scholarly journals Purification and characterization of amylase from local isolate Pseudomonas sp.SPH4

2012 ◽  
Vol 6 (1) ◽  
pp. 69-79
Author(s):  
Hala M. Ali ◽  
Ghazi M. Aziz
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Silver Ions ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Mercury Ions ◽  
Local Isolate ◽  
Optimum Ph

The amylase produced from local isolate Pseudomonas sp. SPH4 was purified by precipitation with 30% saturation ammonium sulphate, followed by ion-exchange chromotography using DEAE-cellulose column, and Gel filtration using Sephacryl S-300 column.The two iso-enzymes (a, b) were purified to (2.83, 3.47) times in the last step with an enzymes yields of (32.36, 76.34)% respectively. Enzyme characterization of the two iso-enzymes indicated that the optimum pH for the two iso-enzymes a and b were (7, 7.5) respectively, while the optimum pH for the iso-enzymes stability were (6.5, 7) respectively. The maximum activity for iso-enzymes (a, b) appeared at 45ºC and stable for 15 min at 30-50ºC and lost approximately 50% of it's activity at rang above 75ºC. Enzyme characterization results showed that the chlorides of silver and mercury had inhibitory effect on enzyme activity, the remaining enzyme activity for the iso-enzymes (a, b) were (46.66, 36.36)% for silver ions and (41.33, 33.63)% for mercury ions at 5 mM respectively, and (28, 28.18)% for silver ions and (25.33, 19.09)% for mercury ions at 10 mM respectively. The iso-enzymes a and b were affected by chelating agent ethylene diamine tetra acetic acid (EDTA) at concentration 2mM the remaining activity (45.33, 43.63)% respectively, and 5mM the remaining activity (28, 28.18)% respectivily, and these iso-enzymes (a, b) refered to metalloenzymes. The iso-enzymes (a, b) were kept their activity when treated by reducing agent (2-mercaptoethanol) at 2 mM the remaining activity (92, 92.72)% respectively, and 5 mM the remaining activity (85.3, 89.09)% respectivily. The iso-enzymes (a, b) were kept their activity when treated by phenyl methyl sulphonyl fluoride (PMSF) at concentration 1mM the remaining activity (93.33, 90.90)% respectivily,and 5 mM the remaining activity (90.66, 87.27)% respectivily, and these indicated that these iso-enzymes didnot referred to serineamylases group.

scholarly journals Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

1992 ◽  
Vol 288 (2) ◽  
pp. 475-482 ◽  
Author(s):  
I Ishii-Karakasa ◽  
H Iwase ◽  
K Hotta ◽  
Y Tanaka ◽  
S Omura
Keyword(s):  
Enzyme Activity ◽  
Culture Medium ◽  
Gel Filtration ◽  
Partial Purification ◽  
Gastric Mucus ◽  
Purification And Characterization ◽  
Streptomyces Sp ◽  
Optimum Ph ◽  
New Type

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

scholarly journals Optimization of Growth Conditions for Purification and Production of L-Asparaginase bySpirulina maxima

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Hanaa H. Abd El Baky ◽  
Gamal S. El Baroty
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Growth Conditions ◽  
Partial Purification ◽  
Specific Enzyme ◽  
Purification And Characterization ◽  
Spirulina Maxima ◽  
Optimum Ph ◽  
Biomass Yields

L-asparaginase (L-AsnA) is widely distributed among microorganisms and has important applications in medicine and in food technology sectors. Therefore, the ability of the production, purification, and characterization of AsnA fromSpirulina maxima(SM) were tested. SM cultures grown in Zarrouk medium containing different N2(in NaNO3form) concentrations (1.25, 2.50, and 5.0 g/L) for 18 days contained a significant various quantity of dry biomass yields and AsnA enzyme levels. MS L-AsnA activity was found to be directly proportional to the N2concentration. The cultures of SM at large scales (300 L medium, 5 g/L N2) showed a high AsnA enzyme activity (898 IU), total protein (405 mg/g), specific enzyme activity (2.21 IU/mg protein), and enzyme yield (51.28 IU/L) compared with those in low N2cultures. The partial purification of crude MS AsnA enzyme achieved by 80% ammonium sulfate AS precipitated and CM-Sephadex C-200 gel filtration led to increases in the purification of enzyme with 5.28 and 10.91 times as great as that in SM crude enzymes. Optimum pH and temperature of purified AsnA for the hydrolyzate were 8.5 and 37 ± 0.2°C, respectively. To the best of our knowledge, this is the first report on L-asparaginase production inS. maxima.

scholarly journals Partial Purification and Characterization of Inulinase Produced from a local isolate of Aspergillus niger J3

2010 ◽  
Vol 4 (2) ◽  
pp. 78-85
Author(s):  
Jasim M. Awdaa
Keyword(s):  
Aspergillus Niger ◽  
Gel Filtration ◽  
Partial Purification ◽  
Mercury Chloride ◽  
Purification And Characterization ◽  
Original Activity ◽  
Local Isolate ◽  
Optimum Ph ◽  
Final Purification

Inulinase was produced from local isolate of Aspergillus niger J3. The inulinase was purified by two steps included precipitation by amonium sulphate at (30-80) % sa-turation and gel filtration on sephadex G100. The final purification folds and the yield of the enzyme were 3.15 times and 28.24%, respectively. The purified enzyme has the following characteristics: The optimum pH of the enzyme activity was 5.5. The enzyme was most stable at pH (4.5 - 6). The optimum temperature for its activity was 45c. The enzyme retained its original activity when incubates at (30-55) c for 20 minutes. Mercury chloride inhibited the enzyme completely at concentration of 10mM, cupper sulphate and calisium chloride inhibited the enzyme at concentrations of 85% and 7% respectively. It was revealed that the enzyme had the efficiency to hydrolyze 87% of 5% inulin solution when treated at 45c for 120 min.

Purification and characterization of exo-β-1,3-glucanase from a hatching supernatant of Strongylocentrotus intermedius

1983 ◽  
Vol 61 (1) ◽  
pp. 54-62 ◽  
Author(s):  
Kiyoshi Takeuchi
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Protein Band ◽  
Divalent Ions ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Single Protein

Exo-β-1,3-glucanase from the sea urchin embryos was purified 114-fold from the initial hatching supernatant by the following procedures: (a) gel filtration on Sephadex G-100; (b) hydrophobic chromatography on 4-phenylbutylamine-Sepharose (PBA-Sepharose); (c) two ion-exchange chromatographic steps on DEAE-cellulose; (d) gel filtration on Ultrogel AcA 34; (e) gel filtration on Sephadex G-100. The purified enzyme contained 2.2% carbohydrate and gave a single protein band corresponding to a molecular weight of 136 000 following electrophoresis on sodium dodecyl sulfate (SDS) – urea – polyacrylamide gel. Gel filtration on Ultrogel AcA 34 with a nondenaturing solvent gave a molecular weight of 130 000 ± 6000. The enzyme displayed an optimum pH at 5.0–5.5 and hydrolysed laminarin and PS(curdlan)-beads at the nonreducing ends, releasing glucose. Although activity of the purified enzyme was not affected by SDS, urea, some divalent ions, and 2-mercaptoethanol, both dithiothreitol and Hg2+ were markedly inhibitory.

scholarly journals Extraction, precipitation and characterization of urease from Vicia faba L.

2020 ◽  
Vol 31 (1) ◽  
pp. 9
Author(s):  
Dalal Subree Bedan
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Vicia Faba ◽  
Urease Activity ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Mineral Salts ◽  
Urease Enzyme ◽  
Vicia Faba L ◽  
Optimum Ph

This study aimed to extract urease enzyme from available plant source which was broad beans (Vicia faba L.) using aqueous solution in a ratio1:3(w:v) .The crude extract appeared enzyme activity 33.3 U/ml. Results of this study revealed the possibility to precipitate urease enzyme after extraction using different precipitation methods consisted of Acetone precipitation; Alcohol precipitation and Ammonium sulfate precipitation, these ways of urease precipitation gave enzyme activities 74.8; 43.6 and 82.2 U/ml respectively .The enzyme precipitation using 60% saturation ratio of Ammonium sulfate gave the maximum activity of urease precipitation among other precipitation methods. Enzyme characterization appeared the optimum pH of urease activity was 8.0 and gave enzyme activity 93.8U/ml, while the optimum pH of urease stability was 6.0; the enzyme maintains its activity. On the other side the optimum temperature of precipitated urease activity was 50°C and the enzyme activity reached 99.3U/ml while the optimal temperature of urease stability was 40°C ,the precipitated urease maintain 100% of activity. The effect of other factors on urease action were studied also, these are consisted of the storage time of enzyme ,urease maintain its activity for 8days and the activity begun to decline after that time; the effect of time reusability was also revealed that the enzyme could be used for six times and maintain its activity. The influence of different mineral salts on precipitated urease were also recorded ;these salts were CuSO4 , MgSO4, MgCl2,KCl,CaCl2and FeSo4, results appeared that magnesium and calcium salts were activator for the precipitated urease while copper ,potassium and ferrous salts were inhibitor of the precipitated urease. Urea concentration as an enzyme substrate were also examined and results appeared the optimal substrate concentration was 30mM; enzyme activity reached to125.2U/ml.

Regulation and Characterization of L-Serine: Pyruvate Aminotransferase in Rat Liver Cytosol and Mitochondria

1977 ◽  
Vol 32 (3-4) ◽  
pp. 249-253 ◽  
Author(s):  
Jiro Hoshino ◽  
Uta Kühne ◽  
Branka Filjak ◽  
Hans Kröger
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
High Protein Diet ◽  
Mitochondrial Fraction ◽  
Protein Diet ◽  
Low Protein ◽  
Rat Liver Cytosol

Abstract Distribution of rat liver serine: pyruvate aminotransferase between cytosol and mitochondria varies considerably with the dietary and hormonal state of animals. Feeding a high-protein diet or fasting the animals results in an increase in the enzyme activity of both fractions but more marked in the mitochondrial fraction. A low-protein diet exerts the reverse effect. A single administration of dibutyryl cyclic AMP causes a rapid elevation of the enzyme activity in both fractions, which is effectively prevented by cycloheximide, actinomycin D and cortisone. The activity in mitochondria increases with a lag of 2 h following injection of the nucleotide inducer, in contrast to the cytosol enzyme, which increases without any lag. Gel filtration and DEAE cellulose chromatography of the enzyme from both fractions revealed the similar pattern and some kinetic constants of these two types of the enzyme were not significantly different from each other. These results indicate that rat liver serine: pyruvate aminotransferase is synthesized in the extra-mitochondrial site and transfered to mitochondria.

scholarly journals Purification, Characterization of L-Methioninase from Candida tropicalis, and Its Application as an Anticancer

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Mohsen Helmy Selim ◽  
El-Zahraa Karm Eldin ◽  
Moataza Mahmoud Saad ◽  
El-Sayed Eliwa Mostafa ◽  
Yosrea Hassan Shetia ◽  
...  
Keyword(s):  
Candida Tropicalis ◽  
Anticancer Agent ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Tumor Cell Lines ◽  
Sds Page ◽  
Optimum Ph ◽  
Ph Range

The aim of the present study is to purify L-methioninase from Candida tropicalis 34.19-fold with 27.98% recovery after ion exchange chromatography followed by gel filtration. The purified enzyme revealed a single band on SDS-PAGE gel with a molecular weight of 46 KDa. Its optimum temperature was 45 to 55 and thermal stability was 55°C for 15 min. The enzyme had optimum pH at 6.5 and stability at a pH range of 5.5 to 7.0 for 24 hr. The maximum activity was observed with substrate concentration of 30 µM and Km was 0.5 mM. The enzyme was strongly inhibited by Cd+2 and Cu+2 while it was enhanced by Na+, Ni+2, and Mg+2 at 10 mM while Ca+2 had slight activation at 20 mM. In addition, the potential application of the L-methioninase as an anticancer agent against various types of tumor cell lines is discussed.

scholarly journals Purification and characterization of a novel laccase from the mushroom Pleurotus nebrodensis.

2012 ◽  
Vol 59 (3) ◽  
Author(s):  
Guo-Ting Tian ◽  
Guo-Qing Zhang ◽  
He-Xiang Wang ◽  
Tzi Bun Ng
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Slight Reduction ◽  
Pleurotus Nebrodensis ◽  
Precipitous Decline ◽  
Ph Range ◽  
Purification Protocol

A novel laccase with a molecular mass of 64 kDa and the N-terminal sequence AIGPDDTINF was isolated from fresh fruiting bodies of the mushroom Pleurotus nebrodensis. The purification protocol comprised ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and gel filtration on Superdex 75. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose, but not on CM-cellulose. It demonstrated an optimal temperature of 70°C. The enzyme activity increased steadily over the temperature range 20°C-70°C. There was only a slight reduction in activity at 80°C. However, all activity disappeared following exposure to 100°C for 10 minutes. The enzyme activity changed only slightly over the pH range 3-5, with the optimum at pH 5, but underwent a precipitous decline when the pH was elevated to 6, and was undetectable at pH 8 and pH 9.

scholarly journals Purification and Characterization of Amyloglucosidase Enzyme from the Thermophilic Endomycopsis fibuligera Using Sago Starch as a Substrate

2018 ◽  
Vol 16 (3) ◽  
pp. 302
Author(s):  
Ahyar Ahmad ◽  
Harningsih Karim
Keyword(s):  
Ammonium Sulphate ◽  
Deae Cellulose ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Degree Of Saturation ◽  
Sago Starch ◽  
Purification And Characterization ◽  
Ammonium Sulphate Fractionation ◽  
Crude Extract Enzyme

An investigation on purification and characterization of amyloglucosidase enzyme from Endomycopsis fibuligera by fermentation of sago starch has been carried out. This enzyme is inductive and can be produced by fermenting sago starch in a medium containing E. fibuligera. Crude enzyme was obtained by centrifuging the medium cultures containing E. fibuligera at 6,000 rpm for 20 min and then adding with 0.15 M acetate buffer (pH 5.0). Enzyme activity was determined using Somogyi-Nelson method by quantifying the released glucose from amyloglucosidase catalysis of starch (0.2%) as substrate. Prepurification process was conducted by ammonium sulphate fractionation and it showed that the ammonium sulphate fractionation with the degree of saturation of 40-60% produced a maximum activity of enzyme. Purification by DEAE-Cellulose and Sephadex G-75 column chromatography produced three and one fractions with purifity 17.4 and 22.5 times, respectively, compared to the crude extract enzyme. Characterization of this enzyme showed the optimum condition at pH 5.0 and 55 °C with 0.2% starch as substrate. The amyloglucosidase activities was strongly increased by addition of Co2+ and Mn2+ ions, whereas the activities were weakly decreased by addition of K+, Mg2+, and Fe3+ ions.

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