG gel filtration column chromatography to obtain a purified Glycyrrhiza polysaccharide named GPS-1. NMR and methylation analysis revealed that GPS-1 is composed of homogalacturonan (HG)-type pectin with 4)-D-GalpA-(1 as the backbone. This study of GPS-1 also examined its significant role in regulating immune activity in vitro and in vivo. As a result, GPS-1 promoted the secretion of IFN-g and IL-4 in mice and increased the proportion of CD3+CD4+ and CD3+CD8+ T lymphocytes in their spleens. Dendritic cells (DCs) treated with GPS-1 showed promotion of DC maturation, antigen presentation, and phagocytic capacity. The results suggest that GPS-1 is a potential immunomodulator that stimulates the immune system by regulating multiple signaling pathways. Combined with our characterization of the primary structure of GPS-1, the present investigation provides the basis for future study of the form-function relationship of polysaccharides. [ABSTRACT FROM AUTHOR]

Probiotics are gaining attention due to their functions of regulating the intestinal barrier and promoting human health. The production of exopolysaccharide (EPS) is one of the important factors for probiotics to exert beneficial properties. This study aimed to screen exopolysaccharidesproducing lactic acid bacteria (LAB) and evaluate the probiotic potential. we obtained three exopolysaccharide fractions (EPS1, EPS2, and EPS3) from Lactobacillus pantheris TCP102 and purified by a combination of ion-exchange chromatography and gel permeation chromatography. The structures of the fractions were characterized by FT-IR, UV, HPLC, and scanning electron microscopy (SEM) analysis. The Mw of EPS1, EPS2, and EPS3 were approximately 20.3, 23.0, and 19.3 kDa, and were mainly composed of galactose, glucose, and mannose, with approximate molar ratios of 2.86:1:1.48, 1.26:1:1, 1.58:1.80:1, respectively. Furthermore, SEM analysis demonstrated that the three polysaccharide fractions differ in microstructure and surface morphology. Additionally, preliminary results for immuneenhancing and anticancer activities reveal that these EPSs significantly induced the production of nitric oxide (NO), TNF-α, and IL-6 in Ana-1 cells and peritoneal macrophage cells. Meanwhile, the EPSs also significantly suppressed the proliferation of HCT-116, BCG-803, and particularly A-2780 cells. The results suggest that the three novel EPSs isolated from Lactobacillus pantheris TCP102 can be regarded as potential application value in functional food and natural antitumor drugs. [ABSTRACT FROM AUTHOR]

Proteins are the second most essential macromolecules after nucleic acids. This article aimed at the comparative analysis of extraction methods and nutritional benefits of milk and dairy products. Proteins form the body mass and perform several crucial tasks that include acting as a catalyst and carrying out different metabolic reactions in the body. Furthermore, protein acts as a transporter, transmits nerve impulses, provides mechanical support or immune protection, and controls growth. Several sources of proteins are present, but milk holds an important place due to its biological activities. The considerable health benefits of milk and its products are due to proteins. Yoghurt and cheese have significant importance among milk products. Proteins of milk and its products can be extracted by pH adjustment through homogenisation, centrifugation, and deproteinisation. There are several techniques for identifying and quantifying milk and product proteins. The Kjeldahl and spectrophotometric methods are the most widely used methods for quantifying proteins in milk and its products. Furthermore, these techniques include electrophoresis and chromatographic methods, including native gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), urea-PAGE, capillary electrophoresis, and isoelectric focusing. A few are chromatographic methods like reverse-phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography, and ion-exchange chromatography. Each technique has its advantages as well as disadvantages. The selection of the process depends upon the type of protein. The extracted proteins from milk and its products have many health or therapeutic effects that exhibit antimicrobial, antiproliferative, antioxidant, antihypertensive, anticancer, antiviral, and immunomodulatory effects. Yoghurt has prime importance among milk products because of its therapeutic effects and more protein. [ABSTRACT FROM AUTHOR]

This study was aimed to use digestive duct of common carp Cyprinus Carpio L. as economic source for fish lipase extraction . The enzyme was extracted using potassium phosphate buffer (pH 7, 0.2 M ). The crude extract then precipitated at the saturation range between 40 – 80 % and dialyzed . Further purified carried out through ion exchange chromatography using DEAE-Cellulose column (2.5 × 30 cm) and gel filtration chromatography by Sephadex G-100 column (1.5 x 60 cm). The purified lipase had a molecular weight of 37.150KDa,and optimum pH and temperature for enzyme activity about 45◦C and 6.5 using olive oil as substrate .The specific activity, number of purification folds and yield of purified Lipase were 146.48U/mg, 9.76 and 40.50% respectively. [ABSTRACT FROM AUTHOR]

In this study, three acidic polysaccharides from different plant parts of Codonopsis pilosula var. Modesta (Nannf.) L. T. Shen were obtained by ion exchange chromatography and gel filtration chromatography, and the yields of these three polysaccharides were different. According to the preliminary experimental results, the antioxidant activities of the polysaccharides from rhizomes and fibrous roots (CLFP-1) were poor, and was thus not studied further. Due to this the structural features of polysaccharides from roots (CLRP-1) and aerial parts (CLSP-1) were the object for this study and were structurally characterized, and their antioxidant activities were evaluated. As revealed by the results, the molecular weight of CLRP-1and CLSP-1 were 15.9 kDa and 26.4 kDa, respectively. The monosaccharide composition of CLRP-1 was Ara, Rha, Fuc, Xyl, Man, Gal, GlcA, GalA in a ratio of 3.8: 8.4: 1.0: 0.8: 2.4: 7.4: 7.5: 2.0: 66.7, and Ara, Rha, Gal, GalA in a ratio of 5.8: 8.9: 8.0: 77.0 in for CLSP-1. The results of structural elucidation indicated that both CLRP-1 and CLSP-1 were pectic polysaccharides, mainly composed of 1, 4-linked galacturonic acid with long homogalacturonan regions. Arabinogalactan type I and arabinogalactan type II were presented as side chains. The antioxidant assay in IPEC-J2 cells showed that both CLRP-1 and CLSP-1 promoted cell viability and antioxidant activity, which significantly increase the level of total antioxidant capacity and the activity of superoxide dismutase, catalase, and decrease the content of malondialdehyde. Moreover, CLRP-1 and CLSP-1 also showed powerful antioxidant abilities in Caenorhabditis elegans and might regulate the nuclear localization of DAF-16 transcription factor, induced antioxidant enzymes activities, and further reduced reactive oxygen species and malondialdehyde contents to increase the antioxidant ability of Caenorhabditis elegans. Thus, these finding suggest that CLRP-1 and CLSP-1 could be used as potential antioxidants. [ABSTRACT FROM AUTHOR]

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

Copyright of Mycosystema is the property of Mycosystema Editorial Board and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

[Objectives]This article aimed to establish the expression and purification system of the effector protein HARP1 from cotton bollworm in vitro to express and purify the HARP1 protein in vitro and lay the foundation for analyzing the molecular mechanism of the interaction between HARP1 protein and JAZ proteins in plant jasmonate signal pathway. [Methods]The codon optimization of harp1 gene sequence was carried out. Protein prediction software was used to predict signal peptide and mature peptide in HARP1. Based on the above information, the expression plasmid of the target protein was constructed and induced to express, and the HARP1 protein was purified by various methods including affinity chromatography, protease digestion, dialysis, ion exchange chromatography and gel filtration chromatography. SDS-PAGE and Western blot were used to analyze and detect the expression and purity of protein. [Results]Through optimization, the codon adaptation index (CAI) of harp1 gene increased from 0.39 to 0.95. The protein prediction software predicted that the signal peptide of HARP1 was composed of the 21 amino acids on the N-terminal. The cleavage site of the signal peptide and the mature peptide was between Alanine21 and Leucine22,and the mature peptide was from Leucine22 to Arginine122. The expression plasmid pETDuet1-His6-MBP-3 C-HARP1（22-122）was constructed, and IPTG could induce the expression of the target protein. The fusion protein His6-MBP-3 C-HARP1（22-122）was purified by Ni-NTA Column. 3 C protease could remove the His6-MBP tag, and the HARP1（22-122）protein was successfully separated from His6-MBP tag using anion exchange column. Superdex 200 increase Gel Filtration Column was used for further purification to obtain monomer HARP1（22-122）protein. [Conclusions]The in vitro expression and purification system of HARP1 was successfully established. [ABSTRACT FROM AUTHOR]

An unusual low molecular weight serine protease (NnP28) has been purified from Indian Cobra (Naja naja) venom from the western region of the Indian sub-continent. We reported the purification and characterization of low molecular NnP28, emphasizing its role on human fibrinogen and anticoagulant property. NnP28 was purified using gel filtration column chromatography followed by ion exchange chromatography. Protein gel electrophoresis revealed its molecular weight ≈28 kDa. The exact molecular mass of NnP28 was found to be 27.12 kDa by mass spectrometry, hydrolyzing casein specifically, inhibited by PMSF suggesting it has a serine protease. NnP28 prolonged the clotting time of re-calcified human citrated plasma and activated partial thromboplastin time (APTT) exhibiting anticoagulant property. NnP28 exhibited fibrinogenolytic activity. Thus, the present study demonstrates the presence of unusual low molecular weight serine protease, emphasizing its importance of region-specific Indian cobra species. [ABSTRACT FROM AUTHOR]

On the other hand, chromatography is a powerful analytical tool for separating individual compounds in foods. Some include column chromatography, thin-layer chromatography (TLC), paper chromatography (PC), gas chromatography (GC), ion exchange chromatography (IEC), gel permeation chromatography (GPC), high-pressure liquid chromatography (HPLC), and affinity chromatography (AC) [[2]]. Spectroscopy is a technique indispensable for evaluating the quality of foods. [Extracted from the article]

Zearalenone (ZEN) is a notorious mycotoxin commonly found in Fusarium -contaminated crops, which causes great loss in livestock farming and serious health problems to humans. In the present work, we found that crude peroxidase extraction from soybean hulls could use H 2 O 2 as a co-substate to oxidize ZEN. Molecular docking and dynamic simulation also supported that ZEN could bind to the active site of soybean hull peroxidase (SHP). Subsequently, SHP extracted from soybean hulls was purified using a combined purification protocol involving ammonium sulfate precipitation, ion exchange chromatography and size exclusion chromatography. The purified SHP showed wide pH resistance and high thermal stability. This peroxidase could degrade 95 % of ZEN in buffer with stepwise addition of 100 μM H 2 O 2 in 1 h. The two main ZEN degradation products were identified as 13-OH-ZEN and 13-OH-ZEN-quinone. Moreover, SHP-catalyzed ZEN degradation products displayed much less cytotoxicity to human liver cells than ZEN. The application of SHP in various food matrices obtained 54 % to 85 % ZEN degradation. The findings in this study will promote the utilization of SHP as a cheap and renewable biocatalyst for degrading ZEN in food. • SHP could directly oxidize ZEN with H 2 O 2 as a co-substrate. • 13-OH-ZEN and 13-OH-ZEN-quinone were the main ZEN oxidation products. • SHP-catalyzed ZEN degradation could be regarded as detoxification. • The application of SHP in food matrices obtained 54 % to 85 % ZEN degradation. [ABSTRACT FROM AUTHOR]

A 200 KDa acid-stable thiol β-amylase, appreciably present in the stem of Paederia foetida (48,000 ± 5,500 Units (U)/100 g fresh wt.) was purified by (NH4)2SO4 precipitation, ion exchange chromatography, size exclusion chromatography and HPLC. The enzyme was optimally active in pH 6.0 at 60 °C, showed a specific activity of 3466 U/mg protein and displayed a Km of 3.5 ± 0.1 mg/ml (soluble starch). Activity was stable in the pH range of 3.0–8.0, retaining 94 ± 1% activity at pH 3. The enzyme was stable up to 55–57 °C, beyond which the activity fell sharply. Hg2+ and Ag+ (1 mM concentration) completely inhibited the enzyme activity. Enzyme was strongly inhibited by DTNB, PCMB, N-ethylmaleimide, iodoacetic acid, iodoacetamide, and was experimentally determined to be a thiol amylase (3 SH group/mole); the DTNB inhibition of activity being released by cysteine. The enzyme efficiently hydrolysed potato starch (DE = 51), corn starch (DE= 46), gelatinised cereal flours (wheat, rice and gram), amylopectin, raw starch and raw cereal flours. The enzyme was determined to be a β-amylase (maltose as the only product) by end product analysis and its inability to hydrolyse β-limit dextrin. Immobilisation of the enzyme (crude) on oxidised bagasse (dialdehyde cellulose) increased the temperature optima (by 10 °C) and thermo-stability (retaining 48 ± 2% and 38 ± 1% activity at 70 and 80 °C respectively). The immobilised enzyme system efficiently produced maltose from cereal and tuber starches, remaining 85 ± 1% and 80 ± 1% active after 10th and 20th cycles respectively. [ABSTRACT FROM AUTHOR]

С-phycocyanin (phycocyanin) is a pigment-protein complex of the lightharvesting phycobiliprotein family that takes part in the primary phase of photosynthesis in lower plants. The phycocyanin content depends on such factors as the species of microalgae, physical state of biomass, extraction techniques, etc. The main methods for obtaining phycocyanin from biomass include chemical, physical and enzyme treatments with the following purification by such methods as precipitation with ammonium sulfate, ion exchange chromatography, and gel filtration chromatography. The commercial value of phycocyanin is directly related to the methods of its obtaining, purification and purity. The ratio of absorbances A620/A280 indicates the grade of phycocyanin and is the principal index of its purity. If the ratio of A620/A280 is greater than 4, phycocyanin can be used for pharmaceutical and analytical studies; at not less than 0.7, phycocyanin can be used for the food industry and at not less than 3.9 phycocyanin can be used as a reagent. The purified phycocyanin has some absorption maxima at the wavelengths of 610-625 nm, 353 nm and 277 nm. Its molecular mass ranges from 110 to 220 kDa. The search for extraction methods is aimed at obtaining a high yield of phycocyanin of an appropriate purity in industrial scale. This will allow a wider introduction of phycocyanin into the food, cosmetics and pharmaceutical industries as a safe product with many positive biological properties, in particular, antioxidant, antitumor, antiplatelet, antibacterial, hypotensive, anti-inflammatory, etc. [ABSTRACT FROM AUTHOR]

Purpose: This study focuses on the effect of length and structure of attached polyethylene glycol (PEG) chain on hydrodynamic radius (Rh) and chromatographic retention of PEGylated protein. To this aim human serum albumin (HSA) as a standard protein was PEGylated site specifically with mPEG-maleimide. Methods: Separated PEG_HSA fractions were analyzed by size exclusion and anion exchange chromatography (AExC). The purity of fractions and the relative mobility of PEGylated and native proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Hydrodynamic radius was determined based on the retention time of fractions on size exclusion chromatography (SEC), and also according to the previously reported equations. Results: A linear relation was shown between the molecular weight of attached PEG and Rh of PEGylated HSA. No significant difference between Rh of proteins modified with linear and branched PEG was shown. In SDS-PAGE, the delaying effect of branched PEG on movement of PEGylated protein was higher than that of linear PEG. Conclusion: As PEGylated HSA and dimer HSA have almost the same size and in SEC they elute at very close retention times, so in this case ion exchange chromatography (IExC) is more effective than SEC in separation of PEGylated HSA. Branched PEG- HSA showed earlier elution on anion exchange chromatography compared to linear PEG-HSA, that this can explain the different shielding effect of various structures of attached PEGs. The smaller size of PEGylated HSA in compare to the sum of the hydrodynamic radiuses of native HSA and attached PEG could be as a result of shielded attachment of polymer around protein. [ABSTRACT FROM AUTHOR]

A novel polysaccharide fraction (HEP) from Hericium erinaceus was successively isolated and purified in the present study. We researched its structure and thermal stabilities, and further studied its antioxidant activities in vitro. The results showed that HEP was an acid heteropolysaccharide, with an average molecular weight of approximately 19.7 kDa by high‐performance gel permeation chromatography. Ion chromatography indicated that HEP was mainly composed of fucose:galactose:glucose:mannose:gluconic acid (Fuc:Gal:Glu:Man:GlcA) in a molar ratio of 1:2.87:0.09:0.12:0.01. Additionally, Fourier‐transformed infrared and NMR spectroscopy further demonstrated that HEP was a pyranose containing α‐configuration, mainly consisting of α‐1‐4‐Fuc and α‐1‐6‐Gal as the main chain, with →3,6)‐α‐D‐Man‐(1→and→1,6)‐Glc was branched, with α‐D‐GlcpA‐(1 as T‐terminal. The specific rotation of HEP was +55°; by the differential scanning calorimetry and the thermal stability measurement of thermogravimetric analysis for HEP showed that the pyrolysis process of HEP was mainly divided into two processes, and its melting point was 75.93℃. In vitro anti‐oxidation experiments showed that HEP had a certain ability to scavenge DPPH, hydroxyl, superoxide anion, and ABTS radicals. It was found that HEP had a strong ability to scavenge DPPH‐free radicals, and the highest scavenging rate could reach 91.72% ± 0.17%, which was basically equivalent to the scavenging ability of Vitamin C (Vc). Therefore, it was revealed that HEP might be used as a natural antioxidant component. Practical applications: A novel polysaccharide (HEP) had a potent activity possibly due to its monosaccharide composition, sugar residues, and physicochemical properties. This research proved the potential of HEP in anti‐oxidation and provided the possibility of developing new natural anti‐oxidation products. [ABSTRACT FROM AUTHOR]

The structures and immunomodulatory activities of two polysaccharides (SDH-WA and SDH-0.2A) from Rehmanniae Radix Praeparata (RRP) were investigated. RRP crude polysaccharide was obtained by water extraction and purified. Ion chromatography, high-performance gel permeation chromatography, Fourier-transform infrared spectroscopy, methylation analysis, gas chromatography–mass spectrometry, and nuclear magnetic resonance were used to characterize the polysaccharides. The main chain of SDH-WA was →6)-α-D-Gal p -(1→6)-α-D-Gal p -(1→5)-α-L-Ara f -(1→3,5)-α-L-Ara f -(1→, terminal sugar residue α-L-Ara f -(1→ linked to residue →3,5)-α-L-Ara f -(1→ on the main chain by an O-3 bond. The other two terminal sugar residues α-D-Gal p -(1→ and →6)-β-D-Gal p were linked to the end of the main chain. The main chain of SDH-0.2A was →2,4)-α-L-Rha p -(1→4)-α-D-Gal p A-(1→. Three branched chains α-D-Gal p -(1→6)-α-D-Gal p -(1→5)-α-L-Ara f -(1→3,5)-α-L-Ara f -(1→, →3,6)-β-D-Gal p -(1→5)-α-L-Ara f -(1→, and →4)-β-D-Gal p -(1→5)-α-L-Ara f -(1→ were linked to the main chain residue →2,4)-α-L-Rha p -(1→ by an O-2 bond. Three terminal sugar residues α-D-Gal p -(1→, α-L-Ara f -(1→, and →6)-β-D-Gal p were linked to the end of the chain. Both polysaccharides showed no cytotoxic effects on and significantly promoted the phagocytic activity of RAW264.7 cells. They dose-dependently improved lysozyme activity and stimulated the production of TNF-α and IL-6 by RAW264.7 cells, but attenuated the secretion of lysozymes, TNF-α, IL-6, IL-1β, and nitric oxide by lipopolysaccharide-induced RAW264.7 cells. The present studies suggest that PRR polysaccharide is a valuable source with immunomodulating. • Two polysaccharides (SDH-WA, SDH-0.2A) were purified from Rehmanniae Radix Praeparata. • Structures of SDH-WA and SDH-0.2A were characterized. • Immunomodulating activities of SDH-WA and SDH-0.2A in RAW264.7 cells were studied. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The results that presented in the study were based on collecting one hundred and ten clinical samples of oral cavity, burns and wounds. Primary and secondary diagnositic showed that 14 out of these isolates belonged to Streptococcus spp.. The ability of these isolates in arginine deiminase enzyme production was screened and the results showed that these isolates were arginine deiminase producers with variable degrees. It has been found that one of these isolates which were Streptococcus mitis S5 had the highest specific activity (0.184 U/mg protein), therefore it was chosen for further study. Results of the optimum conditions for arginine deaminase production reveal that the maximum arginine deaminase production was achieved after supplementation of the production medium (pH 7.5) with 1% maltose as carbon source, 1.2% tryptone as nitrogen source, 30mM as arginine concentration and incubated at 37°C for 18h. Under these conditions, ADI purified in three steps including; precipitation with 70% saturated ammonium sulphate, dialysis then ion exchange chromatography using DEAE- cellulose column and the last step was gel filtration chromatography throughout Sephadex G150 column. Specific activity of the purified enzyme was increased up to 18 U/mg with 5 folds of purification and 70.8 % enzyme recovery. [ABSTRACT FROM AUTHOR]

This study demonstrated the purification of bovine lactoferrin (BLF), caprine lactoferrin (CLF) and ovine lactoferrin (OLF) by ion-exchange chromatography and gel filtration chromatography. The purified lactoferrin (LF) were identified by mass spectrometry and its purity was analyzed by RP-HPLC. LFs with different iron saturations were established, and it was found that iron saturation of Holo-CLF (62.01%) and Holo-OLF (63.74%) were lower than Holo-BLF (83.93%). The effect of iron saturation on the physicochemical properties of LFs were evaluated by circular dichroism, Fourier transform infrared spectroscopy and differential scanning calorimetry. The results showed that both species and iron saturation did not affect the secondary structure of LFs, however, they did influence tertiary structure of LFs. The conformation of Holo-LFs was more compact than that of Apo-LFs. Furthermore, Native-OLF showed the tightest conformation, followed by Native-BLF and Native-CLF. In addition, the increase of iron saturation enhanced the thermal stability of LFs in all three species. Among three species, Apo-OLF was the most thermally stable in among three species due to its more compact structure, while Holo-BLF was the most thermally stable among three species due to its highest iron saturation. Although Native-CLF had a relatively lower iron saturation compared to BLF and OLF, its thermal stability was relatively higher as the main denaturation temperature peak of CLF was located at 85.65 °C. These findings would contribute to further understanding of the information of LFs at different iron saturation levels, which is crucial for the production and application of LFs in the future. [Display omitted] • Separation of lactoferrin based on ion exchange and gel filtration chromatography. • The iron saturation of Holo-CLF and Holo-OLF was lower than that of Holo-BLF. • Iron saturation affects the tertiary structure and thermal stability of lactoferrin. [ABSTRACT FROM AUTHOR]

Lipoxygenase (linoleate:oxygen oxidoreductase, LOX) was extracted from African oil bean seed and purified in three purification steps using ammonium sulphate precipitation, ion exchange chromatography on DEAE-Sephadex A50 column and gel filtration chromatography on Sephadex G 200. The optimum pH and temperature were investigated while the pH and temperature stability were also examined. The effect of metal ions on LOX was also investigated. The molecular weight and kinetic parameters were determined. The single protein band on SDS-PAGE suggested that the enzyme was homogenous. The molecular weight was found to be 77.54 kDa. A purification fold of 32.7 and 10.7% yield of the enzyme was achieved. The optimum temperature of the purified lipoxygenase was 40 °C and the enzyme was thermally stable at 40 °C and rapidly lost activity at 50 °C and above. The purified enzyme had an optimum pH of 6.0 and was stable at pH 5.0 and 6.0. The kinetic parameters K m and V max of the purified lipoxygenase were 0.1 mM and 94.40 μmol/min, respectively. The activity of the enzyme was enhanced in the presence of Ca2+ and Mg2+ while Zn2+, Cu2+ and Fe2+ significantly reduced the enzyme activity. Total inactivation of the lipoxygenase activity was observed at alkaline environment. This study confirmed lipoxygenase activity in African oil bean seed and showed properties that could be explored in improving its utilization in industrial food processing. [Display omitted] • Lipoxygenase was extracted from dormant seeds of African oil bean and purified. • The purified enzyme had temperature and pH optima of 40 °C and pH 6.0, and total inactivation in alkaline environment. • The kinetic parameters Km and Vmax of the purified lipoxygenase were 0.1 mM and 94.40 μmol/min, respectively. • Ca2+ and Mg2+ enhanced activity of the enzyme, while Zn2+, Cu2+ and Fe2+ inhibited the activity. • The African oil bean lipoxygenase could be exploited in industrial processes. [ABSTRACT FROM AUTHOR]

Although the reversed-phase liquid chromatography (RPLC) is the most used separation front for mass spectrometry, many other separation modes are critical for enabling characterization of the protein therapeutics. Specifically, chromatographic separations under native conditions, such as those based on size exclusion chromatography (SEC) and ion-exchange chromatography (IEX), are used for characterizing important biophysical properties of protein variants in drug substance and drug product. Because most native state separation modes use non-volatile buffers with high salt concentration, optical detection has been traditionally used. However, there is an increasing need to understand and identify the optical underlying peaks by mass spectrometry for structure elucidation. For size variant separation by SEC, the native MS helps to understand the nature of the high molecular weight species, as well as clipping sites for low molecular weight fragments. For charge variant separation by IEX, native MS can reveal the post-translational modifications or other important factors contributing to charge heterogeneity at the intact level. Here, we demonstrate the power of native MS by direct coupling of SEC and IEX eluent to a time-of-flight mass spectrometer to characterize bevacizumab and NISTmAb. Our studies exemplify the effectiveness of native SEC-MS for characterizing bevacizumab's high molecular weight species at less than 0.3% (based on SEC/UV peak area%) and analyzing the fragment pathway with single amino acid difference for its low molecular weight species at less than 0.05%. Good IEX charge variant separation was obtained with consistent UV and MS profiles. The identity of separated acidic and basic variants were elucidated by native MS at intact level. We successfully differentiated several charge variants including glycoform variants that have not been reported before. In addition, native MS allowed identification of higher molecular weight species as late eluted variants. Overall, the SEC and IEX separation combined with high resolution and high sensitivity native MS, which is significantly different from the traditional RPLC-MS workflows, can be an effective tool that offers valuable insights for us to understand protein therapeutics at native state. [Display omitted] Separate variants of mAb therapeutics by size exclusion or ion-exchange chromatography. • Enable direct MS detection and identification. • Degradation pathway analysis for SEC size variant <0.05%. • Direct identification of complex charge variants including glycoform and aggregation variant [ABSTRACT FROM AUTHOR]