Ammonium Sulfate Fractionation Research Articles

Comparison of the Exopeptidase Activity of Fractions from Crude Extracts of Octopus Octopus vulgaris Cuvier Hepatopancreas Using Different Fractionation Methods

This study was performed to identify the optimum fractionation method and conditions to obtain exopeptidase-active fractions from octopus hepatopancreas (HP) crude extracts (CEs) using four techniques: solid ammonium sulfate fractionation, polyethylene glycol (PEG) fractionation, anion exchange chromatography, and gel filtration chromatography. The fractions with the highest total activity toward L-leucine-p-nitroanilide (Leu-pNA) were fraction IV from the ammonium sulfate and PEG fractionation, and fraction II in ion exchange and gel filtration chromatography. The total exoprotease activity of these fractions was highest in fraction IV (4,050.20 U) of ammonium sulfate fractionation, followed by fraction II (3,600.28 U) from gel filtration chromatography, fraction IV (2,861.30 U) from PEG fractionation, and fraction II (2,576.28 U) from ion exchange chromatography. These results suggest that ammonium sulfate fractionation using 60-80% ammonium sulfate was the most efficient method for separating the exoprotease active fractions from CEs of octopus HP.

High-level expression, purification and study of bioactivity of fusion protein M-IL-2(88Arg, 125Ala) in Pichia pastoris

M-IL-2(88Arg, 125Ala) is a fusion protein comprising melittin genetically linked to a mutant human interleukin 2(88Arg, 125Ala). In this study, we constructed an expression system of M-IL-2(88Arg, 125Ala) in Pichia pastoris: GS115/pPICZα A/M-IL-2(88Arg, 125Ala), and achieved the high-level expression of the fusion protein. The maximum yield of the fusion protein M-IL-2(88Arg, 125Ala) reached up to 814.5mg/L, higher than the system in Escherichiacoli. The fusion protein was purified by means of ammonium sulfate fractionation, dialysis and nickel ion affinity chromatography. The molecular weight of the fusion protein is about 26kDa, conforming the theoretical value. And M-IL-2(88Arg, 125Ala) possesses strong antigen-specificity by Western blot detection. Bioassay results indicated that the fusion protein could directly inhibit the growth of human ovarian cancer SKOV3 cells and Hela cells in vitro. This study provides an alternative strategy for large-scale production of bioactive M-IL-2(88Arg, 125Ala) using P. pastoris as an expression host and paves the way to clinical practice.

Biochemical and functional characterization of transiently expressed in neural precursor (TENP) protein in emu egg white.

A protein transiently expressed in the neural precursors of developing tissues (TENP) was found to be present in emu (Dromaius novaehollandiae) egg white as one of the major proteins. Nucleotide analysis of its encoding cDNA revealed a sequence of 452 amino acids including a 19 amino acid peptide signal. Phylogenetic analysis determined that emu TENP was clustered within the bactericidal/permeability-increasing protein (BPI) superfamily together with other avian TENPs. RT-PCR analysis revealed that the emu TENP gene was highly expressed in the magnum of the oviduct, indicating that TENP is a major egg white component. Emu TENP was purified by anion exchange chromatography and ammonium sulfate fractionation. Unlike BPI, emu TENP exhibited antibacterial activity against Gram-positive bacteria, including Micrococcus luteus and Bacillus subtilis, but not against Gram-negative bacteria such as Escherichia coli and Salmonella Typhimurium. The results suggest that emu TENP is a potent novel antibacterial protein with a spectrum distinct from that of BPI.

A statistical approach for the enhanced production of thermostable alkaline protease showing detergent compatibility activity from Bacillus circulans

Response surface methodology (RSM) was employed to enhance the production of a thermostable alkaline protease from Bacillus circulans. Significant influences of peptone, yeast extract, and glucose on protease production were noted with a one-variable-at-a-time optimization strategy. Then, a full factorial central composite design (CCD) was applied to study the effects of glucose, peptone, and yeast extract to determine the optimal concentrations of these compounds for protease production by B. circulans under shake flask fermentation conditions. The statistical reliability and significance of the model was validated by an F-test for analysis of variance (ANOVA); enzyme production was improved significantly under optimized conditions. The enzyme was purified by ammonium sulphate fractionation, and gel filtration chromatography. Maximum enzyme activity was observed at 60°C temperature, and at pH 10. Alkaline protease from B. circulans showed excellent compatibility and stability in the presence of commercial detergents like Ariel, Surf Excel, Tide, Rin, Nirma, Wheel, and Doctor and showed excellent blood destaining effectiveness with commercial detergents.

Purification and characterization of an alkaline pectin lyase produced by a newly isolated Brevibacillus borstelensis (P35) and its applications in fruit juice and oil extraction

An alkaline pectin lyase (PNL) (EC 4.2.2.10) secreted by Brevibacillus borstelensis P35 (GenBank Number: FJ417406) was purified using ammonium sulfate fractionation, anion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-150. The pH and temperature optima of the enzyme were found to be 8.0 and 60 °C. The enzyme does not loose activity up to 60 °C if exposed for 1 h. The values of K m and V max of the enzyme were 0.625 mg/mL and 126.32 s−1, respectively. The molecular weight was found to be 36 ± 01 kDa. The presence of 10 mM concentration of Ca2+, Cu2+, Mn2+, Mg2+, Zn2+, Hg2+, Fe2+ and EDTA, l-cystein, ascorbic acid significantly enhanced the PNL of the purified enzyme. In the course of the laboratory trials, it was demonstrated that PNL from B. borstelensis (P35) could be successfully applied to the production and clarification of fruit juice and oil extraction.

Purification and characterization of arylacetonitrile‐specific nitrilase of Alcaligenes sp. MTCC 10675

Arylacetonitrile-hydrolyzing nitrilase (E.C. 3.5.5.5) of Alcaligenes sp. MTCC 10675 has been purified by up to 46-fold to homogeneity and 32% yield using ammonium sulfate fractionation, Sephacryl S-300 gel permeation, and anion exchange chromatography. The molecular weight of the native enzyme was estimated to be 520 ± 60 kDa. The subunit has a molecular weight of 60 ± 14 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the purified enzyme were 6.5 and 50 °C, respectively. The purified arylacetonitrilase has a half-life of 3 H 20Min at its optimum temperature. The value for Vmax, Km , kcat , and ki of enzyme for mandelonitrile as a substrate was 50 ± 05 µmol/Min/mg, 13 ± 02mM, 26 ± 03 Sec(-) , and 32.4 ± 03mM, respectively. Alcaligenes sp. MTCC 10675 arylacetonitrilase amino acid sequence has variations from other reported arylacetonitrilase, namely, A11G, N21H, D149N, S170T, P171R, S179A, Q180N, and S191A, and it has a high thermal stability and catalytic rate as compared with the already purified arylacetonitrilase.

Expression and Purification of Bioactive High-Purity Recombinant Mouse SPP1 in Escherichia coli

Secreted phosphoprotein 1 (SPP1) is a phosphorylated acidic glycoprotein. It is broadly expressed in a variety of tissues, and it is involved in a number of physiological and pathological events, including cancer metastasis, tissues remodeling, pro-inflammation regulation, and cell survival. SPP1 has shown its function of protecting tissues and organs against injury and wound, giving itself potentials to become a therapy target or giving its antibodies of other counter-acting reagents potentials to become drug candidates. Non-tagged (native) recombinant SPP1 would be valuable in therapeutic and pharmaceutical researches. In our study, mouse Spp1 DNA fragment without signal peptide was built in pET28a(+) vector and transformed into Escherichia coli BL21 (DE3). The recombinant mouse SPP1 (rmSPP1) was then expressed in bacteria upon induction by isopropyl β-D-thiogalactopyranoside (IPTG). The abundance of rmSPP1 was increased using isoelectric precipitation and ammonium sulfate fractionation methods, and anion and cation exchange chromatography was employed to further purify rmSPP1. Finally, we got rmSPP1 product with 12.8 % productivity, 97 % purity, satisfactory bioactivity, and low endotoxin content.

Partial Purification and Properties of Adenosine Triphosphatase (ATPase) from Liver Fluke Fasciola hepatica

The adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3.;ATPase) is a membrane -bound enzyme which transport protons across the plasma membrane using ATP as an energy source. The adenosine triphosphatase (ATPase ; EC: 3.6.1.3) was extracted from membrane preparations of adult Fasciola hepatica by chloroform treatment and purified by means of ammonium sulphate fractionation, gel filtration on sephadex G-200 and DEAE- Cellulose chromatography. The molecular weight was calculated to be 305.000 dalton by gel filtration. Kinetic experiments demonstrated a biphasic linear lineweaver - burk relationship (km=0.142 and 1.66 mM) thus revealing the existence of two substrate binding enzyme sites. In our study revealed that partial inhibition of Mg²⁺ dependent purified enzyme by oligomycin suggest the absence of mitochondrial ATPase in F. hepatica.

Purification and Biochemical Characterization of Lipase from Tunisian Euphorbia peplus Latex

AbstractLipases from vegetable sources have been the focus of intense and growing research. The use of enzymes from plants has the advantage of employing industrial waste products. The lipase activity of Euphorbia peplus L. (Euphorbiaceae) was investigated for the first time. The Euphorbia peplus latex lipase (EpLL) was purified after ammonium sulfate fractionation and anion exchange chromatography on a DEAE‐Cellulose column leading to 12.57‐fold purification. The EpLL displayed a probable molecular weight of about 40 kDa. The lipase activity was optimum at a temperature of 40 °C and pH 8, the specific activities of EpLL were found to be 249 ± 12.45 and 161.4 ± 8.07 U/mg when tributyrin (TC4) and olive oil were used as substrate respectively. The enzyme retained 80 % of its activity when incubated for 1 h at 50 °C. The EpLL was strongly destabilised by divalent metal ions (Fe2+, Mg2+, Zn2+ and Cu2+). Lipase was slightly stimulated by Triton X‐100 and Tween‐80, while strongly inhibited by sodium dodecyl sulfate. A good stability of the enzyme in the presence of organic solvents was reveled suggesting its industrial utility.

PURIFICATION OF RUMEN MICROBIAL XYLANASE WITH IMMOBILIZED SEPHAROSE-CIBACRON BLUE F3GA IN COLUMN CHROMATOGRAPHY

Sepharose-Cibacron blue (CB) F3GA for pseudo-affinity adsorption of bioproducts was synthesized and was subjected to rumen microbial enzyme evaluation through batch binding and column chromatography of xylanase. Based on equilibrium adsorption data, the best temperature, contact time, and elution agent were 30°C, 15 min, and 0.5 M NaCl (pH 6), respectively. The results show that homogenizing method had better performance in the liberation of enzyme, so that the amount of enzyme in rumen liquor approximately doubled. In pre-concentration methods, it was shown that freeze drying and precipitation of enzyme using ammonium sulfate were the best. Microbial xylanase was purified from rumen liquor to 1.85- and 11.80-fold by ammonium sulfate fractionation and Sepharose-CB column chromatography, respectively.

Identification of perfluorooctane sulfonate binding protein in the plasma of tiger pufferfish Takifugu rubripes

It is well known that perfluorooctane sulfonate (PFOS) preferentially accumulates in the plasma of wildlife and humans. Although earlier studies have suggested that this was due to binding of PFOS to a plasma protein, definite characterization of the protein in in vivo exposure studies was not conducted thus far. In this study, we conducted both in vitro and in vivo experiments to identify PFOS binding protein in the plasma of fish. For the in vivo studies, PFOS was administered intraperitoneally to tiger pufferfish, Takifugu rubripes, and the plasma was separated by ammonium sulfate fractionation. High concentrations of PFOS were found in the 65–70 percent ammonium sulfate fraction (190ng/mL). After SDS-PAGE and N-terminal amino acid sequence analysis, the PFOS-binding protein was identified as an apolipoprotein A–I, which was confirmed on the basis of a significant correlation to the PFOS concentration in each fraction. The plasma samples fractionated by ammonium sulfate from untreated pufferfish were subjected to PFOS binding assay by the equilibrium dialysis method. The results further confirmed that the 60–65 percent ammonium sulfate fraction showed a high PFOS-binding ratio, similar to that found from in vivo studies. We demonstrated that PFOS is likely bound to an apolipoprotein A–I in the plasma of tiger pufferfish in in vivo and in vitro studies.

Purification and characterization of two agarases from Agarivorans albus OAY02

Two extracellular β-agarases were purified from Agarivorans albus OAY2, which was isolated from seaweed collected in Qingdao, Shandong, China. The fermentation process was optimized with enzyme activity improving from 1.06U/ml to 2.65U/ml. Agarases were purified by ammonium sulfate fractionation and ion-exchange chromatography. Purification resulted in a 107-fold increase in agarase-a activity and a 52-fold increase in case of agarase-b, with high specific activities of 2715 and 1338U/mg, respectively. The molecular masses of the agarases were estimated to be 50kDa and 107kDa, respectively. The optimum temperatures for the activities of agarase-a and agarase-b were 40°C and 50°C, respectively. Agarase-a was stable at 30°C, while agarase-b was stable at 50°C. The optimum pH values for agarase-a and agarase-b were both 9.0. Agarase-a was inhibited by Cu2+, Mn2+, Co2+ and EDTA, while agarase-b was inhibited by Cu2+, Mn2+ and Fe2+. The results of 13C NMR and TLC showed that hydrolysis of agarose by agarase-a produced neoagarobiose (NA2), neoagarotetraose (NA4) and neoagarohexaose (NA6). Agarase-b hydrolyzed agarose to yield neoagarobiose (NA2) and neoagarotetraose (NA4). Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) and the enzymatic product results indicated that agarase-a and agarase-b might be novel agarases.

Purification and Characterization of a Zinc-Dependent Cinnamyl Alcohol Dehydrogenase from Leucaena leucocephala, a Tree Legume

A cinnamyl alcohol dehydrogenase (CAD) from the secondary xylem of Leucaena leucocephala has been purified to homogeneity through successive steps of ammonium sulfate fractionation, DEAE cellulose, Sephadex G-75, and Blue Sepharose CL-6B affinity column chromatographies. CAD was purified to 514.2 folds with overall recovery of 13% and specific activity of 812. 5nkat/mg. Native and subunit molecular masses of the purified enzyme were found to be ∼76 and ∼38kDa, respectively, suggesting it to be a homodimer. The enzyme exhibited highest catalytic efficiency (Kcat/Km 3.75μM(-1)s(-1)) with cinnamyl aldehyde among all the substrates investigated. The pH and temperature optima of the purified CAD were pH8.8 and 40°C, respectively. The enzyme activity was enhanced in the presence of 2.0mM Mg(2+), while Zn(2+) at the same concentration exerted an inhibitory effect. The inclusion of 2.0mM EDTA in the assay system activated the enzyme. The enzyme was inhibited with caffeic acid and ferulic acid in a concentration-dependent manner, while no inhibition was observed with salicylic acid. Peptide mass analysis of the purified CAD by MALDI-TOF showed a significant homology to alcohol dehydrogenases of MDR superfamily.

Purification and characterization of a novel marine Arthrobacteroxydans KQ11 dextranase

Dextranases can hydrolyze dextran deposits and have been used in the sugar industry. Microbial strains which produce dextranases for industrial use are chiefly molds, which present safety issues, and dextranase production from them is impractically long. Thus, marine bacteria to produce dextranases may overcome these problems. Crude dextranase was purified by a combination of ammonium sulfate fractionation and ion-exchange chromatography, and then the enzyme was characterized. The enzyme was 66.2kDa with an optimal temperature of 50°C and a pH of 7. The enzyme had greater than 60% activity at 60°C for 1h. Moreover, 10mM Co2+ enhanced dextranase activity (196%), whereas Ni2+ and Fe3+ negatively affected activity. 0.02% xylitol and 1% alcohol enhanced activity (132.25% and 110.37%, respectively) whereas 0.05% SDS inhibited activity (14.07%). The thickness of S. mutans and mixed-species oral biofilm decreased from 54340nm to 36670nm and from 64260nm to 43320nm, respectively.

Molecular Cloning and Function Characterization of a New Macrophage-Activating Protein from Tremella fuciformis

Silver ear mushroom ( Tremella fuciformis ) is an edible fungus with health benefits. In this study, we purified a new T. fuciformis protein (TFP) and demonstrated its ability to activate primary murine macrophages. The isolation procedure involved ammonium sulfate fractionation and ion exchange chromatography. TFP naturally formed a 24 kDa homodimeric protein and did not contain glycan residues. The TFP gene was cloned using the rapid amplification of cDNA ends method, and the cDNA sequence of TFP was composed of 408 nucleotides with a 336 nucleotide open reading frame encoding a 112 amino acid protein. TFP was capable of stimulating TNF-α, IL-1β, IL-1ra, and IL-12 production in addition to CD86/MHC class II expression, mRNA expression of M1-type chemokines, and nuclear NF-κB accumulation in murine peritoneal macrophage cells. Furthermore, TFP failed to stimulate TLR4-neutralized and TLR4-knockout macrophages, suggesting that TLR4 is a required receptor for TFP signaling on macrophages. Taken together, these results indicate that TFP may be an important bioactive compound from T. fuciformis that induces M1-polarized activation through a TLR4-dependent NF-κB signaling pathway.

Isolation and characterization of thermostable phycocyanin from Galdieria sulphuraria

Phycocyanin is a highly valuable pigmented protein synthesized by several species of cyanobacteria and red alga. In this study we demonstrate the production of thermostable phycocyanin from the unicellular red alga Galdieria sulphuraria. Phycocyanin was extracted by repeated freeze-thaw cycles and purified in a two-step process using ammonium sulfate fractionation, at 25% and 50% concentrations. Purified phycocyanin exhibited maximum absorbance at 620 nm, and the purity ratio (A620/A280) was found to be greater than 4. The recovery efficiency of phycocyanin from the crude extract was above 80%. In total, approximately 19 milligram pure phycocyanin was obtained from 3 g of wet cell mass of Galdieria sp. Subunits α and β of the protein were separated by SDS-PAGE and analyzed by MALDITOF mass spectrometry for identification, which confirmed that the isolated protein is phycocyanin. The molecular weight of α and β subunits of phycocyanin was found to be 17.6 and 18.4 kDa, respectively.

Biochemical Characterization of Two Polyphenol Oxidase Purified from Edible Yam (Dioscorea cayenensis-rotundata cv. Kponan) Cultivated in Côte d’Ivoire

Yam (Dioscorea cayenensis-rotundata cv. Kponan) is a staple food in many tropical regions. A biochemical characterization study of crude polyphenol oxidase from yam (PPO) was carried out to provide useful information for food processing operations. Two polyphenol oxidases of edible yam (Dioscorea cayenensis-rotundata cv. Kponan) cultivated in Cote d’Ivoire were purified to homogeneity. The purification procedure consisted of ammonium sulphate fractionation, ion-exchange, size exclusion and hydrophobic interaction chromatography. The enzymes designated PPO1 and PPO2 had native molecular weights of approximately 113.7±0.34 and 115.65 ±1.78 kDa, respectively, and functioned as dimeric (PPO2) and monomeric (PPO1) structures. The two isoforms isolated had different optimum pHs and temperatures. The maximal activity of PPO1 occurred at 35oC and pH 6.0. On the other hand, PPO2 had a maximum activity at 25oC and pH 6.6. The enzymes were stable at their optimal temperatures (25oC and 35oC) and their pH stability was in the range of 5.6–7.0. Polyphenol oxidases (PPO1 and PPO2) remained their full activity in the presence of ion Mn, Fe, Na and Cu but were Original Research Article Annual Research & Review in Biology, 4(23): 3532-3548, 2014 3533 inhibited strongly by the reducing agents as beta-mercaptoethanol, L-ascorbic acid, sodium bisulphite and L-cysteine. The values Vmax/Km showed that the enzymes have the greatest reactivity towards dopamine among the substrates used. The present work is therefore the first report on the purification to homogeneity of a yam tuber PPOs

EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF COLD-ADAPTED INORGANIC PYROPHOSPHATASE FROM PSYCHROPHILIC Shewanella sp. AS-11

In the presence of divalent cations, inorganic pyrophosphatase is activated to hydrolyze inorganic pyrophosphate to inorganic phosphate. Here, we clone, express, purify, and characterize inorganic pyrophosphatase from the psychrophilic Shewanella sp. AS-11 (Sh-PPase). The recombinant Sh-PPase was expressed in Escherichia coli BL21 (DE3) at 20°C using pET16b as an expression vector and purified from the cell extracts by a combination of ammonium sulfate fractionation and anion-exchange chromatography. Sh-PPase was found to be a family II PPase with a subunit molecular mass of 34 kD that preferentially utilizes Mn2+ over Mg2+ ions for activity. The functional characteristics of Sh-PPase, such as activity, temperature dependency, and thermal inactivation, were greatly influenced by manganese ions. Manganese ion activation increased the enzyme's activity at low temperatures; therefore, it was required to gain the cold-adapted characteristics of Sh-PPase.

Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Pigeon Pea (Cajanus cajan) Seeds

Glucose-6-phosphate dehydrogenase has been purified from pigeon pea (Cajanus cajan) seeds and subjected to characterization. The enzyme was purified 123.69 fold with a yield of 21.37% by ammonium sulphate fractionation, PEG-4000 precipitation, CM cellulose column chromatography and DEAE cellulose column chromatography. The catalytically active enzyme is a dimer of 113 KDa with a subunit molecular weight of 55 KDa. Thermal inactivation of enzyme follows first order kinetics at 30&#176C and 40&#176C with half life of 6 and 1.5 min respectively. Km value for glucose-6-phosphate and NADP+ was found to be 2.68 mM and 0.75 mM respectively whereas Vmax value was found to be 0.11 U/mL and 0.13 U/mL respectively. The enzyme shows more affinity towards NADP+ than glucose-6-phosphate. The pKa value was found to be 10.41 indicating that the amino acid residue at active site might be lysine. The enzyme exhibited maximum catalytic activity at pH 8.2. The enzyme was found to be highly thermosensitive with gradual loss of activity above 30&#176C temperature.

Preparative studies on the isolation of an enzyme associated with carthamin synthesis in Carthamus tinetorius L.

Preliminary operations for the isolation of an enzyme associated with cathamin synthesis (carthamin-synthesizing enzyme) were done with a soluble extract of safflower seedlings. Ethanol and acetone seriously affected the solubility and the activity of the enzyme. Ammonium sulfate fractionation, calcium acetate precipitation and protamine sulfate treatment were all useful techniques for removing inert protein from the crude extract however, the activity of the carthamin-synthesizing enzyme could not be separated from that of native polyphenol oxidase or peroxidase. Partial purification of the carthamin-synthesizing enzyme could be achieved by filtration of a crude extract through Sephadex G-100 gel after treating it with ammonium sulfate, calcium acetate and protamine sulfate. -the purified enzyme reacted possitively with precarthamin at pH 4.8 in 50.0 mM acetate buffer. The synthesized product was identified as carthamin by checking its R<sub>f</sub> values on silica gel plates developed with three different developing solvents and UV absorption spectral pattern in methanol. The enzyme sample was unstable on storage and stabilization of the sample was tested by applying many procedures. Carthamin-synthesizing enzyme had low affinity to celite 535. No carthamin was found in an incubation medium containing precarthamin and an authentic sample of polyphenol oxidase or peroxidase. Carthamin could not be converted to any blackish catabolites through polyphenol oxidase- or peroxidase-catalyzed oxidative reaction. Changes in the activities of three different enzymes, carthamin-synthesizing enzyme, polyphenol oxidase and peroxidase, were investigated in the extract from both etiolated and green seedlings of safflower and the data were discussed in relation to the catalytic properties of the enzymes.