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 PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

2017 ◽  
Vol 18 (2) ◽  
pp. 1-10 ◽  
Author(s):  
Dzun Noraini Jimat ◽  
Intan Baizura Firda Mohamed ◽  
Azlin Suhaida Azmi ◽  
Parveen Jamal
Keyword(s):  
Specific Activity ◽  
Hot Spring ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Bacillus Sp ◽  
Sds Page ◽  
Fast Flow ◽  
Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60°C.

scholarly journals Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants (Camellia sinensis (L.) O. Kuntze)

2005 ◽  
Vol 37 (6) ◽  
pp. 363-370 ◽  
Author(s):  
Ye-Yun Li ◽  
Chang-Jun Jiang ◽  
Xiao-Chun Wan ◽  
Zheng-Zhu Zhang ◽  
Da-Xiang Li
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Optimum Activity ◽  
Development Of Resistance ◽  
Sds Page ◽  
C 50 ◽  
Tea Plants

Abstractβ-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity, with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50 °C and was stable at temperatures lower than 40 °C. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.

scholarly journals Purification and characterization of cephalosporin 7α-hydroxylase from Streptomyces clavuligerus

1991 ◽  
Vol 280 (2) ◽  
pp. 471-474 ◽  
Author(s):  
X Xiao ◽  
S Wolfe ◽  
A L Demain
Keyword(s):  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
Exchange Chromatography ◽  
Cephalosporin C ◽  
Hydroxylase Activity ◽  
Purification And Characterization ◽  
Major Band ◽  
Sds Page ◽  
Optimum Ph

Cephalosporin 7 alpha-hydroxylase, which catalyses the conversion of cephalosporins into their 7 alpha-hydroxy derivatives, was purified nearly 390-fold from Streptomyces clavuligerus through ion-exchange chromatography, (NH4)2SO4 fractionation, gel filtration and dye chromatography, with the use of h.p.l.c. to monitor enzyme activity. The nearly pure enzyme migrates as a single major band, with an Mr of 32,000 in SDS/PAGE. Its optimum pH is in the range 7.3-7.7. Under our conditions the reaction was fastest at temperatures in the range 20-30 degrees C. The Km for cephalosporin C is 0.72 mM, and the Vmax. is 15.4 mumol of cephalosporin C hydroxylated/min per mg. Cephalosporin 7 alpha-hydroxylase did not show any deacetoxycephalosporin C synthase or deacetoxycephalosporin C hydroxylase activity.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

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 Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

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 Characterization of a Robust and pH-Stable Tannase from Mangrove-Derived Yeast Rhodosporidium diobovatum Q95

Marine Drugs ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. 546
Author(s):  
Jie Pan ◽  
Ni-Na Wang ◽  
Xue-Jing Yin ◽  
Xiao-Ling Liang ◽  
Zhi-Peng Wang
Keyword(s):  
Gallic Acid ◽  
Tannic Acid ◽  
Specific Activity ◽  
Maximum Activity ◽  
Sds Page ◽  
Rhodosporidium Diobovatum ◽  
Bacteria And Fungi ◽  
Ph Range ◽  
First Time

Tannase plays a crucial role in many fields, such as the pharmaceutical industry, beverage processing, and brewing. Although many tannases derived from bacteria and fungi have been thoroughly studied, those with good pH stabilities are still less reported. In this work, a mangrove-derived yeast strain Rhodosporidium diobovatum Q95, capable of efficiently degrading tannin, was screened to induce tannase, which exhibited an activity of up to 26.4 U/mL after 48 h cultivation in the presence of 15 g/L tannic acid. The tannase coding gene TANRD was cloned and expressed in Yarrowia lipolytica. The activity of recombinant tannase (named TanRd) was as high as 27.3 U/mL. TanRd was purified by chromatography and analysed by SDS-PAGE, showing a molecular weight of 75.1 kDa. The specific activity of TanRd towards tannic acid was 676.4 U/mg. Its highest activity was obtained at 40 °C, with more than 70% of the activity observed at 25–60 °C. Furthermore, it possessed at least 60% of the activity in a broad pH range of 2.5–6.5. Notably, TanRd was excellently stable at a pH range from 3.0 to 8.0; over 65% of its maximum activity remained after incubation. Besides, the broad substrate specificity of TanRd to esters of gallic acid has attracted wide attention. In view of the above, tannase resources were developed from mangrove-derived yeasts for the first time in this study. This tannase can become a promising material in tannin biodegradation and gallic acid production.

Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation

1994 ◽  
Vol 40 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Andreas Prokop ◽  
Peter Rapp ◽  
Fritz Wagner
Keyword(s):  
Molecular Mass ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glucanase Activity ◽  
Sds Page ◽  
S 100 ◽  
Reduced Activity

Production of extracellular β-1, 3-glucanase activity by a monokaryotic Schizophyllum commune strain was monitored and results indicated that the β-glucanase activity consisted of an endo- β-1, 3-glucanase activity, besides a negligible amount of β-1, 6-glucanase and β-glucosidase activity. Unlike the β-1, 3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the β-1, 3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo- β-1, 3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing β-1, 3-linkages, including lichenan, a β-1, 3-1, 4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular β-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo- β-1, 3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass β-1, 3-/β-1, 6-glucan. The Km of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50 °C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50 °C. The enzyme was completely inhibited by 1 mM Hg2+. Growth of the monokaryotic S. commune strain was not affected by its constitutive endo- β-1, 3-glucanase formation.Key words: endo- β-1, 3-glucanase, Schizophyllum commune, monokaryon, constitutive endo- β-1, 3-glucanase formation.

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.

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