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2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Dawei Wu ◽  
Yanrong Zhang ◽  
Dawei Wang ◽  
Tingting Liu ◽  
Shanshan Zhang ◽  
...  

In this study, hot water was used to extract Inonotus obliquus oligosaccharide. DEAE-cellulose and Sepharose G-200 were used to purify Inonotus obliquus oligosaccharide. Inonotus obliquus oligosaccharide IOP-2A was obtained. Its molecular weight Mw is about 1000 Da. The monosaccharide composition and molar ratio were glucose : xylose : galactose : mannose = 54.1 : 13.6 : 13.2 : 6.7. In addition, it also contains a small amount of galactose, gluconic acid, rhamnose, and fucose. IOP-2A contained mainly β-glycosidic bonds. Among them, 1,4-glycosidic bonds accounted for 9.2%, and 1,6-glycosidic bonds accounted for 85.1%. Oligosaccharide macromolecules formed a layered structure. Mouse experiments showed that IOP-2A had the function of preventing hyperlipidemia. At the same time, IOP-2A had a certain protective effect on the liver and kidney. The mechanism of IOP-2A in preventing hyperlipidemia was obtained from the perspective of mouse intestinal flora.


2021 ◽  
Vol 2 (4) ◽  
pp. 32-42
Author(s):  
Sabah Gazal ◽  
Susan Jamil Ali . ◽  
Perry Habib Saifullah .

Acetyle CoA Carboxylase-1was purified from serum of premenop- ausal women with breast cancer (after Mastectomy or treatment ) by Gel Filtration using Sephadex G-100 and by Ion Exchange using DEAE-Cellulose A-50, also the molecular weight was estimated by the Electrophoresis on Acrylamide in the absence of denaturing elements . The result showed that a single band was obtained at 210 KD by Gel Filtering while Ion Exchange showed one band at 210 KD. The optimum temperature of purified Acetyle CoA Carboxylase-1 was 40 , optimal pH at 7.5 and the optimum substrate concentration at 1.8mM. Michaelis-Menten constant (km) was 0.3mM and Vmax was 23mM.min-1.


2021 ◽  
Vol 83 (3) ◽  
pp. 72-80
Author(s):  
O.B. Balko ◽  

According to our previous results, S-type bacteriocins of Pseudomonas aeruginosa are characterized by high activity against phytopathogenic Pseudomonas syringae strains. In addition to these pyocins producing strains are able to synthesize microcin-II-like bacteriocins. Presence of interaction between these two killer factors can determine methods of their use and activity increase of bacteriocins with antiphytopathogenic properties. The aim of the work was to test possibility of interaction between S-type pyocins and microcin-II-like bacteriocins of P. aeruginosa. Methods. The objects of the study were pyocins produced by 6 P. aeruginosa strains. Killer factors in composition of induced lysates were concentrated by 70% ammonium sulphate precipitation, dialyzed through dialysis membrane with molecular weight cut-off (MWCO) 3.5 kDa. Then ion-exchange chromatography with DEAE-cellulose, gel filtration with Sephadex G-75 and ultracentrifugation at 215.000 g for 1 and 4 hours were used for their separation. Protein concentration and antimicrobial activity were determined in obtained fractions. Visualization of proteins in active fraction composition was conducted by electrophoresis according to the Laemmli method. Results. Under ion-exchange chromatography with DEAE-cellulose application elution of bacteriocins available in lysate composition occurs simultaneously. The highest indices of activity and protein concentration were in the 4th fraction, containing two protein bands with molecular weight near 58 and 9 kDa, which are typical for S5 pyocin and microcin-II-like bacteriocins of P. aeruginosa. Further gel filtration of sampled fractions through Sephadex G-75 allowed to separate noted killer factors and obtaine purified fraction containing microcin-II-like pyocins only. Application of ultracentrifugation during 1 hour didn’t precipitate studied bacteriocins, whereas during 4 hours – lead to their separation. At the same time a twofold increase of activity indices for S-type pyocins in precipitates and for microcin-IIlike killer factors – in supernatants were observed. However achieved concentration was characterized by short-term effect, since in 14 days activity of supernatants decreased by 4–16 times, and for precipitates – by 80–640 times. Then revealed tendency for activity decrease continued. Conclusions. S-type pyocins and microcin-II-like bacteriocins of P. aeruginosa interact with each other, that ensures their stabilization and protects again destruction. Application of methods that cause separation of these killer factors is inexpedient, since it results into considerable decrease of bacteriocin activity indices.


2021 ◽  
Vol 14 (3) ◽  
pp. 30-38
Author(s):  
I. I. Patalakh ◽  

Current large-scale production of blood-derived pharmacological preparations is aimed at expanding the list of products and deeper extraction of target proteins especially at the pre-purification stage. In particular, this problem becomes critical for the isolation of proteins like protein C (PC), which is present in plasma in trace amounts. Aim. We aimed to improve the buffer composition to minimize the interaction of PC with other proteins and lipids that are inevitably present in the stock material. Methods. The content of protein C in plasma and its derivatives was assessed by the amidolytic activity to the chromogenic substrate S2366. A decrease in homologous impurities and plasma enrichment with protein C was provided by selective bulk adsorption on DEAE-cellulose. Results. Here we describe that an equimolar mixture of two amino acids (L-arginine and L-glutamic acid) essentially increased the content of protein C at the stage of cryo-depleted plasma pre-purification, including initial dilution and subsequent enrichment of plasma with protein C due to selective bulk adsorption on DEAE- cellulose. Additionally, it was revealed that solutions of these amino acids, when combined, inhibit the induced amidolytic activity of protein C and increase its solubility (in contrast to other plasma proteases). Conclusion. Pre-adding of a mixture of amino acids L-arginine and L-glutamic acid to cryo-depleted plasma significantly optimizes the pre-purification stage of protein C, providing a 5-fold increase in its yield after elution from DEAE-cellulose.


2021 ◽  
Author(s):  
Yves Mann Elate Lea Mbassi ◽  
Marie Solange Evehe Bebandoue ◽  
Wilfred Fon Mbacham ◽  
John Payne Muluh

Abstract A peroxidase isoenzyme (named A6 in a previous study) was purified from radicles of a Vigna species by a combination of gel filtration on Sephadex G-100, heat treatment, CM-cellulose chromatography and DEAE-cellulose chromatography. It has been successfully separated from other anionic isoperoxidases expressed in the same tissue. It has a molecular weight of about 41 kDa and exhibits a great activity toward the oxidation of O-dianisidine, ABTS, TMB, DAB and OPD at optimum pH (pH 3 for ABTS, pH 4 for OPD and pH 6 for the others) and toward the reduction of H2O2. Its very acid optimum pH for the oxidation of ABTS is not a characteristic of other peroxidases except African oil palm tree peroxidase. Apparent Km values for these substrates were respectively 3.50 mM, 0.12 mM, 1.81 mM, 0.05 mM, 17.22 mM and 2.53 mM; catalytic efficiencies were 5.12×104 mM-1.min-1, 2.22×106 mM-1.min-1, 1.59×105 mM-1.min-1, 1.82×105 mM-1.min-1, 3.17×105 mM-1.min-1and 1.79×106 mM-1.min-1. It has an optimum temperature of activity around 60°C, and its heat inactivation fit to the first-order kinetics, with half-lives of 3.06 weeks, 13.5 hours, 15 min and 3.5 min at 50°C, 70°C, 80°C and 90°C respectively. The calculated activation energy (E) for its thermal inactivation was found to be 221.5 KJ/mol at pH8. This peroxidase isoenzyme is stable for 4 months at room temperature, loosing only 5% of its initial activity over this period. Mg2+ inhibits the activity of the enzyme. The Ca2+ ions greatly increase the stability of this peroxidase at 80°C, while Mn2+ and Zn2+ reduce it. The enzyme is inhibited by sodium azide at concentrations above 1 µM with an IC50 value around 10 µM. This inhibition, in addition to the RZ value (A403nm/A280nm) evaluated at 2.4 confirms the presence at the active site of the enzyme of a heme group common to class III peroxidases. The unusual catalytic and thermal characteristics of A6 could make it a potent tool in several biotechnological applications, especially as part of kit for enzyme immunoassays and clinical diagnosis.


BMC Chemistry ◽  
2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Lei Guo ◽  
Hongwei Dai ◽  
Jiayu Ma ◽  
Junmin Wang ◽  
Yan Hua ◽  
...  

Abstract Background Fungal polysaccharides belong to a very important class of biological macromolecules in nature, and have complex monosaccharide composition and structure. These studies on structure and biological activity of fungal polysaccharides have become one of the research hotspots of scholars at home and abroad. Results This study was performed in order to understand the structural characteristics and antioxidant activity of polysaccharides from Lenzites betulina (LBPs). The LBPs were deproteinized using sevag method, and further purified by DEAE cellulose-52 column and Sephadex G-100 column chromatographies, then the two refined polysaccharides were obtained and named LBPs-5 and LBPs-6. Fourier transform infrared spectrometry (FT-IR) showed that LBPs-5 and LBPs-6 are typical β-pyranose with characteristic peaks of polysaccharides. The molecular weight of the two water-soluble polysaccharides were estimated to be 3.235 × 103 Da and 6.196 × 103 Da by HPGPC, respectively. HPLC with PMP derivatization analysis indicated that the monosaccharide compositions of LBPs-5 were mannose, glucuronic acid, glucose, and galactose in a molar ratio of 0.05:0.15:0.76:0.04. The monosaccharide compositions of LBPs-6 were mannose, glucuronic acid, and glucose, in a molar ratio of 0.04:0.17:0.79. Furthermore, the two water-soluble polysaccharides demonstrated strong scavenging effects on DPPH·, ABTS·+, ·OH and weak total reducing power, especially LBPs-6 was significantly stronger in scavenging rate than that of LBPs-5. Conclusions The outcome of the study indicated that LBPs had good potential as medicine and food.


2021 ◽  
Vol 43 ◽  
pp. e49082
Author(s):  
José Ariévilo Gurgel Rodrigues ◽  
Ana Luíza Gomes Quinderé ◽  
Norma Maria Barros Benevides

The structural complexity of the agaran type-sulfated polysaccharides (SPs) found in Acanthophora muscoides limits its investigation as anticoagulant alternative to heparin which induces clot complications. This study was extended to evaluate the properties of a SPs fraction and its alkali/desulfated derivatives on an intrinsic pathway-induced thrombin generation (TG) continuous model using 60-fold diluted normal or serpins-depleted human plasma. 0.75 M NaCl-eluted SPs fraction by DEAE-cellulose chromatography containing sulfate (35.20%), total sugars (55.97%) and no proteins showed charge homogeneity and heterogeneous molecular weight by agarose/polyacrylamide gel electrophoresis, respectively, using sequential staining with toluidine blue and Stains-All. Fourier Transform Infrared spectroscopy confirmed agaran-structure. Intact fraction poorly acted on the activated partial thromboplastin time (3.10 IU) than heparin (193 IU), but there was a preponderance of the serpin-independent effect than serpin-dependent one in TG assay comparing both systems was continually recorded. Heparin abolished plasma TG, but was inactive in depleted human plasma. While desulfated derivative of the respective fraction anticipated and induced thrombin formation vs. untreated plasma. The results suggested that sulfated sugars residues in the sacharide units of the polymer appear to be important to attenuate TG in vitro.


2021 ◽  
Vol 2 (1) ◽  
pp. 10-15
Author(s):  
Thaís Barboni Alves ◽  
Gabriela Molinari Roberto ◽  
Maria Sol Brassesco ◽  
Luis Henrique Souza Guimarães

Different fungal species, especially from the genus Aspergillus, have been reported as producers of small molecules, including proteins, with biological activity and a better understanding of their sources, structure, function and toxicity is essential for their biotechnological applications. According to this, our aim was to evaluate the cytotoxic activity of the extracellular filtrate produced by A. niveus. The crude filtrate obtained in YPD medium containing 18 kDa protein, after cultivation for 120 h, was selected for cytotoxic assay, assessed by Giemsa staining, against different human tumor cell lines. Crude filtrate inhibited (from 27% to 50%) the ONS-76 (medulloblastoma), HT144T (melanoma), HOS (osteosarcoma), T98G (glioblastoma) human tumor cell lines and MRC-5 (fibroblasts) human normal cells, at 20 µg/mL for 72 h treatment. According to this, the 18 kDa protein band and the fractions obtained after DEAE-Cellulose procedure were evaluated through mass spectrometry (MS/MS) analysis, revealing the presence of peptides with similarity to the alpha-sarcin, mitogillin and Aspf1 ribotoxins described for other Aspergillus species. In conclusion, the A. niveus extracellular filtrate containing ribotoxin-like proteins reduced, in vitro, the growth of human tumor cell lines indicating their biotechnological potential, indicating a possible future application in the elaboration of immunotoxins.


Author(s):  
Doaa A. Darwish ◽  
Hassan M. M. Masoud ◽  
Mohamed M. Abdel-Monsef ◽  
Mohamed S. Helmy ◽  
Hind A. Zidan ◽  
...  

Abstract Background Honey bee venom contains various enzymes with wide medical and pharmaceutical applications. Results The phospholipase A2 (PLA2) has been apparently purified from the venom of Egyptian honey bee (Apis mellifera lamarckii) 8.9-fold to a very high specific activity of 6033 U/mg protein using DEAE–cellulose and Sephacryl S-300 columns. The purified bee venom PLA2 is monomeric 16 kDa protein and has isoelectric point (pI) of 5.9. The optimal activity of bee venom PLA2 was attained at pH 8 and 45 °C. Cu2+, Ni2+, Fe2+, Ca2+, and Co2+ exhibited a complete activating effect on it, while Zn2+, Mn2+, NaN3, PMSF, N-Methylmaleimide, and EDTA have inhibitory effect. Conclusions The purified bee venom PLA2 exhibited anti-platelet aggregation and anti-coagulation activities which makes it promising agent for developing novel anti-clot formation drugs in future.


Author(s):  
Dibyendu Sekhar Mahanty ◽  
Sautrik Basu ◽  
Jukta Adhikari

Altered salinity is one the most important perils encountered by marine plants inclusive of algae. Under hyper saline condition plants accumulate several stress relieving osmolytes including myo-inositol, the most widespread cyclitol in plants. The present communication reports the occurrence of myo-inositol biosynthesis in six different Rhodophycean seaweeds growing under stressful intertidal habitats of the Okha coast (Gujarat, India), on the basis of a study conducted on two marker enzymes of myo-inositol biosysnthesis [L-myo-inositol-1-phosphate synthase and D/L-myo-inositol-1-phosphate phosphatise]. Both enzymes were partially purified from Halymenia venusta to about 27 and 39 folds respectively over the homogenate following low-speed centrifugation, 30-75% ammonium sulphate fractionation, successive chromatography through DEAE-cellulose / CM-Cellulose, Sephadex G-200 and BioGel 0.5m / UltrogelAcA 34 columns. The temperature and pH optima for both the enzymes were similar and were recorded to be 350C and 7.5 respectively. For MIPS, D-glucose-6-phosphate and NAD were the exclusive substrate and coenzyme respectively and D/L-MIP was the sole substrate for MIPP. The Km values for D-glucsoe-6-phosphate and β-NAD were recorded to be 3.599 mM and 0.2366 mM respectively, while the Km value for D-MIP was found to be 0.4070 mM. Monovalent cations K+ had slight stimulatory, Li+ was strong inhibitory for both the enzymes. Divalent cations Ca2+ exhibited slight stimulatory and Cd2+ reduced MIPS and MIPP activities. MIPP was stimulated by Mg2+. Cu2+ and Hg2+ were strong inhibitors of both the enzymes. A steady and proportionate increase in the content of free myo-inositol was observed along with elevated levels of recorded salinity.


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