scholarly journals Immunological Characterization of Acid Phosphatase in an Endophytic Fungus

2007 ◽  
Vol 7 ◽  
pp. 77 ◽  
Author(s):  
Rajani Malla ◽  
Md Zeyaullah ◽  
Utprekshya Pokharel ◽  
Ajit Varma
Keyword(s):  
Acid Phosphatase ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Phosphate Metabolism ◽  
Immunoblotting Analysis ◽  
Native Page ◽  
Selective Precipitation ◽  
Denatured Protein ◽  
Sds Page

Piriformospora indica produced only one form of intracellular acid phosphatase irrespective of the phosphate concentration and was purified. The enzyme was possibly a constitutive enzyme showing molecular mass of 66kDa as separated by SDS PAGE. Antibodies raised against cytosolic acid phosphatase of P. indica using gel band in native PAGE after selective precipitation of ammonium sulfate followed by gel filtration and ion exchange chromatography, gave productive antibody and immunoblotting analysis. Its reaction with native protein as well as denatured protein was significant. The antibody immunoprecipitated a single band of approximately 66kDa protein in SDS gel. The antibody localized the enzyme on the polyphosphate granules, cell-wall, vacuoles and cytoplasm of the mycelium indicating the possible sites of phosphate metabolism. <i> Nepal Journal of Science and Technology</i> Vol. 7, 2006

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 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.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

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.

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.

Purification and characterization of an acid phosphatase from peanut (Arachis hypogaea) seed

1984 ◽  
Vol 62 (2) ◽  
pp. 385-391 ◽  
Author(s):  
Sheikh Mehboob Basha
Keyword(s):  
Molecular Weight ◽  
Acid Phosphatase ◽  
Arachis Hypogaea ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Arachis Hypogaea L ◽  
Nitrophenyl Phosphate ◽  
Phosphorylated Sugars

An acid phosphatase (EC 3.1.3.2) from peanut (Arachis hypogaea L.) seed has been purified 433-fold by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. The purified preparation was found to be homogeneous by electrophoresis and gel filtration. The molecular weight of the enzyme was estimated to be approximately 240 000 and it was found to be composed of six identical subunits, each with an apparent molecular weight of 42 500. Following isoelectric focusing, the isolectric point (pI) of the enzyme was found to be around pH 5.6. The apparent Km value with p-nitrophenyl phosphate as substrate was 2 ? 10−1 μM. The enzyme was inhibited by Hg2+, Fe2+, Cu2+, Zn2+, Pb2+, and F−. Higher concentrations (2–50 μM) and long incubation periods (60–90 min) with Ca2+ and Mg2+ ions were shown to activate the enzyme. This enzyme showed no effect toward phosphorylated sugars but appear to hydrolyze ATP, ADP, AMP, and β-glycerophosphate.

scholarly journals Characterization of the antibodies detected by the microscopic agglutination test for bovine leptospirosis

1974 ◽  
Vol 73 (3) ◽  
pp. 425-432 ◽  
Author(s):  
J. A. Morris ◽  
S. N. Hussaini
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Agglutination Test ◽  
Microscopic Agglutination Test ◽  
Density Gradient Ultracentrifugation ◽  
Selective Precipitation ◽  
Bovine Leptospirosis

SummaryThe nature of the antibodies detectable by the microscopic agglutination test for bovine leptospirosis was examined. Density gradient ultracentrifugation, gel filtration and disulphide-bond-reduction experiments indicated that antileptospiral agglutinating activity was present in both IgM and IgG immunoglobulin fractions. This was confirmed by selective precipitation of specific antibody classes and ion-exchange chromatography.

scholarly journals Pemurnian Parsial dan Karakterisasi Enzim Xilanase dari Bakteri Laut Bacillus safencis strain LBF P20 Asal Pulau Pari Jakarta

2017 ◽  
Vol 37 (1) ◽  
pp. 31
Author(s):  
Fitria Fitria ◽  
Nanik Rahmani ◽  
Sri Pujiyanto ◽  
Budi Raharjo ◽  
Yopi Yopi
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Centrifugal Filter ◽  
Sds Page ◽  
Enzyme Specific Activity ◽  
Hydrolysis Of

Enzyme xylanase (EC 3.2.1.8) is widely used in various industrial  fields for the hydrolysis of xylan (hemicellulose) into xylooligosaccharide and xylose. The aims of this study were to  conduct partial purification and characterization of xylanase from marine Bacillus safencis strain LBF P20 and to obtain the  xylooligosaccharide types from xylan hydrolysis by this enzyme.  Based on this research, the optimum time for enzyme production  occurred at 96 hours with the enzyme activity of 6.275 U/mL and  enzyme specific activity of 5.093 U/mg. The specific activities were  obtained from precipitation by amicon® ultra-15 centrifugal filter devices, gel filtration chromatography and anion exchange chromatography that were increased by 15.07, 34.7, and 96.0  U/mg. The results showed that the highest activity at pH 7, temperature of 60 °C, and stable at 4 °C. Type of  xylooligosaccharide produced by this study were xylohexoses, xylotriose, and xylobiose. SDS-PAGE analysis and zimogram  showed that the molecular weight of xylanase protein were about  25 kDa. ABSTRAKEnzim xilanase (EC 3.2.1.8) digunakan dalam hidrolisis xilan  (hemiselulosa) menjadi xilooligosakarida dan xilosa. Penelitian  ini bertujuan untuk melakukan purifikasi parsial dan karakterisasi xilanase dari bakteri laut Bacillus safencis strain LBF P20 serta uji  hidrolisis untuk mengetahui jenis xilooligosakarida yang  dihasilkan oleh enzim tersebut. Berdasarkan hasil penelitian, waktu optimum untuk produksi enzim terjadi pada jam ke 96  dengan aktivitas enzim sebesar 6,275 U/mL dan aktivitas spesifik enzim sebesar 5,093 (U/mg). Aktivitas spesifik enzim hasil  pemekatan dengan amicon® ultra-15 centrifugal filter devices,  kromatografi filtrasi gel dan kromatografi penukar anion  mengalami peningkatan berturut-turut sebesar 15,1; 34,7 dan96,0 U/mg. Hasil karakterisasi menunjukkan aktivitas  tertinggi pada pH 7, suhu 60 °C dan stabil pada suhu 4 °C. Analisis SDS-PAGE dan zimogram menunjukkan berat molekul protein xilanase berkisar 25 kDa. Jenis gula reduksi yang  dihasilkan yaitu xiloheksosa, xilotriosa, dan xilobiosa.

scholarly journals Purification and characterization of an endoxylanase from Trichoderma koningii G-39

1991 ◽  
Vol 278 (2) ◽  
pp. 329-333 ◽  
Author(s):  
L Huang ◽  
T H Hseu ◽  
T T Wey
Keyword(s):  
Specific Activity ◽  
Xylanase Activity ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Crystalline Cellulose ◽  
Trichoderma Koningii ◽  
Sds Page ◽  
Immunological Relationship ◽  
Oat Spelt

Trichoderma koningii G-39 produced xylanases in submerged culture using oat spelt xylan or crystalline cellulose, Avicel, as the sole carbon source. A low-Mr xylanase was purified from the culture filtrate by ion-exchange chromatography on SP-Trisacryl-M and gel filtration on Fractogel TSK HW-50F. It was homogeneous on SDS/PAGE and isoelectric focusing. A typical procedure provided about 11-fold purification with 4.5% protein yield and 50% activity recovery. The purified enzyme has an Mr value of about 21,500 and a pI of 8.9. Its specific activity was 6100 units/mg of protein, with optimal activity towards 0.5% xylan at about pH 5.5 and 60 degrees C. The purified enzyme had no activity against CM-cellulose with a degree of substitution of 0.63. It also showed no beta-xylosidase activity. The Km and Vmax. values, as determined with the soluble fraction of oat spelt xylan as substrate, were 0.70 mg/ml and 1.85 x 10(6) mumol/min per mg of enzyme respectively. Hg2+ (1 mM) and SDS (10 mM) completely inhibited xylanase activity, whereas Ca2+ showed no significant effect on the enzyme activity at 1 mM, but gave 80% inhibition at 10 mM. The enzyme contained about 4.4% carbohydrate and showed an immunological relationship to a cellobiohydrolase from the same fungal strain.

scholarly journals A Boophilus microplus vitellin-degrading cysteine endopeptidase

Parasitology ◽  
2003 ◽  
Vol 126 (2) ◽  
pp. 155-163 ◽  
Author(s):  
A. SEIXAS ◽  
P. C. DOS SANTOS ◽  
F. F. VELLOSO ◽  
I. DA SILVA VAZ ◽  
A. MASUDA ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Boophilus Microplus ◽  
Hard Tick ◽  
Purification And Characterization ◽  
Basic Residue ◽  
Cysteine Endopeptidase ◽  
Sds Page

Here we describe the purification and characterization of a vitellin (VT) degrading cysteine endopeptidase (VTDCE) from eggs of the hard tick Boophilus microplus. A homogeneous enzyme preparation was obtained by chromatographic fractionation on ion-exchange and gel filtration columns and an autolysis step. This step consisted of incubation of a semipurified enzyme (after the first ion-exchange chromatography) at pH 4·0 that dissociated the enzyme from VT, to which VTDCE is naturally tightly associated. The enzyme purity was confirmed by capillary and native gel electrophoresis, and SDS–PAGE suggested the enzyme is a dimer of 17 and 22 kDa. VTDCE was active upon several synthetic substrates, with a preference for a hydrophobic or a basic residue in P1, and a hydrophobic residue in P2. VTDCE also hydrolysed haemoglobin, albumin, gelatin and vitellin. VTDCE is inactive in the absence of DTT and was totally inhibited by E-64, indicating it is a cysteine endopeptidase. Our results suggest that VTDCE is a major enzyme involved in yolk processing during B. microplus embryogenesis.

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