scholarly journals A Novel Aspartic Protease with HIV-1 Reverse Transcriptase Inhibitory Activity from Fresh Fruiting Bodies of the Wild MushroomXylaria hypoxylon

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Qing-Xiu Hu ◽  
Guo-Qing Zhang ◽  
Rui-Ying Zhang ◽  
Dan-Dan Hu ◽  
He-Xiang Wang ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Deae Cellulose ◽  
Aspartic Protease ◽  
Fruiting Bodies ◽  
Extraction Step ◽  
Sds Page ◽  
Terminal Amino ◽  
Ph Range ◽  
Pepstatin A ◽  
Hiv 1

A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroomXylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession), and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6–8 and in the temperature range 35–60°C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50value of 8.3 μM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities.

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.

Human Leucocyte Urokinase Inhibitor - Purification, Characterization and Comparative Studies Against Different Plasminogen Activators

1985 ◽  
Vol 54 (04) ◽  
pp. 750-755 ◽  
Author(s):  
M Kopitar ◽  
B Rozman ◽  
J Babnik ◽  
V Turk ◽  
D E Mullins ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Inhibitory Activity ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Plasminogen Activator Inhibitor ◽  
Exchange Chromatography ◽  
Sds Page ◽  
Peripheral Leukocytes ◽  
Ph Range ◽  
Activator Inhibitor

SummaryA plasminogen activator inhibitor (PA-I) which inhibits primarily plasminogen activator of the urokinase type (u-PA) was isolated from the cytosol of human peripheral leukocytes. The inhibitor was isolated using ion exchange chromatography, gel filtration and FPLC. This inhibitor has an apparent molecular weight of 45 kDa, determined by SDS-PAGE, and a pi of 5.5-5.7. The inhibitor is a fast reacting inhibitor, is thermally unstable and is inactivated outside the pH range 7-9. Treatment of cytosol to pH 9 for 30 min at 37° C resulted in a large increase in inhibitory activity. Antibodies against human placental UK-I completely quenched the inhibitory activity of human leucocyte UK-I.

scholarly journals Highly-Expressed Aspartic Protease from Aspergillus Fumigatus-Mediated Enzymolysis of Silkworm (Bombyx Mori) Pupae Protein

2021 ◽  
Author(s):  
Richard A. Herman ◽  
Chen Xie ◽  
Zi-Qian Zha ◽  
Zong-Nan Li ◽  
Jin-Zheng Wang ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Bombyx Mori ◽  
Specific Activity ◽  
Aspartic Protease ◽  
Degree Of Hydrolysis ◽  
Protease Gene ◽  
Silkworm Pupae ◽  
Sds Page ◽  
Ph Range ◽  
Hydrolysis Of

Abstract Aspartic protease emerges as an optimistic hydrolytic agent to obtain several protein hydrolysates. An aspartic protease gene from Aspergillus fumigatus Af293 was successfully expressed in Pichia pastoris (GS115) and its hydrolytic potentials on silkworm (Bombyx mori) pupae protein were determined. It was optimum at pH 4.0 and 50 °C and stable over pH range 4.0-5.0 and temperatures 45-55 °C with a specific activity of 8408.9 ± 305.6 U/mg. SDS-PAGE analysis revealed the molecular weight of the recombinant protease to be 45 kDa. The half-life (t1/2) of the recombinant protease at 40, 50, 60, and 70 °C was 30, 25, 35, and 20 min, respectively. The protease showed enhanced activity in the presence of Cu2+, Pb2+ and SDS. Its substrate specificity studies were revealed in the order of cleaving ability to Bovine Serum Albumin (BSA) > Silkworm pupae powder (SPP) > Casein > Casein sodium salt (CSS). Upon hydrolysis of silkworm pupae protein, it showed enhanced and plausible hydrolytic potentials, increasing the degree of hydrolysis to 50 ± 6.1% at 6 h, increased solubility by 80%, and improved functional properties. The stable characteristics and hydrolytic performance of the recombinant aspartic protease qualify it for industrial application, especially within the food and related industries.

scholarly journals Antitumor and HIV-1 Reverse Transcriptase Inhibitory Activities of a Hemagglutinin and a Protease Inhibitor from Mini-Black Soybean

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Xiu Juan Ye ◽  
Tzi Bun Ng
Keyword(s):  
Breast Cancer ◽  
Reverse Transcriptase ◽  
Inhibitory Activity ◽  
Breast Cancer Cells ◽  
Deae Cellulose ◽  
Defense Proteins ◽  
Black Soybean ◽  
Trypsin Inhibitory Activity ◽  
Inhibitory Activities ◽  
Hiv 1

Protease inhibitors (PIs) and hemagglutinins are defense proteins produced by many organisms. From Chinese mini-black soybeans, a 17.5-kDa PI was isolated using chromatography on Q-Sepharose, SP-Sepharose, and DEAE-cellulose. A 25-kDa hemagglutinin was purified similarly, but using Superdex 75 instead of DEAE-cellulose in the final step. The PI inhibited trypsin and chymotrypsin (IC50= 7.2 and 8.8 μM). Its trypsin inhibitory activity was stable from pH 2 to pH 13 and from 0∘C to 70∘C. The hemagglutinin activity of the hemagglutinin was stable from pH 2 to pH 13 and from 0∘C to 75∘C. The results indicated that both PI and hemagglutinin were relatively thermostable and pH-stable. The trypsin inhibitory activity was inhibited by dithiothreitol, signifying the importance of the disulfide bond to the activity. The hemagglutinating activity was inhibited most potently by D (+)-raffinose and N-acetyl-D-galactosamine, suggesting that the hemagglutinin was specific for these two sugars. Both PI and hemagglutinin inhibited HIV-1 reverse transcriptase (IC50= 3.2 and 5.5 μM), proliferation of breast cancer cells (IC50= 9.7 and 3.5 μM), and hepatoma cells (IC50= 35 and 6.2 μM), with relatively high potencies.

scholarly journals A Novel Lectin with Antiproliferative and HIV-1 Reverse Transcriptase Inhibitory Activities from Dried Fruiting Bodies of the Monkey Head MushroomHericium erinaceum

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Yanrui Li ◽  
Guoqing Zhang ◽  
Tzi Bun Ng ◽  
Hexiang Wang
Keyword(s):  
Reverse Transcriptase ◽  
Deae Cellulose ◽  
Amino Acid Sequences ◽  
Mitogenic Activity ◽  
Fruiting Bodies ◽  
Murine Splenocytes ◽  
Mouse Splenocytes ◽  
Minimum Concentration ◽  
Hericium Erinaceum ◽  
Hiv 1

A lectin designated asHericium erinaceumagglutinin (HEA) was isolated from dried fruiting bodies of the mushroomHericium erinaceumwith a chromatographic procedure which entailed DEAE-cellulose, CM-cellulose, Q-Sepharose, and FPLC Superdex 75. Its molecular mass was estimated to be 51 kDa and its N-terminal amino acid sequences was distinctly different from those of other isolated mushroom lectins. The hemagglutinating activity of HEA was inhibited at the minimum concentration of 12.5 mM by inulin. The lectin was stable at pH 1.9–12.1 and at temperatures up to C, but was inhibited by , , and ions. The lectin exhibited potent mitogenic activity toward mouse splenocytes, and demonstrated antiproliferative activity toward hepatoma (HepG2) and breast cancer (MCF7) cells with an of 56.1 M and 76.5 M, respectively. It manifested HIV-1 reverse transcriptase inhibitory activity with an of 31.7 M. The lectin exhibited potent mitogenic activity toward murine splenocytes but was devoid of antifungal activity.

scholarly journals Purification and Characterization of Carboxymethyl Cellulase from Bacillus sp. Isolated from a Paddy Field

2012 ◽  
Vol 61 (1) ◽  
pp. 51-55 ◽  
Author(s):  
PONNUSWAMY VIJAYARAGHAVAN ◽  
S.G. PRAKASH VINCENT
Keyword(s):  
Paddy Field ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Relative Molecular Mass ◽  
Bacillus Sp ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Ph Range

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.

scholarly journals A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake MushroomLentinus edodes

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Guo-Qing Zhang ◽  
Ying-Ying Wu ◽  
Tzi-Bun Ng ◽  
Qing-Jun Chen ◽  
He-Xiang Wang
Keyword(s):  
Sequence Similarity ◽  
Gel Filtration ◽  
Residual Activity ◽  
Deae Cellulose ◽  
Maximal Activity ◽  
Phytase Activity ◽  
Ph Tolerance ◽  
Phosphorylated Compounds ◽  
Terminal Amino ◽  
Ph Range

A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroomLentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate >β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+at a tested concentration of 5 mM.

scholarly journals A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa.

2011 ◽  
Vol 58 (4) ◽  
Author(s):  
Jian Sun ◽  
Yongchang Zhao ◽  
Hongmei Chai ◽  
Hexiang Wang ◽  
Tzi Bun Ng
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fruiting Bodies ◽  
Wild Mushroom ◽  
Sds Page ◽  
Human Hepatoma Hepg2 Cells

A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 µM. The protease did not have antifungal or ribonuclease activity.

Characterization of Glycoprotein Fraction from Carp Pituitaries Isolated Using Concanavalin A as the Affinity Ligand

1998 ◽  
Vol 63 (3) ◽  
pp. 434-440 ◽  
Author(s):  
Irena Hulová ◽  
Jana Barthová ◽  
Helena Ryšlavá ◽  
Václav Kašička
Keyword(s):  
Concanavalin A ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Gel Chromatography ◽  
Affinity Ligand ◽  
Sds Page ◽  
Terminal Amino ◽  
Α And Β Subunits

Glycoproteins that have affinity to Concanavalin A were isolated from the acetone-dried pituitaries of common carp (Cyprinus carpio L.). Two fractions of glycoproteins were separated using gel chromatography on Superdex 75HR. The fraction with lower molecular weight (30 000) corresponding to the carp gonadotropin cGtH II was composed of two subunits as determined using SDS-PAGE. This protein fraction was further divided into four components using reversed-phase HPLC. Two fractions were pure α and β subunits of cGtH II as follows from immunodetection and from determination of N-terminal amino acid sequences. The other two were a mixture of α and β subunits as was also revealed by N-terminal analysis. Capillary electrophoresis was also used for characterization of isolated glycoproteins.

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