scholarly journals Purification of immunostimulatory glucose homopolymer from broccoli (Brassica oleracea var. italica)

2021 ◽  
Author(s):  
Atsushi Miyashita ◽  
Keiko Kataoka ◽  
Toshio Tsuchida ◽  
Akihiko Ano Ogasawara ◽  
Hiroshi Hamamoto ◽  
...  
Keyword(s):  
Brassica Oleracea ◽  
Column Chromatography ◽  
Active Substance ◽  
Specific Activity ◽  
Gel Filtration ◽  
Hot Water ◽  
Composition Analysis ◽  
Anion Exchange Column ◽  
Molecular Weights ◽  
Wide Range

We prepared broccoli (Brassica oleracea var. italica) neutral polysaccharides (flow-through fractions of anion exchange column chromatography from hot water extracts) from different broccoli cultivars and compared their immunostimulatory effects in the silkworm muscle contraction assay. The cultivars showed a wide range of activity, with the cultivar 'Winter dome' showing the highest specific activity (more than 100 times higher than curdlan). Furthermore, the active substance was purified by gel filtration column chromatography. The active substance showed heterogeneous molecular weights of more than 270 kDa. Sugar composition analysis of the purified fraction revealed that more than 95% of its sugar component was glucose, suggesting that the immunostimulatory neutral polysaccharide from broccoli cultivar Winter dome was a homopolymer of glucose. The purified fraction also induced TNF production in cultured mouse macrophage cells. These results suggest that the glucose homopolymer in broccoli has an immunostimulatory effect on both arthropod and mammalian immune system.

scholarly journals Partial Purification and Characterisation of Pectinase Produced by Aspergillus niger LFP-1 Grown on Pomelo Peels as a Substrate

2021 ◽  
Vol 32 (1) ◽  
pp. 1-22
Author(s):  
Mohd Taufiq Mat Jalil ◽  
◽  
Darah Ibrahim ◽  
◽  
Keyword(s):  
Aspergillus Niger ◽  
Column Chromatography ◽  
Metal Ion ◽  
Specific Activity ◽  
Gel Filtration ◽  
Citrus Fruit ◽  
Crude Enzyme ◽  
Partial Purification ◽  
Anion Exchange Column ◽  
C 50

In the present study, pectinase was produced by local fungal isolate, Aspergillus niger LFP-1 grown on pomelo peels as a sole carbon source under solid-state fermentation (SSF). The purification process begins with the concentration of crude enzyme using ammonium sulfate precipitation and followed by purification using anion-exchange column chromatography (DEAE-Sephadex) and subsequently using gel filtration column chromatography (Sephadex G-100). On the other hand, the molecular weight of the purified enzyme was determined through SDS-PAGE. The findings revealed the crude enzyme was purified up to 75.89 folds with a specific activity of 61.54 U/mg and the final yield obtained was 0.01%. The molecular mass of the purified pectinase was 48 kDa. The optimum pH and temperature were 3.5 and 50°C, respectively. This enzyme was stable at a range of pH 3.5 to 4.5 and a relatively high temperature (40°C–50°C) for 100 min. The Km and Vmax were found to be 3.89 mg/mL and 1701 U/mg, respectively. Meanwhile, pectin from citrus fruit and the metal ion (Co2+) were the best substrate and inducer to enhance pectinase yield, respectively.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

scholarly journals Purification and Characterization ofα -l-Arabinopyranosidase andα -l-Arabinofuranosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium Metabolizing Ginsenoside Rb2 and Rc

2003 ◽  
Vol 69 (12) ◽  
pp. 7116-7123 ◽  
Author(s):  
Ho-Young Shin ◽  
Sun-Young Park ◽  
Jong Hwan Sung ◽  
Dong-Hyun Kim
Keyword(s):  
Ammonium Sulfate ◽  
Column Chromatography ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bifidobacterium Breve ◽  
Ammonium Sulfate Fractionation ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Ginsenoside Rb2

ABSTRACT Two arabinosidases, α-l-arabinopyranosidase (no EC number) and α-l-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. α-l-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 μmol/min/mg.α -l-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 μmol/min/mg. The molecular mass ofα -l-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that ofα -l-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. α-l-Arabinopyranosidase and α-l-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40°C and pH 4.5 and 45°C, respectively. Both purified enzymes were potently inhibited by Cu2+ and p-chlormercuryphenylsulfonic acid.α -l-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinopyranoside, followed by ginsenoside Rb2. α-l-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-β-galactopyranoside or p-nitrophenyl-β-d-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified α-l-arabinosidases. This is the first reported purification ofα -l-arabinopyranosidase from an anaerobic Bifidobacterium sp.

scholarly journals Purification and characterization of fat body lipase from the Greater Wax Moth Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Author(s):  
Rahma R.Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M.S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

AbstractLipid mobilization and transport in insects is under investigation, especially lipases and lipophorin because of their roles in energy production and transport of lipids at flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from last larval instar of Galleria mellonella. Purification methods by combination of ammonium sulfate precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633±0.000551 mg/ml and 1.5754±0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore biochemical characterization of fat body lipase was carried out through testing its activities against several factors such as; different temperatures, pH ranges, metal ions and inhibitors ending by determination of their kinetic parameters with the use of p-Nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35-37°C and 37-40°C and pH ranges of 7-9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+ and Na+ metal ions indicating that fat body lipase is metalloproteinase. Additionally, lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfony fluoride (PMSF), ethylene-diaminetetractic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA) providing an evidence of presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 301.95mM Km and 0.316 Umg−1 Vmax. By considering the purification of fat body lipase from larvae and using some inhibitors especially ion chelating agents, it is suggested to develop this study by using lipase inhibitors to reach a successful control of Galleria mellonella in the near future.

Purine catabolism in man: characterization of placental microsomal 5′-nucleotidase

1976 ◽  
Vol 54 (5) ◽  
pp. 462-469 ◽  
Author(s):  
Irving H. Fox ◽  
Pamela J. Marchant
Keyword(s):  
Specific Activity ◽  
Ph Optimum ◽  
Electrophoretic Variants ◽  
Molecular Weights ◽  
Wide Range ◽  
Stokes Radius ◽  
Michaelis Constants ◽  
Light Form ◽  
Molecular Weight Form

Human placental microsomal 5′-nucleotidase (EC 3.1.3.5) was prepared free of alkaline phosphatase by isoelectric focusing. A total of seven electrophoretic variants were isolated during the preparation of six placentas. Only three to six variants were found in a single placenta. The isoelectric pH's were 6.70, 6.44, 6.23, 6.02, 5.76, 5.63 and 5.44. These were found to be composed of variable quantities of a large, medium and low molecular weight form. The apparent molecular weights of the medium and light form of the enzyme were 86 500 and 43 500, respectively, as estimated from Stokes radius and sedimentation velocity determinations. The electrophoretic variants were not distinguishable with respect to specific activity and Michaelis constants for AMP, GMP or CMP or inhibition by ATP, CTP or adenosine. These electrophoretic variants appeared to be pseudoisozymes based upon different states of aggregation of a common primary sequence.There was a wide range of substrate specificity among nucleoside 5′-monophosphates which included 2-deoxyribose compounds. With AMP as 100, substrate activity was: CMP, 122; NMN, 74; GMP, 68; IMP, 63; XMP, 28 and UDP–glucose, 68. The Michaelis constants for AMP, GMP and CMP ranged from 12–18 μM, from 33–67 μM and from 170–250 μM, respectively. Although 5′-nucleotidase was active in the absence of divalent cation, 5 mM MgCl2 stimulated the enzyme activity to 234% of control and shifted the pH optimum of 9.8 to a plateau from pH 7.4–9.8.

On the purification of a fatty acid synthetase complex from an insect

1976 ◽  
Vol 54 (8) ◽  
pp. 1397-1399 ◽  
Author(s):  
S. N. Thompson ◽  
J. S. Barlow
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Column Chromatography ◽  
De Novo ◽  
Gel Filtration ◽  
Fatty Acid Synthetase ◽  
De Novo Synthesis ◽  
Anion Exchange Column ◽  
Malonyl Coa ◽  
Salt Fractionation

The fatty acid synthetase complex of the blowfly, Lucilia sericata, catalyzing the de novo synthesis of fatty acids from acetyl- and malonyl-CoA (EC 2.3.1.9 and EC 2.3.1.39 respectively) and requiring NADPH, was isolated from whole-insect homogenates by ultracentrifugation. Purification was carried out by salt fractionation, anion exchange column chromatography on ECTEOLA (epichlorohydrin triethanolamine) cellulose, and gel filtration column chromatography on Sephadex®.

Purification and characterization of the esterases involved in aflatoxin biosynthesis in Aspergillus parasiticus

1996 ◽  
Vol 42 (8) ◽  
pp. 804-810 ◽  
Author(s):  
Ken-Ichi Kusumoto ◽  
Dennis P. H. Hsieh
Keyword(s):  
Aspergillus Parasiticus ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Preparative Chromatography ◽  
Aflatoxin Biosynthesis ◽  
Anion Exchange Column ◽  
Versiconal Hemiacetal Acetate

The esterases from the cell-free extracts (CFEs) of Aspergillus parasiticus ATCC15517, an aflatoxin-producing strain, catalyzing the hydrolytic conversion of versiconal hemiacetal acetate (VHA) to versiconal was biochemically studied. The specific activity of the enzymes increased 2.5-fold during incubation of mycelia through 40–55 h. No metal ions were required for enzyme stability, but EDTA at 1 mM and dithiothreitol at 0.5–5 mM increased its stability. Three peaks of VHA esterase activity were resolved when the proteins in the CFEs prepared from the mycelia of different ages were separated by anion-exchange column chromatography, suggesting that at least three VHA esterases were present in the eluate of this purification step. One of these esterases extracted from the mycelia of a 55-h culture was partially purified in five steps by means of preparative chromatography and fast protein liquid chromatogaphy. The partially purified enzyme when reacted with [14C]diisopropylfluorophosphate followed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis gave a single radiolabelled band, which corresponded to a protein of 32 kDa. The molecular mass of the partially purified VHA esterase determined with gel filtration was around 60 kDa. The results suggested that the enzyme consists of two isomeric subunits.Key words: aflatoxin biosynthesis, esterase, versiconal hemiacetal acetate, Aspergillus parasiticus.

scholarly journals The Isolation of Aggregates of Spectrin From Bovine Erythrocyte Membranes

1975 ◽  
Vol 28 (3) ◽  
pp. 259 ◽  
Author(s):  
GB Ralston
Keyword(s):  
Ionic Strength ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Sedimentation Equilibrium ◽  
Erythrocyte Membranes ◽  
Bovine Erythrocyte ◽  
Molecular Weights ◽  
Wide Range

Aggregated states of spectrin from bovine erythrocyte membranes can be detected in sedimentation velocity experiments. These aggregates have been isolated by means of gel filtration on columns of 4 % agarose. They appear to be stable over a wide range of pH and ionic strength, although they are dissociated by sodium dodecyl sulphate. Sedimentation equilibrium measurements yielded values of 960000 and 480000 for the molecular weights of the major aggregates, corresponding to a tetramer and dimer, respectively. The presence of different aggregated states in spectrin preparations may explain the wide variation in the reported physica~ properties of spectrin.

scholarly journals Purification and Characterization of a Novel Thermostable α-l-Arabinofuranosidase from a Color-Variant Strain of Aureobasidium pullulans

1998 ◽  
Vol 64 (1) ◽  
pp. 216-220 ◽  
Author(s):  
Badal C. Saha ◽  
Rodney J. Bothast
Keyword(s):  
Column Chromatography ◽  
Metal Ion ◽  
Aureobasidium Pullulans ◽  
Specific Activity ◽  
Gel Filtration ◽  
Variant Strain ◽  
Color Variant ◽  
C 50 ◽  
Hydrolysis Of

ABSTRACT A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced α-l-arabinofuranosidase (α-l-AFase) when grown in liquid culture on oat spelt xylan. An extracellular α-l-AFase was purified 215-fold to homogeneity from the culture supernatant by ammonium sulfate treatment, DEAE Bio-Gel A agarose column chromatography, gel filtration on a Bio-Gel A-0.5m column, arabinan-Sepharose 6B affinity chromatography, and SP-Sephadex C-50 column chromatography. The purified enzyme had a native molecular weight of 210,000 and was composed of two equal subunits. It had a half-life of 8 h at 75°C, displayed optimal activity at 75°C and pH 4.0 to 4.5, and had a specific activity of 21.48 μmol · min−1· mg−1 of protein againstp-nitrophenyl-α-l-arabinofuranoside (pNPαAF). The purified α-l-AFase readily hydrolyzed arabinan and debranched arabinan and released arabinose from arabinoxylans but was inactive against arabinogalactan. TheKm values of the enzyme for the hydrolysis of pNPαAF, arabinan, and debranched arabinan at 75°C and pH 4.5 were 0.26 mM, 2.14 mg/ml, and 3.25 mg/ml, respectively. The α-l-AFase activity was not inhibited at all byl-arabinose (1.2 M). The enzyme did not require a metal ion for activity, and its activity was not affected byp-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM).

scholarly journals The purification and properties of a ribonucleoenzyme, o-diphenol oxidase, from potatoes

1970 ◽  
Vol 118 (1) ◽  
pp. 15-23 ◽  
Author(s):  
K. Balasingam ◽  
W. Ferdinand
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Disc Electrophoresis ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Major Form ◽  
Purification And Properties ◽  
Minimal Molecular Weight

1. o-Diphenol oxidase was isolated from potato tubers by a new approach that avoids the browning due to autoxidation. 2. There are at least three forms of the enzyme, of different molecular weights. The major form, of highest molecular weight, was separated from the others in good yield and with high specific activity by gel filtration through Bio-Gel P-300. 3. The major form is homogeneous by disc electrophoresis but regenerates small amounts of the species of lower molecular weight, as shown by rechromatography on Bio-Gel P-300. 4. There is an equal amount of RNA and protein by weight in the fully active enzyme. The RNA cannot be removed without loss of activity, and is not attacked by ribonuclease. 5. The pH optimum of the enzyme is at pH5.0 when assayed with 4-methylcatechol as substrate. It is ten times more active with this substrate than with chlorogenic acid or catechol. The enzyme is fully active in 4m-urea. 6. A minimal molecular weight of 36000 is indicated by copper content and amino acid analysis of the protein component of the enzyme. 7. The protein contains five half-cystinyl residues per 36000 daltons, a value similar to that found in o-diphenol oxidase from mushrooms. It also contains tyrosine residues although, when pure, it does not turn brown by autoxidation.

Sign in / Sign up

Export Citation Format

Share Document