scholarly journals Protease from Courgette (Luffa Acutangula L (Roxb)): Isolation, Purification, and Some Characteristics

2019 ◽  
Vol 5 (2) ◽  
pp. 64-71
Author(s):  
MIKE PERMATA SARI ◽  
DWIRINI RETNO GUNARTI ◽  
MOHAMAD SADIKIN
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Thiol Group ◽  
Exchange Chromatography ◽  
Enzyme Replacement ◽  
Protease Enzyme ◽  
Minute Duration ◽  
Luffa Acutangula ◽  
Ammonium Sulphate Saturation

The Courgette or oyong (Luffa acutangula L. (Roxb)) is member of Cucurbitaceae mainly used as vegetable. Beside used as vegetables, courgette aslo used as keratolytic agent. This fact is supposed that this vegetables contain protease. This research is succeed to purified courgette’s protease by four step. That was precipitate by 70% ammonium sulphate saturation, purification using DEAE cellulose ion exchange chromatography and gel filtration chromatography using sephadex G-100 and G-75. Purified courgette’s protease had 81,922 U/mg for specific activity and 34 kDa molecular weight. This enzyme had the characteristic such as activated optimally at 37oC, pH 7 and 10 minute duration time. This enzyme activity can decrease by PMSF and H2O2, its remarkable that courgette protease is serine protease and had the thiol group in its structure. The ability to digest food proteins materials like boiled meat and boiled white egg by courgette protease proves that the courgette protease enzyme is could be used in enzyme replacement therapy in mild digestion problem. 

scholarly journals PURIFICATION AND CHARACTERIZATION OF SALIVARY AMYLASE INHIBITOR EXTRACTED FROM BARLEY

2016 ◽  
Vol 47 (4) ◽  
Author(s):  
Abood & Hakeem
Keyword(s):  
Ammonium Sulfate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Alpha Amylase ◽  
Ionic Exchange ◽  
Inhibition Activity ◽  
Amylase Inhibitors

Amylase inhibitors were purified by many sequential steps included concentration by gradual addition of ammonium sulfate at  saturation ratios. ranged from 0 to 90% . The best ratio of saturation was found to be 70% as the specific activity and inhibition activity toward Human alpha-amylase(HAS)  were the highest ( 8 U/mg and 6 U/ml respectively as compared to those of the rest ratios, the ratio of saturation with ammonium sulfate 60 % and then 50%, (5.8 ,5.5  )U/ml and( 7.7 ،7 )U/mg respectively for inhibition activity and specific activity and for  40% ,30%20%  saturation  the inhibition activity and specific activity were(5 ،4.8 ،4 ) u/ml (6.6 ،6 ،5.8) u/mg respectively .The precepitation step was followed by ionic exchange chromatography technique by DEAE-cellulose column( 3×11 )cm and the results showed that there was one peak with inhibition activity toward (HAS). Further  purification steps were conducted using gel filtration on Sephacryl S-200 column    (1.5  ×  60)cm; the purification folds was5.59 times with outcome of 46.5%.The results of alpha-amylase inhibitors characterization showed that the molecular weight was about 23.44 and 22.9  kDa  as determined by electrophoresis and gel filteration respectively.                                         

scholarly journals Purification and Characterization of Melanogenic Enzyme Tyrosinase from Button Mushroom

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Kamal Uddin Zaidi ◽  
Ayesha S. Ali ◽  
Sharique A. Ali
Keyword(s):  
Agaricus Bisporus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Edible Mushroom ◽  
Total Activity ◽  
Protein Band ◽  
Exchange Chromatography ◽  
Ammonium Sulphate Precipitation ◽  
Key Enzyme

Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

scholarly journals Two acid RNases from Dactylis glomerata seeds. Purification, properties and effect of polyamines and lectins on their activity

2014 ◽  
Vol 54 (3) ◽  
pp. 241-253 ◽  
Author(s):  
Janina Wiśniowska ◽  
Bronisława Morawiecka
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Dactylis Glomerata ◽  
Hydrolytic Activity ◽  
Relative Activity ◽  
Exchange Chromatography ◽  
Lentil Lectin ◽  
Optimum Ph ◽  
Rnase Ii

Two glycoproteidic acid RNases (RNase I and RNase II) were obtained and purified from the seeds of <em>Dactylis glomerata</em> by extraction with acetate buffer, fractionation with ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, DEAE-Sphadex, affinity chromatography on Con A-Sepharose and gel filtration on Bio-Gel P60. RNase I with a specific activity of 2582 U•mg<sup>-1</sup> protein and an optimum pH of 4.9 and RNase II with a specific activity of 1928 U• mg<sup>-1</sup> protein and optimum pH of 4.6, were isolated. They lacked nuclease, phosphodi- and monoesterase activities. Both forms of the enzyme hydrolyzed pyrimidine homopolymers with a preference for poly U and exhibited a low specificity for purine homopolymers (poly G and poly A). RNase I acted with a 3-fold higher hydrolytic activity on poly C homopolymer than RNase IL The hydrolytic activity of both enzymes was inhibited by Zn<sup>+2</sup>, Fe<sup>+2</sup>, Cu<sup>+2</sup> ions when yeast RNA was the substrate. The amines spermine, spermidine and tyramine at a concentration of 0.1 mM increased the enzymatic activity of both RNases by 20 to 60% of the relative activity. The hydrolytic activity of RNases I and II was stimulated by the presence of lentil lectin (LL), soybean lectin (SBA) and potato lectin (STA), and inhibited by the presence of concanavalin A. The 20-200% stimulation and 40-60% inhibition depended on the proportion, on a weight basis, of enzyme to lectin and were reversible in the presence of receptor sugars.

scholarly journals A biological study of chitinase produced by clinical isolates of Pseudomonas aeruginosa and detection of ChiA responsible gene

2020 ◽  
Vol 11 (2) ◽  
pp. 1318-1330
Author(s):  
Tahreer Hadi Saleh ◽  
SabaTalib Hashim ◽  
Raghad Abdulatif Abdulrazaq Al-Obaidi ◽  
Bahaa Abdullah Laftaah AL-Rubaii
Keyword(s):  
Liver Cell ◽  
Cytotoxic Effect ◽  
Specific Activity ◽  
Morphological Changes ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Clinical Isolates ◽  
Biological Study ◽  
Variable Degree

All isolates in this study were diagnosed as P. aeroginosa according to the 16srRNA gene. Only two isolates were produced chitinase on chitin agar medium and were positive to the chiA gene. Most of the isolates exhibited high sensitivity (95%) and (90%) to Imipenem and Carbenicillin, respectively, and the resistance to Amoxicillin +Clavulanic acid was shown (80%), while revealed a variable degree in their response to others antibiotics. The crude extract activity and specific activity for extracted chitinase enzymes were 33 U/ml and 18.644U/mg, respectively. The enzyme was purified by different steps include: precipitating with saturation 70% of ammonium sulfate and then applied on ion-exchange chromatography using DEAE- cellulose column and then employ the Sephadex G-200 column for gel filtration chromatography. The purification fold and yield was 28.5%. The molecular weight of the purified enzyme was determined by SDS-PAGE method, and it appeared at 50 kDa. The results of the MTT assay showed that the chitinase has a cytotoxic effect on cancer liver cell lines at a concentration of 100 µg /ml and increased gradually at a concentration of 600 µg /ml, while it showed no or less cytotoxic effect on normal embryonic liver cell line (WRL-68). Chitinase enzyme appeared a higher antibacterial activity at concentration 600 µg/ml and a lower activity at concentration 400 towards clinical isolates of S. aurous and E. coli. The study of histopathological effects was exhibited little morphological changes on cells of liver tissues.

scholarly journals Caldolase, a chelator-insensitive extracellular serine proteinase from a Thermus spp

1989 ◽  
Vol 262 (2) ◽  
pp. 409-416 ◽  
Author(s):  
G A Saravani ◽  
D A Cowan ◽  
R M Daniel ◽  
H W Morgan
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Serine Proteinase ◽  
Gel Permeation Chromatography ◽  
Exchange Chromatography ◽  
Acetate Buffer ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Low Ionic Strength

An extracellular alkaline serine proteinase from Thermus strain ToK3 was isolated and purified to homogeneity by (NH4)2SO4 precipitation followed by ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex, affinity chromatography on N alpha-benzyloxycarbonyl-D-phenylalanyl-triethylenetetraminyl-Sepha rose 4B and gel-filtration chromatography on Sephadex G-75. The purified enzyme had a pI of 8.9 and an Mr determined by gel-permeation chromatography of 25,000. The specific activity was about 37,700 proteolytic units/mg with casein as substrate, and the pH optimum was 9.5. Proteolytic activity was inhibited by low concentrations of di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride, but was unaffected by EDTA, EGTA, o-phenanthroline, N-ethyl-5-phenylisoxazolium-3′-sulphonate, N alpha-p-tosyl-L-phenylalanylchloromethane, N alpha-p-tosyl-L-lysylchloromethane, trypsin inhibitors and pepstatin A. The enzyme contained approx. 10% carbohydrate and four disulphide bonds. No Ca2+, Zn2+ or free thiol groups were detected. It hydrolysed several native and dye-linked proteins and synthetic chromogenic peptides and esters. The enzyme was very thermostable (half-life values were 840 min at 80 degrees C, 45 min at 90 degrees C and 5 min at 100 degrees C). The enzyme was unstable at low ionic strength: after 60 min at 75 degrees C in 0.1 M-Tris/acetate buffer, pH 8, only 20% activity remained, compared with no loss in 0.1 M-Tris/acetate buffer, pH 8, containing 0.4 M-NaCl.

scholarly journals Characterization and Cytotoxic Activity of Cytosine Deaminase Enzyme Purified from Locally Isolated Escherichia coli

2018 ◽  
Vol 15 (3) ◽  
pp. 262-269
Author(s):  
Baghdad Science Journal
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Cell Line ◽  
Cancer Cell ◽  
Specific Activity ◽  
Gel Filtration ◽  
Cytosine Deaminase ◽  
Deae Cellulose ◽  
Cancer Cell Line ◽  
Exchange Chromatography

This research was aimed to the purification and characterization of cytosine deaminase as a medically important enzyme from locally isolated Escherichia coli; then studying its cytotoxic anticancer effects against colon cancer cell line. Cytosine deaminase was subjected to three purification steps including precipitation with 90% ammonium sulfate saturation, ion exchange chromatography on DEAE-cellulose column, and gel filtration chromatography throughout Sephadex G-200 column. Specific activity of the purified enzyme was increased up to 9 U/mg with 12.85 folds of purification and 30.85% enzyme recovery. Characterization study of purified enzyme revealed that the molecular weight of cytosine deaminase produced by E. coli was about 48 KDa, the highest enzyme activity at pH 8.5, and is most stable at pH 7.5 - 9, the enzyme also showed a full activity at a range of temperatures between 45-60 0C. Enzyme activity was strongly inhibited in the presence of mercuric chloride and copper sulphate, when added individually at a constant concentration. However, calcium chloride, manganese chloride and ferric chloride caused a little increase in enzyme activity while sodium azide had no effect on enzyme activity. Upon cytotoxic effect study through micro-cultured tetrazolium assay (MTT) against Caco-2 cell line. Purified cytosine deaminase was found to inhibit the growth of Caco-2 cancer cell line with an IC50 of 242.5 ?g/ml in a comparison to an IC50 of 1864 ?g/ml for crude enzyme. Besides, the enzyme didn’t show significant effect on WRL normal cell line.

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.

scholarly journals Production and comparison of mature single-domain ‘trefoil’ peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58

1995 ◽  
Vol 308 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
M P Chadwick ◽  
F E B May ◽  
B R Westley
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Gel Filtration ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Exchange Chromatography ◽  
Single Domain ◽  
Trefoil Peptides ◽  
Trefoil Peptide

The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the ‘trefoil’ family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.

Kinetic and Thermodynamic Characterization of Lignin Peroxidase Isolated from Agaricus bitorqus A66

2012 ◽  
Vol 10 (1) ◽  
Author(s):  
Ismat Bibi ◽  
Haq Nawaz Bhatti
Keyword(s):  
Lignin Peroxidase ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Gel Filtration ◽  
Veratryl Alcohol ◽  
Exchange Chromatography ◽  
Different Temperatures ◽  
Scale Experiment ◽  
Menten Kinetic

This study deals with purification and characterization of lignin peroxidase (LiP) isolated from Agaricus bitorqus A66 during decolorization of NOVASOL Direct Black dye. A laboratory scale experiment was conducted for maximum LiP production under optimal conditions. Purification & fractionation of LiP was performed on DEAE-Sepharose ion exchange chromatography followed by Sephadex G-50 gel filtration. The purified LiP has a specific activity of 519 U/mg with 6.73% activity recover. The optimum pH and temperature of purified LiP for the oxidation of veratryl alcohol were 6.8 and 45 °C, respectively. Michaelis-Menten kinetic constants (Vmax and Km) were determined using different concentrations of veratryl alcohol (1-35 mM). The Km and Vmax were 16.67 mM and 179.2 U/mL respectively, for veratryl alcohol oxidation as determined from the Lineweaver-Burk plot. Thermal inactivation studies were carried out at different temperatures to check the thermal stability of the enzyme. Enthalpy of activation decreased where Free energy of activation for thermal denaturation increased at higher temperatures. A possible explanation for the thermal inactivation of LiP at higher temperatures is also discussed.

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