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 Purification and Characterization of Endoglucanase from local isolate of Aspergillus flavus

2013 ◽  
Vol 10 (3) ◽  
pp. 844-853
Author(s):  
Baghdad Science Journal
Keyword(s):  
Aspergillus Flavus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Stability ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Original Activity ◽  
A Value ◽  
Optimum Ph

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.

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.                                         

Catabolism of (R)-Amygdalin and (R)-Vicianin by Partially Purified ß-Glycosidases from Prunus serotina Ehrh. and Davallia trichomanoides

1984 ◽  
Vol 39 (3-4) ◽  
pp. 232-239 ◽  
Author(s):  
Gary Kuroki ◽  
Pauline A. Lizotte ◽  
Jonathan E. Poulton
Keyword(s):  
Ion Exchange ◽  
Deae Cellulose ◽  
Hydrolytic Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Prunus Serotina ◽  
Artificial Substrates ◽  
Black Cherry ◽  
Significant Activity ◽  
Optimum Ph

Mature black cherry (Prunus serotina Ehrh.) seeds accum ulate high levels of the cyanogenic disaccharide (R)-amygdalin. Extracts from these seeds contain two β-glycosidases which have been identified and completely resolved by DEAE-cellulose ion-exchange chromatography. Amygdalin hydrolase hydrolyzed (R)-am ygdalin at an optimum pH of 5.5, releasing (R)-prunasin and D-glucose. This enzyme showed highest activity towards (R)-am ygdalin and failed to hydrolyze (R)-prunasin. linamarin, β-gentiobiose and cellobiose. A distinct β-glycosidase, prunasin hydrolase, displayed a pronounced preference for (R)-prunasin, hydrolyzing this cyanogenic monosaccharide at an optimum pH of 6.5 to mandelonitrile and D-glucose. Prunasin hydrolase was inactive towards (R)-am ygdalin, linamarin, and β-gentiobiose. Both enzymes showed significant activity towards the artificial substrates β-ONPGlu and β-PNPGlu but did not hydrolyze α-PNPGlu. In view of the pronounced specificity of these enzymes towards endogenous cyanogens, it is concluded that upon disruption of black cherry seeds (R)- amygdalin is catabolized to mandelonitrile in a stepwise manner (the sequential mechanism) by amygdalin hydrolase and prunasin hydrolase with (R)-prunasin serving as intermediate. Young fronds of Davallia trichomanoides are rich sources of (R)-vicianin (the β-vicianoside of (R)-mandelonitrile). A β-glycosidase, vicianin hydrolase, has been partially purified from frond extracts by ion-exchange chromatography. At the optimum pH of 6.0, this enzyme showed highest hydrolytic activity with (R)-vicianin, although both (R)-am ygdalin and (R)-prunasin could be hydrolyzed at approximately 15% of the rate observed with (R)-vicianin. It failed to hydrolyze β-gentiobiose, cellobiose, linamarin and α-PNPGlu. Closer exam ination revealed that (R)-vicianin and (R)-amygdalin were hydrolyzed at the aglycone-disaccharide bond (the simultaneous mechanism) yielding mandelonitrile and the respective disaccharides vicianose and β-gentiobiose

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.

Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

1982 ◽  
Vol 47 (4) ◽  
pp. 1139-1148 ◽  
Author(s):  
Karel Hauzer ◽  
Linda Servítová ◽  
Tomislav Barth ◽  
Karel Jošt
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

scholarly journals Acid phosphatases and ribonucleases from Dactylis glomerata seeds. I. Chromatographic and electrophoretic heterogeneity of the enzymes

2015 ◽  
Vol 47 (4) ◽  
pp. 441-453 ◽  
Author(s):  
Eleonora Wieczorek ◽  
Janina Wiśniewska ◽  
Bronisława Morawiecka
Keyword(s):  
Acid Phosphatase ◽  
Sodium Acetate ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Dactylis Glomerata ◽  
Molecular Forms ◽  
Acid Phosphatases ◽  
Sodium Acetate Buffer ◽  
Optimal Activity ◽  
Optimum Ph

Acid phosphatase and ribonuclease extracted with 0.1 M sodium acetate buffer, pH 5.1 from Dactylis glomerata seeds, and partially purified by means of 70% ethanol precipitation showed electrophoretic and Chromatographic heterogeneity. After chromatography on DEAE-cellulose acid phosphatase and ribonuclease were separated into four peaks. Nonadsorbing acid phosphatase on DEAE-cellulose (peak I) was separated into four peaks on CM-cellulose. The highest activity (11 units/mg) was found in fraction b (acid phosphatase Ib). The enzyme was activated by Mg<sup>2+</sup>, Ca<sup>2+</sup>, Li<sup>+</sup>, Cs<sup>+</sup>, K<sup>+</sup> ions and inhibited by Cu<sup>2+</sup>, Zu<sup>2+</sup>, F<sup>-</sup> and Mo<sup>-6</sup> at optimum pH 5.0. Strong absorbing ribonuclease on DEAE-cellulose (peak IV) was further separated on G-200 Sephadex into two molecular forms: RN-asa1 and RN-ase2. Ribonuclease l, a thermolabile enzyme with specific activity 807 units/mg, showed an optimal activity at pH 4.8-5.1.

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%.

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