scholarly journals A Novel Laccase with Potent Antiproliferative and HIV-1 Reverse Transcriptase Inhibitory Activities from Mycelia of MushroomCoprinus comatus

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Shuang Zhao ◽  
Cheng-Bo Rong ◽  
Chang Kong ◽  
Yu Liu ◽  
Feng Xu ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Ph Value ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
High Sequence Similarity ◽  
Immunodeficiency Virus ◽  
Filtration Step ◽  
Hiv 1

A novel laccase was isolated and purified from fermentation mycelia of mushroomCoprinus comatuswith an isolation procedure including three ion-exchange chromatography steps on DEAE-cellulose, CM-cellulose, and Q-Sepharose and one gel-filtration step by fast protein liquid chromatography on Superdex 75. The purified enzyme was a monomeric protein with a molecular weight of 64 kDa. It possessed a unique N-terminal amino acid sequence of AIGPVADLKV, which has considerably high sequence similarity with that of other fungal laccases, but is different from that ofC. comatuslaccases reported. The enzyme manifested an optimal pH value of 2.0 and an optimal temperature of 60°C using 2,2′-azinobis(3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) as the substrate. The laccase displayed, at pH 2.0 and 37°C,Kmvalues of 1.59 mM towards ABTS. It potently suppressed proliferation of tumor cell lines HepG2 and MCF7, and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with an IC50value of 3.46 μM, 4.95 μM, and 5.85 μM, respectively, signifying that it is an antipathogenic protein.

scholarly journals A Laccase with Antiproliferative and HIV-I Reverse Transcriptase Inhibitory Activities from the Mycorrhizal FungusAgaricus placomyces

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Jian Sun ◽  
Qing-Jun Chen ◽  
Qing-Qin Cao ◽  
Ying-Ying Wu ◽  
Li-Jing Xu ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Mycorrhizal Fungus ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Hep G2 ◽  
Immunodeficiency Virus ◽  
Terminal Amino ◽  
Hiv 1 ◽  
Mcf 7

A novel 68 kDa laccase was purified from the mycorrhizal fungusAgaricus placomycesby utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature forA. placomyceslaccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7′-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2,Kmvalues of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC50of 1.8 μM, 1.7 μM, and 1.25 μM, respectively, signifying that it is an antipathogenic protein.

scholarly journals Novel galactonic acid-binding hexameric lectin from Hibiscus mutabilis seeds with antiproliferative and potent HIV-1 reverse transcriptase inhibitory activities.

2009 ◽  
Vol 56 (4) ◽  
Author(s):  
Sze Kwan Lam ◽  
Tzi Bun Ng
Keyword(s):  
Reverse Transcriptase ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Inhibitory Activities ◽  
Binding Lectin ◽  
Hiv 1 ◽  
Hepatoma Hepg2 ◽  
Mcf 7

A hexameric 150-kDa lectin was isolated from dried Hibiscus mutabilis seeds using a chromatographic protocol that involved ion exchange chromatography on SP-Sepharose, and gel filtration on Superdex 75 and Superdex 200. The lectin was not adsorbed on SP-Sepharose and was eluted from the Superdex 75 column in the void volume. It was eluted in the first peak from Superdex 200. It was strongly adsorbed on DEAE-cellulose and Q-Sepharose and could not be easily desorbed. The hemagglutinating activity of the lectin, which was stable at pH 4-7 and up to 50 degrees C, could be inhibited by 25 mM galactonic acid. This is the first report of a galactonic acid-binding lectin. It potently inhibited HIV-1 reverse transcriptase with an IC(50) of 0.2 microM. It exhibited weak antiproliferative activity towards both hepatoma HepG2 cells (40% inhibition) and breast cancer MCF-7 cells (50% inhibition) at 100 microM concentration of the lectin. It did not inhibit mycelial growth of a number of fungi tested.

scholarly journals A Laccase with HIV-1 Reverse Transcriptase Inhibitory Activity from the Broth of Mycelial Culture of the MushroomLentinus tigrinus

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
LiJing Xu ◽  
HeXiang Wang ◽  
TziBun Ng
Keyword(s):  
Reverse Transcriptase ◽  
Inhibitory Activity ◽  
Laccase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Mycelial Culture ◽  
Lentinus Tigrinus ◽  
Terminal Amino ◽  
Hiv 1

A 59 kDa laccase with inhibitory activity against HIV-1 reverse transcriptase (IC50=2.4 μM) was isolated from the broth of mycelial culture of the mushroomLentinus tigrinus. The isolation procedure involved ion exchange chromatography on DEAE-cellulose and CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was adsorbed on both types of ion exchangers. About 95-fold purification was achieved with a 25.9% yield of the enzyme. The procedure resulted in a specific enzyme activity of 76.6 U/mg. Its N-terminal amino acid sequence was GIPDLHDLTV, which showed little similarity to other mushroom laccase and otherLentinus tigrinusstrain laccase. Its characteristics were different from previously reported laccase of otherLentinus tigrinusstrain. Maximal laccase activity was observed at a pH of 4 and at a temperature of 60°C, respectively. This study yielded the information about the potentially exploitable activities ofLentinus tigrinuslaccase.

scholarly journals Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2

1993 ◽  
Vol 295 (2) ◽  
pp. 463-469 ◽  
Author(s):  
S A Freeman ◽  
K Peek ◽  
M Prescott ◽  
R Daniel
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
High Sequence Similarity

The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70 degrees C, 43 h at 80 degrees C and 90 min at 90 degrees C were found in the presence of 5 mM CaCl2. At 90 degrees C increasing the CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 30 to 130 min. Half-lives of 19.4 min at 100 degrees C and 4.4 min at 105 degrees C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activators such as Ca2+, Mg2+ and Zn2+.

scholarly journals Purification and Characterization of a Lectin fromPhaseolus vulgaris cv.(Anasazi Beans)

2009 ◽  
Vol 2009 ◽  
pp. 1-9 ◽  
Author(s):  
Arishya Sharma ◽  
Tzi Bun Ng ◽  
Jack Ho Wong ◽  
Peng Lin
Keyword(s):  
Sequence Similarity ◽  
Gel Filtration ◽  
Peritoneal Macrophages ◽  
Exchange Chromatography ◽  
Hemagglutinating Activity ◽  
Murine Splenocytes ◽  
Ph Range ◽  
Hiv 1 ◽  
Mcf 7

A lectin has been isolated from seeds of thePhaseolus vulgaris cv.“Anasazi beans” using a procedure that involved affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The lectin was comprised of two 30-kDa subunits with substantial N-terminal sequence similarity to otherPhaseoluslectins. The hemagglutinating activity of the lectin was stable within the pH range of 1–14 and the temperature range of 0–80∘C. The lectin potently suppressed proliferation of MCF-7 (breast cancer) cells with anIC50of 1.3 μM, and inhibited the activity of HIV-1 reverse transcriptase with anIC50of 7.6 μM. The lectin evoked a mitogenic response from murine splenocytes as evidenced by an increase in [3H-methyl]-thymidine incorporation. The lectin had no antifungal activity. It did not stimulate nitric oxide production by murine peritoneal macrophages. Chemical modification results indicated that tryptophan was crucial for the hemagglutinating activity of the lectin.

Phaseococcin, an antifungal protein with antiproliferative and anti-HIV-1 reverse transcriptase activities from small scarlet runner beans

2005 ◽  
Vol 83 (2) ◽  
pp. 212-220 ◽  
Author(s):  
Patrick H.K Ngai ◽  
T B Ng
Keyword(s):  
Reverse Transcriptase ◽  
Gel Filtration ◽  
Leukemia Cell ◽  
Deae Cellulose ◽  
Antifungal Protein ◽  
Bacillus Species ◽  
Mouse Splenocytes ◽  
Antifungal Proteins ◽  
Reticulocyte Lysate ◽  
Hiv 1

From the seeds of small scarlet runner beans (Phaseolus coccineus 'Minor'), an antifungal protein with an N-terminal sequence homologous to those of defensins was isolated. The antifungal protein bound to Affi-gel blue gel and Mono S but it did not bind to DEAE-cellulose. It was further purified by gel filtration on a Superdex peptide column. It exhibited a molecular mass of 5422 Da as determined by mass spectrometry. The protein, designated as phaseococcin, suppressed mycelial growth in a number of fungi including Botrytis cinerea, Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, Physalospora piricola, and Rhizoctonia solani. It also inhibited proliferation in several Bacillus species and the leukemia cell lines HL60 and L1210 and curtailed the activity of HIV-1 reverse transcriptase. It did not affect proliferation of mouse splenocytes and neither did it inhibit protein synthesis in a cell-free rabbit reticulocyte lysate system.Key words: antifungal proteins, runner beans, antiproliferative.

scholarly journals An Inulin-Specific Lectin with Anti-HIV-1 Reverse Transcriptase, Antiproliferative, and Mitogenic Activities from the Edible Mushroom Agaricus bitorquis

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Guo-Qing Zhang ◽  
Qing-Jun Chen ◽  
Jing Hua ◽  
Zi-Lu Liu ◽  
Yue Sun ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Gel Filtration ◽  
Leukemia Cell ◽  
Edible Mushroom ◽  
Exchange Chromatography ◽  
Human Type ◽  
Agaricus Bitorquis ◽  
Terminal Amino ◽  
Anti Hiv ◽  
Hiv 1

A novel lectin (ABL) was purified from the dried fruiting bodies of Agaricus bitorquis. An efficient 3-step purification protocol involved two consecutive steps of ion exchange chromatography on Q-Sepharose and SP-Sepharose and gel filtration by FPLC on Superdex 75. ABL is a monomeric protein with the molecular mass of 27.6 kDa, which is different from other lectins from genus Agaricus. Its N-terminal amino acid sequence is EYTISIRVYQTNPKGFNRPV which is unique and sharing considerably high similarity of other mushroom lectins. The hemagglutinating activity of the lectin was inhibited by inulin. Based on hemagglutination tests, ABL prefers rabbit, human type A, and AB erythrocytes to human type B and O erythrocytes. The lectin inhibits the activity of HIV-1 reverse transcriptase and the proliferation of leukemia cell (L1210) with an IC50 value of 4.69 and 4.97 μM, respectively. Furthermore, ABL demonstrates the highest mitogenic activity with a response of 24177.7 ± 940.6 [3H-methyl] thymidine counts per minute (CPM) at a concentration of 0.91 μM.

scholarly journals Extraction, and Purification of Peroxidase Enzyme from Peganum harmala Seeds

2019 ◽  
Vol 9 (04) ◽  
pp. 689-692
Author(s):  
Hayder Nasser Al-Mentafji ◽  
Mahmoud Hamid Al-Fahdawi ◽  
Albab Fawwaz Al-farras
Keyword(s):  
Ion Exchange ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Peganum Harmala ◽  
Enzyme Recovery ◽  
Extraction And Purification ◽  
Peroxidase Enzyme ◽  
Filtration Step

The aim of this study was to extract peroxides enzyme from Peganum harmala seeds; peroxides was extracted by using different extraction buffer solutions, then it was purified by three steps of purification includes precipitation with ammonium sulfate in a saturation ratio of 70 %, ion exchange chromatography through DEAE-Cellulose, and gel filtration chromatography throughout Sephadex G-100, and determine the optimum condition for extraction. This was performed by controlling the type and concentration of buffer, pH of the buffer used, and the ratio of extraction. The Sodium acetate buffer with 0.2mM and pH 5.0 was found to be the best buffer for the extraction of peroxidase. By using the extraction ratio for a plant of 1:3 (W/V), the specific activity was 195 U/mg protein. These three purification steps raised the specific activity to 235U/mg protein in the precipitation step with purification fold 2.3 and enzyme recovery 69%; the specific activity was increased to 243U/mg protein in Ion exchange step with purification fold 2.4 and enzyme recovery 23%, also the specific activity doubled after gel filtration step to 447U/mg protein with purification fold 4.4 and enzyme recovery 15%.

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

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