Acetone Precipitation Research Articles

BIOSYNTHESIS AND DEVELOPMENT OF NOVEL METHOD FOR COMMERCIAL PRODUCTION OF BIOSURFACTANT UTILIZING WASTE POTATO PEELS

The aim of this research work was to create compelling strategy for biosurfactant generation by waste potato peels and pulp utilizing mutated B.subtilis DDU20161. Major parameters like creation of medium, fomentation, and pH were kept up in the investigation. The ideal biosurfactant yield by acetone extraction technique was 253.79 mg L-1 with biomass of 3.56 gL-1 at 40 h. The produced biosurfactant was characterised by studying surface tension, emulsification index, TLC, CMC and FTIR. The highest emulsification index was 75.05% in benzene, Critical micelle concentration(CMC) was found to be 0.028μM, and FT-IR study revealed presence of peptide bond at 3188cm-1 (=N-H). The results of stability study indicated that the produced biosurfactant is stable at 300-1000C without any significant change in surface tension properties of biosurfactant. The shaped biosurfactant was found to be stable in alkaline pH. The present work was focussed on developing a production methods utilizing acetone precipitation system.

Sequestration and purification of essential enzyme from Pleurotus florida compost waste and its application studies

Spent oyster mushroom (Pleurotus florida) compost as tremendous source for isolation of industrial significant enzymes such as Amylase, Protease and Cellulase. This study was conducted in order to achieve efficient extraction of lignocellulolytic enzymes amylase (EC 3.2.1.1), Cellulase (EC 3.2.1.4) and Protease (EC 3.4.21.14) from spent oyster mushroom (Pleurotus florida) compost waste. Optimal enzyme recovery was achieved when spent oyster mushroom compost wastes and concentrated by acetone precipitation. The purification was performed by column chromatography. The enzymes such as cellulase, amylase and protease released from oyster mushroom (Pleurotus florida) compost waste were showed activities of 15.78 U/ml, 3.42 U/ml and 0.042 U/ml respectively. These were utilized in various industrial and environmental applications such as Starch processing in potato waste from food industry using amylase and Biotreatment of cotton waste using cellulase.

Microbial Bioremediation of Feather Waste for Keratinase Production: An Outstanding Solution for Leather Dehairing in Tanneries.

In leather industries and tanneries, large amount of wastes has been disposed; which polluting water, soil, and atmosphere and causing serious human health problems. In particular, chemical dehairing process of leather industries produces fair amount of toxic wastes. It is, thus, urgently needed to use alternative processes free from pollution. As more than 90% of keratin is contained in feather, it is desirable to develop bioremediation process using keratinolytic microorganisms. In the present investigation, therefore, we first identified Bacillus cereus and Pseudomonas sp. to be able to produce keratinase. Then, the optimization was performed to maximize the keratinase activity with respect to cultivation temperature, pH, and incubation time. Moreover, the effects of metal ions and various substrates on keratinase activity were also investigated. The result indicates that keratinase activity became maximum at 50°C for both strains, whereas the optimal pH was 10.0 for B. cereus and 7.0 for Pseudomonas sp. The highest keratinase activity of 74.66 ± 1.52 U/mL was attained by B. cereus, whereas 57.66 ± 2.52 U/mL was attained by Pseudomonas sp. Enzymatic dehairing efficiency of leathers was also compared with chemical dehairing (Na2S and CaO), where complete dehairing was achieved by treating them with crude keratinase. Partial enzyme purification was performed by acetone precipitation. Batch cultivation of B. cereus using 1 L fermentor indicates a potential candidate for large-scale keratinase production. Thus, keratinase enzyme by degrading poultry wastes (feather) can be an alternative approach to chemical dehairing in leather industries, thus preventing environmental pollution through bioremediation.

Inactivation kinetics and thermodynamic properties of polygalacturonase produced by Aspergillus awamori CICC 2040 on pretreated orange and plantain peels

This study investigated the effect of pretreatment of orange and plantain peels on the inactivation kinetics and thermodynamic properties of polygalacturonase (PG) produced by Aspergillus awamori CICC 2040. Orange and plantain peel powders were subjected to microwave-assisted NaOH pretreatment and used as substrates for PG production. Un-treated peels served as controls. The PG was purified using acetone precipitation and column chromatography, and the inactivation kinetics, temperature dependency, and thermodynamic properties of the crude and purified PGs were determined. Higher inactivation rate constant was obtained for crude PG produced using pretreated orange peel (CPOF) and plantain peel (CPPF) compared to PG produced using untreated orange peel (Uo) and plantain peel (Up). At all the temperatures considered, higher half-life and decimal reduction time were recorded for CPOF and CPPF compared to Uo and Up. The highest half-life (45.60 min) and decimal reduction time (151.49 min) were recorded for CPOF at 60 ?C. Lower half-life and decimal reduction time were obtained for purified PGs compared to the crude PG. Polygalacturonase produced from pretreated peels had lower activation energy than those produced from untreated ones. The higher activation energy was recorded for the PG produced using orange peel compared to the one from plantain peels. The enthalpy of CPOF and CPPF was slightly lower than Uo and Up. The pretreatment of the peels resulted in a reduction of Gibbs free energy (?G ) and entropy (?S) of crude and purified PG. Higher ?G and ?S were recorded for the purified PG compared to the crude PG. Negative entropy and enthalpy were recorded for all the PGs. The findings from this study showed that the kinetic and thermodynamic properties of PG, produced by Aspergillus awamori CICC 2040, were enhanced by the pretreatment of orange and plantain peels.

Influence of sulfated polysaccharides from Ulva lactuca L. upon Xa and IIa coagulation factors and on venous blood clot formation

The interest in bioactive compounds from natural sources, such as marine organisms, has increased considerably in recent years. Among these compounds, sulfated polysaccharides from seaweed exhibit a broad spectrum of biological activities. Sulfated polysaccharides from green algae are still poorly investigated. For this reason, in this study, using unusual methodologies, such as extraction conditions, FACE, and RAMAN, we investigated the structural features of F50UL and F70UL sulfated polysaccharides from Ulva lactuca L. and their distinct in vitro anticoagulant and in vivo antithrombotic activities. Sulfated polysaccharides of U. lactuca were obtained by enzymatic proteolysis with ALCALASE® and fractionated by acetone precipitation. F50Ul and F70Ul sulfated polysaccharides with higher yield were partially chemically characterized by Fluorophore-assisted carbohydrate electrophoresis (FACE) and RAMAN spectroscopy analysis and submitted to an in vitro screening by APTT, PT, TT, and anti-factor Xa and IIa tests. The venae cavae ligature experimental model for the analysis of in vivo antithrombotic activity of F50Ul and F70Ul sulfated polysaccharides were also performed. The U. lactuca L. sulfated polysaccharides characterization by FACE and RAMAN showed a typical ulvans structure that contains as principal component rhamnose, but other monosaccharides (uronic acid, glucose, and galactose) are present. F50Ul (0.1–1.0 μg/μl) showed anticoagulant activity in vitro. However, F70Ul that has a similar composition did not present these effects. Also, only F50Ul sulfated polysaccharides (≥5 μg/g) showed a great in vivo antithrombotic concentration-dependent and time-dependent activity. In summary, we demonstrate the use of unusual extraction and characterization analysis procedures for U. lactuca L. sulfated polysaccharides and the ability of F50Ul to reduce the weight of thrombus in rats probably by the association with factors Xa and IIa inhibition. These results provide strong evidence of the anticoagulant potential of these sulfated polysaccharides isolated from Ulva lactuca L.

Extraction, Purification, and Characterization of Trypsin Obtained from the Digestive System of Yellowfin Seabream (Acanthopagrus latus).

The development of the marine aquaculture industry has led to the generation of significant amounts of fish wastes. Marine farm wastes exert adverse effects on the surrounding area of the cages. On the other hand, wastes of fish and other aquatic animals are regarded as major sources of valuable natural bioactive compounds, including enzymes, proteins, bioactive peptides, oil, amino acids, collagen, gelatin, calcium, biopolymers, and water-soluble minerals. To investigate the potential of marine fish waste, the whole digestive system of yellowfin seabream (Acanthopagrus latus) was extracted for extraction and identification of trypsin enzyme. Fish (179.93±93.67 g; 184±28.17 cm) were caught from the Persian Gulf and stored at -20 °C. Yellowfin seabream were dissected and their whole digestive systems were removed. Samples were thoroughly washed with distilled water and purified through defatting using acetone and ammonium sulfate precipitation. The following issues were assessed: the total and specific activity of trypsin, protein determination, molecular weight, enzyme activity and stability in different pH values and temperatures. The obtained results indicated that specific activity and protein content of trypsin enzyme were 4.4 U and 3.4 mg/ml, respectively. The molecular weight of 23 kDa was reported for trypsin using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method. Maximum activity and stability of trypsin were observed at 60°C and 45°C, respectively. Trypsin demonstrated maximum activity and stability at a pH value of 8.0. In general, the results of the current study suggested that trypsin extracted from the digestive system of yellowfin seabream has considerable potential for industrial applications, such as the food industry, owing to its characteristics and stability under alkaline conditions.

Fluoride reactivation-enabled sensitive quantification of tabun adducts on human serum albumin by GC-MS/MS via isotope dilution.

Organophosphorus nerve agents inhibit the cholinesterase activity by phosphylation of the active site serine. The resulting phosphylated cholinesterase and adducts on human serum albumin (HSA) are appropriate biomarkers for nerve agents exposure. Several methods have been developed for the detection of nerve agents, including fluoride reactivation or alkaline cleavage. It was previously thought that some nerve agents adducts to HSA could not be detected via fluoride regeneration. In our study, the results showed that tabun (GA) adducts of HSA could be detected by fluoride regeneration. The sample preparation included acetone precipitation, washing and SPE. Deuterated tabun (d5-GA) was applied as the internal standard. The product of regenerated fluorotabun is detected with a good linearity (R2 > 0.997) in the concentration range from 0.02 to 100.0 ng/ml, small relative standard deviation (≤6.89%) and favorable recoveries between 94.8 and 106.3%. The established preparation confirmed the fluorotabun was regenerated from the GA-HSA adducts.

A Solvent-tolerant Alkaline Lipase from Bacillus sp. DM9K3 and Its Potential Applications in Esterification and Polymer Degradation

Solvent-tolerant lipase produced by Bacillus sp. DM9K3 has been isolated from the hypersaline area, White Rann of Kachchh, Gujarat, India. The strain initially showed lipase activity of 11.1 U/mL in a basal medium which increased to 52.0 U/mL under optimized culture conditions. Bacillus sp. DM9K3 exhibited stability at 7% salinity, pH 9.0 and 50°C. The extracellular lipase was partially purified by acetone precipitation followed by DEAE-cellulose resulting in 39-fold purification with 40% yield. Metals ions such as Mg2+, Ca2+ and K+ showed enhanced enzyme activity. EDTA did not have a significant effect on activity suggesting that lipase is not metalloenzyme. The lipase under study showed the highest activity when palmitate (C16) was used as a substrate and was also highly stable in organic solvents such as cyclooctane and benzene. The partially purified enzyme was immobilized for increasing the efficiency of the ethyl caprylate (an orange flavored ester) synthesis in the presence of cyclooctane. Additionally, lipase of Bacillus sp. DM9K3 was explored for biodegradation of polycaprolactone microspheres and showed promising results for potential applications in drug delivery system.

Bromelain plus peroxidase reduces non-Hodgkin lymphoma progression in invivo via up-regulation of antioxidant enzymes and modulating apoptotic protein expression

Aim: Pineapple (Ananas comosus (L.) Merr.) is a good source of bromelain (B) and also contain peroxidase. The objective of this study is isoaltion of bromelain plus peroxidase (BP) from the pineapple fruit to evaluate the anticancer activity of BP from the pineapple fruit of Tripura, compared to commercial bromelain against ascitic Dalton’s lymphoma cells (DLA) in mice. Methods: By acetone precipitation BP was isolated from the pineapple. Animals bearing DLA, receive B and BP orally for 15 alternative days. Apoptotic proteins are assayed using western blot. Results: BP treated mice showed recover of hemoglobin and WBC count compared to control lymphoma animal. The animal showed significant reduction of body weight due to reduced tunor load and elevated reactive oxygen species (ROS) production, elevated levels of vitamin C and vitamin E and other antioxidants in blood after BP treatment. Histology of liver and kidney also shows restored architecture in BP treated animal compared to only B treated group. BP treatment upregulates the cytochrome C, BAD, and BAX protein and downregulates the Bcl-2 and NF-kβ occuring upon BP treatment in the DLA cells collected from lymphoma animal. This induce the apoptosis of DLA cells in lymphoma animal and reduce the tumor load. Conclusion: The present findings suggest that BP from pineapple improves the survival of the induced lymphoma animal compared to only B which may be used as therapeutic target.

Production of an alkali-stable xylanase from Bacillus pumilus K22 and its application in tomato juice clarification

ABSTRACTA xylanase-producing bacterium Bacillus pumilus K22 was isolated from hot spring located in Manghopir area, Karachi, Pakistan. The enzyme was purified to homogeneity by acetone precipitation and size exclusion chromatography. The molecular weight was determined to be approximately 24 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified xylanase retained its activity at wide range of temperature (20-70°C) and pH (5.0–10.0) with maximum stability at 50°C and pH 8.0. Xylanase activity was inhibited by Zn2+, Cu2+, and Hg2+ metal ions while it remained stable in the presence of Mn2+, Ca2+ and Mg2+. The activity was also inhibited by chemical reagents such as EDTA, PMSF, and β-mercaptoethanol. The enzyme was stable in the presence of all organic solvents (20%) except formaldehyde and glycerol, which inhibited its activity. Treatment of tomato pulp with purified xylanase-increased juice yield (30%), clarity (9%), and reducing sugars (69%), indicating an improvement in physico-chemical characteristics of the tomato pulp.

Antioxidant Fucoidans Obtained from Tropical Seaweed Protect Pre-Osteoblastic Cells from Hydrogen Peroxide-Induced Damage.

Some antioxidant compounds decrease the amount of intracellular reactive oxygen species (ROS) and consequently reduce the deleterious effects of ROS in osteoblasts. Thus, these compounds fight against osteoporosis. Brown seaweeds are a rich source of antioxidant fucose-containing sulfated polysaccharides (fucans and fucoidans). We obtained six fucoidans (FRFs)—F0.3, F0.5, F0.7, F1.0, F1.5, and F2.1—from Dictyota mertensii by proteolytic digestion followed by sequential acetone precipitation. Except for F0.3, all FRFs showed antioxidant activity in different in vitro tests. In pre- osteoblast-like cells (MC3T3-L1) exposed to H2O2-oxidative stress, caspase-3 and caspase-9 were activated, resulting in apoptosis of the cells. We also observed a decrease in superoxide dismutase (SOD) and alkaline phosphatase (ALP) activity. The antioxidant FRFs protected the cells from the oxidative damage caused by H2O2, decreasing intracellular ROS and caspase activation, and increasing SOD activity. The most effective protection against damage was provided by F0.7, F1.5, and F2.1. At 0.5 mg/mL, these FRFs also suppressed the H2O2-mediated inhibition of ALP activity. The data indicated that FRFs F0.7, F1.5, and F2.1 from D. mertensii were antioxidants that protected bone tissue from oxidative stress and could represent possible adjuvants for the treatment of bone fragility through counteracting oxidative phenomena.

DdeL, a novel thermostable lectin from Dypsis decaryi seeds: Biological properties

Abstract A novel thermostable lectin, called DdeL, was obtained from the extract of Dypsis decaryi seeds. The extract of the tegument had the highest specific hemagglutination activity (196.03 HA/mg) when subjected to 70% acetone precipitation and purification on Superdex G-75 and exhibited a single peak (38.8 kDa). DdeL, a glycoprotein, had agglutinated blood types and was inhibited only by glycoproteins (albumin, azoalbumin and azocasein). Biochemical properties showed higher activity at acid pH (pH 5–6) than alkaline pH and activity was observed at all temperatures (up to 100 °C). DdeL exhibited bacteriostatic activity against Providencia stuartii and Staphylococcus aureus ATCC 29213, which presented minimum inhibitory concentration (MIC) of 750.0 μg/mL. When tested against fungi Sporotrichum schenckii and Candida albicans showed excellent MIC50 at 0.52 and 2.06 μg/mL, respectively. DdeL showed no cytotoxic effect on macrophages (J774.A1) and renal embryonic cells (HEK-293). in vitro acaricide activity of DdeL against Rhipicephalus showed high activity in low concentration. Therefore, we conclude that DdeL may be of interest for potential biotechnological applications.

Improved Method for the Determination of Polychlorinated Dibenzo-p-dioxins and Dibenzofurans (PCDD/Fs) in Sanitary Napkins

An improved method was developed for determining highly toxic 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo-p-furans (PCDD/Fs) in sanitary napkins. Samples were extracted by Soxhlet (50% dichloromethane/hexane) and sonication (hexane), and a multi-layer silica column was used for clean-up after acetone precipitation to separate the superabsorbent polymers. Improved separation efficiency of PCDD/Fs from sanitary napkins with a high level of superabsorbent polymers was achieved using the acetone precipitation method. Hexane was then applied as an alternative solvent to remove residual interfering substances including sticky components contained on sanitary napkins via alumina column chromatography in the second clean-up step. Recoveries approached 100% with an average relative standard deviation of less than 15%, and recoveries of internal standards were from 70.5 to 114.8% and 0.6 to 13.6% for the two extraction methods. The mean method detection limit was 0.075–0.968 pg/g for Soxhlet extraction and 0.0032–0.091 pg/g for sonication extraction. The established method meets the quality criteria for the screening of dioxins stated in EU Regulations 589/2014 and 709/2014, and therefore provides a suitable alternative for the determination of dioxins in sanitary napkins containing superabsorbent polymers.

Molecular characteristic of α-AMYLASE enzymes producing from Bacillus lichenformis (JQ946317) using solid state fermentation

Abstract Amylases are enzymes which hydrolyze starch molecules to give diverse products including dextrin and progressively smaller polymers composed of glucose units. In the present studies bacterial strain were isolated from the soil samples and identified as Bacillus lichenformis (JQ946317). Maximum a-amylase activity was obtained at pH 7.0, 35 °C after 48 h of incubation. The enzyme was purified using acetone precipitation and DEAE- Sphadex A50 column chromatography and the molecular weight of 63 kDa was confirmed by SDS-PAGE. This enzyme exhibited maximum activity at pH 7.0, performed stability over a broad range of pH 6–8 and was optimally active at 35 °C. The enzyme activity was highest at 48 h of incubation as a as a carbon sources preparation had a strong digesting ability towards various agricultural waste and wheat bran showed highest enzyme. The purification and characterization of a-amylase from a newly isolated Bacillus lichenformis (JQ946317).

Abstract 241: An efficient cell-based assay for quantification of cellular polysialic acid in neuroblastoma

Abstract Introduction Polysialic acid (polySia) decorates the surface of NCAM (neuronal cell adhesion molecule) on neuroendocrine tumors, notably neuroblastoma and small cell lung cancer, and is strongly associated with poor prognosis and aggressive disease in patients in the clinic [1]. PolySia modulates tumor cell-cell and cell-matrix adhesion, migration, invasion and metastasis. SiRNA knockdown of polysialyltransferase (polyST) ST8SiaII, the enzyme primarily responsible for polySia synthesis in tumors, abrogates tumor cell migration and invasion. PolyST is a selective and largely unexplored therapeutic target for neuroblastoma dissemination [1]. Methods and Results We describe the development of a cell-based assay that quantifies the formation of polySia on the cell surface of neuroblastoma cells. This is a sensitive assay that detects the polySia that is released after pH adjustment, organic precipitation and acid hydrolysis. PolySia was first separated from cell lysate by mild acid hydrolysis and acetone precipitation. It was then purified by ethanol precipitation. We further analysed the role of lactonisation of polySia during organic precipitation. The purified polySia is further hydrolysed into sialic acid monomers, followed by DMB labelling and RPLC-fluorescence analysis. This method was validated by analysis of polySia in C6-ST8SiaII cells, following treatment with Endo-NF in a various concentrations. This method proved to be useful to assess polySia changes after treatment of cells with novel polyST inhibitors. The effect of lactonisation of polySia during organic precipitation was also investigated. It was found that lactonised polySia was mainly soluble in 90% acetone/90% ethanol, while natural polySia (non-lactonised) can be precipitated by ethanol in the presence salts. Based on the different solubility of polySia, a cost-efficient assay was designed to purify polySia released from cells with a low level of non-polySia-derived sialic acid contamination before further acid hydrolysis. With this method we quantified the different levels of cell-surface polySia-derived sialic acid between polySia-positive and polySia-negative cell lines. Furthermore, we successfully validated this method by Endo-NF treatment in C6-ST8SiaII cells: the EC50 of Endo-NF digestion of polySia in C6-ST8SiaII cells was approximately 5.4 pM. The assay has been successfully utilised to evaluate novel polyST inhibitors in vitro. [1] Falconer, R.A. et al., Curr. Cancer Drug Targets, 2012, 12, 925-939; [2] Al-Saraireh YMJ et al., PLoS ONE, 2013, 8:e73366. Citation Format: Xiaoxiao Guo, Francis Mprah Barnieh, Jodie Malcolm, Amanda D Race, Goreti Ribeiro Morais, Steven D Shnyder, Paul M Loadman, Laurence H Patterson, Robert A. Falconer. An efficient cell-based assay for quantification of cellular polysialic acid in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 241.

Influence of different parameters on reverse micelle extraction combined with acetone precipitation to purify sn-1,3 extracellular lipase from Aspergillus niger GZUF36

There are few reports on the feasibility of combined reverse micelle extraction and acetone precipitation to obtain electrophoretic pure enzymes. We aimed to purify a sn-1,3 extracellular lipase from a novel Aspergillus niger GZUF36 through this combination in this work. This lipase preliminarily purified by controlling the volume ratio (1:2.5) of crude enzyme solution and acetone. Then, we studied effects of different parameters on reverse micelle extraction. The suitable surfactant, pH, salt and cosolvent and extraction time for forward extraction were 125mM cetyl trimethylammonium bromide (CTAB), 9.0, 0.075M NaCl, 10% n-hexanol and 30min, respectively. Under these conditions, the forward extraction rate reached 90.3% ± 3.2%. The suitable salt, pH, extraction time and short chain alcohol for backward extraction were consecutively 1.5M KCl, 6.5, 60min and 10% ethanol. Adding 10% ethanol shows a significant advantage of improvement the extraction rate. Under these optimal conditions, the total extraction rate and purification factor of lipase reached 76.8% and 10.14, respectively. SDS-PAGE showed that molecular weight of the pure protein was 42.7kDa and TLC exhibited sn-1,3 selectivity of this lipase. LC-MS/MS analysis revealed that the lipase had 297 amino acid residues and was likely to glycosylate. Through the study of different parameters, it demonstrated that the new and simple combination of reverse micelle extraction using CTAB as surfactant and n-hexanol as cosolvent for forward extraction and adding ethanol for backward extraction and acetone precipitation is a promising method to get almost an electrophoretically pure sn-1,3 lipase.

Phytochemical Profile of Andrographis paniculata Extract from Solvent Partition and Precipitation

A rapid and simple method to profile intense phytochemicals in Andrographis paniculata was carried out in this study. The intense phytochemicals could be the main contributors for the previously reported biological activities of the herb. A. paniculata was extracted using 70 % ethanol in a reflux system for 2 hours. The crude extract was further fractionated by organic solvents such as ethyl acetate, butanol and chloroform with the aim to recover terpene derived phytochemicals. The increase of saponin content in the organic fractions could be seen and estimated using vanillin assay catalyzed by strong acids. Data LC-DAD-MS/MS revealed that the solvent systems could recover apigenin glucuronide, andrographolide and dehydroandrographolide dimer, but butanol fraction could recover additional compound for 4-deoxyphloridzin. Although butanol fraction was also found to have the lowest saponin content, the fraction could recover andrographolide and saponin via acetone precipitation effectively. It was found that only apigenin glucuronide could be precipitated if crude extract was directly used in acetone precipitation without solvent partition. The findings of this study could be used as a guideline for those researchers who would like to concentrate the biologically active phytochemicals such as andrographolide and its derivatives in samples for high-end product formulation.

Large-scale quantitative isolation of pure protein N-linked glycans

Glycoproteins are biologically active proteins of which the attached glycans contribute to their biological functionality. Limited data is available on the functional properties of these N-glycans in isolation, without the protein core. Glycan release, typically performed with the PNGase F enzyme, is achieved on denatured proteins in the presence of detergents which are notoriously difficult to be completely removed. In this work we compared two methods aiming at recovering N-glycans in a high yield and at high purity from a PNGase F glycoprotein digest of bovine lactoferrin. Detergents were removed from the digest by two separate approaches. In the first approach, protein and glycans were precipitated with acetone and the detergent containing supernatant was discarded. In the second approach, detergent was removed by adsorption onto a polystyrene resin. Following detergent removal, the glycans were further purified by a sequence of solid phase extraction (SPE) steps. Both approaches for detergent removal yielded a final glycan purity above 85%. Recovery of the glycans from lactoferrin was, however, much lower when utilizing acetone precipitation versus the polystyrene resin; 52% versus 85% respectively. A more detailed analysis of the acetone precipitation step revealed a loss of shorter oligomannose structures specifically. A loss of glycans of lesser complexity (oligomannose and biantennary structures) was also observed for other glycoproteins (RNase B, porcine thyroglobulin, human lactoferrin). These results indicate that acetone precipitation, a commonly used step for small-scale glycan purification, is not suitable for all target glycoproteins. The polystyrene resin detergent removal step conserved the full N-glycan profile and could be applied to all mammalian glycoproteins tested. Using this optimized protocol, large-scale quantitative isolation of N-glycan structures was achieved with sufficient purity for functional studies.

Solvent Fractionation and Acetone Precipitation for Crude Saponins from Eurycoma longifolia Extract.

Eurycoma longifolia is a popular folk medicine in South East Asia. This study was focused on saccharide-containing compounds including saponins, mainly because of their medical potentials. Different organic solvents such as ethyl acetate, butanol, and chloroform were used to fractionate the phytochemical groups, which were consequently precipitated in cold acetone. Solvent fractionation was found to increase the total saponin content based on colorimetric assay using vanillin and sulfuric acid. Ethyl acetate fraction and its precipitate were showed to have the highest crude saponins after acetone precipitation. The samples were shown to have anti-proliferative activity comparable with tamoxifen (IC50 = 110.6 µg/mL) against human breast cancer cells. The anti-proliferative activities of the samples were significantly improved from crude extract (IC50 = 616.3 µg/mL) to ethyl acetate fraction (IC50 = 185.4 µg/mL) and its precipitate (IC50 = 153.4 µg/mL). LC-DAD-MS/MS analysis revealed that the saccharide-containing compounds such as m/z 497, 610, 723, 836, and 949 were abundant in the samples, and they could be ionized in negative ion mode. The compounds consisted of 226 amu monomers with UV-absorbing property at 254 nm, and were tentatively identified as formylated hexoses. To conclude, solvent fractionation and acetone precipitation could produce saccharide-containing compounds including saponins with higher anti-proliferative activity than crude extract against MCF-7 cells. This is the first study to use non-toxic solvents for fractionation of bioactive compounds from highly complex plant extract of E. longifolia.

Purification and Characterization of Xanthine Oxidase from Liver of the Sheep (Ovis Aries)

Xanthine oxidase is a commercially important enzyme with wide area of medical applications to develop diagnostic kits. Xanthine oxidase was extracted, purified and characterized from sheep liver (SLXO). The purification procedure involved acetone precipitation and chromatography on DEAE-cellulose and Sephacryl S-300 columns. The sheep liver xanthine oxidase was homogeneously purified 31.8 folds with 3.5 U/mg specific activity and 24.1% recovery. SLXO native molecular weight was 150 kDa and on SDS-PAGE appeared as single major band of 75 kDa representing a homodimer protein. Isoelectric focusing of the purified SLXO resolved into two closely related isoforms with pI values of 5.6 and 5.8. The apparent Km for xanthine oxidase at optimum pH 7.6 was found to be 0.9 mM xanthine. FeCl2 and NiCl2 increased the activity of SLXO, while CuCl2 and ZnCl2 were found to be potent inhibitors of the purified enzyme. Allopurinol inhibits SLXO competitively with one binding site on the purified molecule and Ki value of 0.06 mM.