Purification of the NADP+: F420oxidoreductase ofMethanosphaera stadtmanae

2000 ◽  
Vol 46 (11) ◽  
pp. 998-1003 ◽  
Author(s):  
Dwayne A Elias ◽  
David F Juck ◽  
Karin A Berry ◽  
Richard Sparling
Keyword(s):  
Gel Filtration ◽  
Total Activity ◽  
Purification Process ◽  
Oxidoreductase Activity ◽  
Native Page ◽  
Sds Page ◽  
Methanosphaera Stadtmanae ◽  
Low Levels ◽  
The Stability ◽  
Optimal Ph

Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420oxidoreductase activity. The NADP+:F420oxidoreductase, the enzyme which catalyses the electron transfer between NADP+and F420in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420reduction was 6.0, and 8.5 for NADP+reduction. During the purification process, it was noted that precipitation with (NH4)2SO4increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of α, β, and γ subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Kmvalues were 370 µM for NADP+, 142 µM for NADPH, 62.5 µM for F420, and 7.7 µM for F420H2. These values were different from the Kmvalues observed in the cell-free extract.Key words: methanogen, NADP:F420oxidoreductase, NADP reductase, F420, NADP+.

Production and purification of an extracellular β-galactosidase from the Dutch elm disease fungus Ophiostoma novo-ulmi

1997 ◽  
Vol 43 (11) ◽  
pp. 1011-1016 ◽  
Author(s):  
Thomas Binz ◽  
Colette Gremaud ◽  
Giorgio Canevascini
Keyword(s):  
Enzyme Activity ◽  
Culture Filtrate ◽  
Galacturonic Acid ◽  
Gel Filtration ◽  
Total Activity ◽  
Dutch Elm Disease ◽  
Optimal Temperature ◽  
Ophiostoma Ulmi ◽  
Sds Page ◽  
Optimal Ph

The causal agents of Dutch elm disease, Ophiostoma ulmi (isolate H200) and Ophiostoma novo-ulmi (isolate CKT-11), secreted similar amounts of β-galactosidase in liquid shake cultures when grown on galacturonic acid or sodium pectate (1.45 ± 0.16 and 1.03 ± 0.24 nkat∙mL−1 for O. ulmi, respectively, and 1.30 ± 0.08 and 1.28 ± 0.26 nkat∙mL−1 for O. novo-ulmi, respectively). Rhamnose and pectin also stimulated secretion but to a lesser extent, whereas on glucose, enzyme activity was barely detectable (≤0.01 nkat∙mL−1). Ophiostoma novo-ulmi was shown by Q-Sepharose chromatography to form two β-galactosidases, named β-galactosidases I and II. In cultures grown on galacturonic acid β-galactosidase I accounted for approximately 75% of the total activity in the culture filtrate. β-Galactosidase I was further purified to apparent electrophoretic homogeneity by means of Sephacryl gel filtration chromatography, chromatofocusing, and Superdex75 gel filtration. The molecular mass of the enzyme was 135 kDa by SDS–PAGE and 123 kDa by gel filtration. Its isoelectric point, determined by chromatofocusing, was 4.9. The optimal pH for enzyme activity was 5.8 and the optimal temperature was 50 °C. The Km values for p-nitrophenyl β-D-galactopyranoside and lactose were 7.52 and 14.23 mM, respectively, and the maximum velocities for these substrates were 1733 and 355 nkat∙mg protein−1, respectively. The Ki value for D(−)-galactonic acid γ-lactone was 2.29 mM.Key words: Dutch elm disease, β-galactosidase, Ophiostoma ulmi, Ophiostoma novo-ulmi.

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 Laundry Detergent Compatibility of Papain Like Protease Purified From Piper Betel Leaves

2018 ◽  
Vol 7 (3.3) ◽  
pp. 132
Author(s):  
A Mousami Shankar ◽  
Dr G.V.D. Sirisha ◽  
Dr K. Vijaya Rachel
Keyword(s):  
Laundry Detergent ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Native Page ◽  
Chromatographic Techniques ◽  
Detergent Compatibility ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
The Stability ◽  
Better Than

Enzymes have wide applications in detergent industry from early 1900’s. Mostly, clothes are soiled by protein based grime. Most of the detergents have either amylase / protease. Various sources were scrutinized for potent protease activity and Betel leaves were selected, the enzyme was then isolated, purified to homogeneity by ammonium sulphate precipitation, DEAE-Cellulose and gel permeation chromatographic techniques. The enzyme was monomeric in nature with a molecular mass of 38kDa as determined by native PAGE and SDS-PAGE. The enzyme shows maximum activity at 60oC and pH 4.0. The Km and Vmax of the enzyme were 4x10-3M and 54µmol/min/mg respectively. The enzyme was categorically inhibited by PCMB and iodo-acetamide suggesting it to have papain like nature. The stability of the enzyme is assessed over the stretch of alkaline pH and temperature. This evaluation validates the stability of the enzyme and its use in detergent formulations. It was evident that after adding the enzyme preparation the stains (tea, chocolate, blood) were removed much better than that of the controls, which affirms that papain like enzyme from betel leaves, enhances detergent activity.  

Purification of sorbitol dehydrogenase from diapause eggs of Dysdercus cingulatus (fabricus 1775)

2017 ◽  
Vol 51 (03) ◽  
Author(s):  
A. S. Shahjahan ◽  
Enciy R. Martin
Keyword(s):  
Low Temperature ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Total Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sorbitol Dehydrogenase ◽  
Purification Technique ◽  
Dysdercus Cingulatus ◽  
Sds Page

Sorbitol Dehydrogenase (SDH) is important enzymes responsible for the protection of eggs from harsh condition as it convert fructose to sorbitol which serves as polyol and stabilize the soluble proteins and lipid bilayer. The enzyme SDH (d-Idiol NAD oxidoreductase EC 1.1.1.14) is purified from the eggs of red cotton bug, Dysdercus cingulatus exposed to low temperature. The SDH from eggs were purified by using different protein purification technique viz, ion exchange chromatography, gel filtration and SDS–PAGE. The ion exchange chromatography showed 0.185U total activity, with a purification fold of 1.48 and yield 3.36%. Gel filtration chromatography showed an increase in purification of SDH activity by 2.73 with 0.095 U total activity and yield of 1.73%. SDS-PAGE revealed that SDH molecular weight of 38 KDa. The Km value of Fructose: NADH is 0.4:0.01 and the Vmax was 0.6 and 0.03. Thus low temperature increases the activity of SDH which convert fructose to sorbitol which protects the cell during diapause condition.

Characterization of the Esterases of Feline Serum

1974 ◽  
Vol 52 (11) ◽  
pp. 1073-1078 ◽  
Author(s):  
D. J. Ecobichon
Keyword(s):  
Close Association ◽  
Gel Filtration ◽  
Spectrophotometric Analysis ◽  
Specific Substrate ◽  
Starch Gel Electrophoresis ◽  
Kinetic Characteristics ◽  
Starch Gel ◽  
Low Levels ◽  
Optimal Ph

Various techniques, including electrophoresis, gel filtration, titrimetric, and spectrophotometric analysis with specific and nonspecific substrates and inhibitors, were used to characterize the serum esterases of male and female cats. Starch-gel electrophoresis yielded five bands of activity: three butyrylcholinesterase bands and two bands of nonspecific carboxylesterase activity. Gel filtration on Sephadex G-200 yielded two distinct peaks of activity, one containing the cholinesterase bands and the other, the carboxylesterase activity. Kinetic characteristics determined for feline serum esterases included: (1) optimal pH; (2) optimal substrates for cholinesterase and carboxylesterase activities; (3) Km values for the choline, α-naphthyl, and p-nitrophenyl esters. With the organophosphate paraoxon as a specific substrate for arylesterase activity, low levels of this enzyme could be detected in close association with the carboxylesterase.

scholarly journals Protease Inhibitors Purified from the Canola Meal Extracts of Two Genetically Diverse Genotypes Exhibit Antidiabetic and Antihypertension Properties

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 2078
Author(s):  
Saira Hussain ◽  
Ata-ur Rehman ◽  
David J. Luckett ◽  
S.M. Saqlan Naqvi ◽  
Christopher L. Blanchard
Keyword(s):  
Protease Inhibitors ◽  
Serine Proteases ◽  
Plant Pathogens ◽  
Gel Filtration ◽  
Ace Inhibition ◽  
Inhibitory Effect ◽  
Canola Meal ◽  
Native Page ◽  
Dpp Iv ◽  
Sds Page

Valorization of vegetable oil waste residues is gaining importance due to their high protein and polyphenol contents. Protease inhibitors (PIs), proteins from these abundantly available waste residues, have recently gained importance in treating chronic diseases. This research aimed to use canola meal of genetically diverse Brassica napus genotypes, BLN-3347 and Rivette, to identify PIs with diverse functionalities in therapeutic and pharmacological applications. The canola meal PI purification steps involved: native PAGE and trypsin inhibition activity, followed by ammonium sulfate fractionation, anion exchange, gel filtration, and reverse-phase chromatography. The purified PI preparations were characterized using SDS-PAGE, isoelectric focusing (IEF), and N terminal sequencing. SDS-PAGE analysis of PI preparations under native reducing and nonreducing conditions revealed three polymorphic PIs in each genotype. The corresponding IEF of the genotype BLN-3347, exhibited three acidic isoforms with isoelectric points (pI) of 4.6, 4.0, and 3.9, while Rivette possessed three isoforms, exhibiting two basic forms of pI 8.65 and 9.9, and one acidic of pI 6.55. Purified PI preparations from both the genotypes displayed dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibition activities; the BLN-3347 PI preparation exhibited a strong inhibitory effect with lower IC50 values (DPP-IV 37.42 µg/mL; ACE 129 µg/mL) than that from Rivette (DPP-IV 67.97 µg/mL; ACE 376.2 µg/mL). In addition to potential human therapy, these highly polymorphic PIs, which can inhibit damaging serine proteases secreted by canola plant pathogens, have the potential to be used by canola plant breeders to seek qualitative trait locus (QTLs) linked to genes conferring resistance to canola diseases.

scholarly journals Production, purification and characterization of an exo-polygalacturonase from Penicillium janthinellum sw09

2016 ◽  
Vol 88 (suppl 1) ◽  
pp. 479-487 ◽  
Author(s):  
YUPING MA ◽  
SIWEN SUN ◽  
HUI HAO ◽  
CHUNPING XU
Keyword(s):  
Gel Filtration ◽  
Maximal Activity ◽  
Growth Conditions ◽  
Purification And Characterization ◽  
Penicillium Janthinellum ◽  
Soil Isolate ◽  
Sds Page ◽  
The Stability ◽  
Ph Range

ABSTRACT A soil isolate, Penicillium janthinellum sw09 has been found to produce significant amounts of an extracellular pectinase subsequently characterized as exo-polygalacturonase (exo-PG). By optimizing growth conditions, P. janthinellum sw09 produced high amount of exo-PG (16.54 units/mL). The crude enzyme was purified by gel filtration chromatography and two exo-PG activity peaks (designated as PGI and PGII) were revealed. On SDS-PAGE analysis, purified PGII using DEAE-Sepharose FF column, was found to be a single band with a molecular mass of 66.2 kDa. The purified PGII exhibited maximal activity at the temperature of 45 oC and pH 5.0. The stability profiles show that PGII is more stable in the pH range of 4.0-8.0 and below 60 oC. The Km and Vmax for the enzyme was 1.74 mg/mL and 18.08 μmol/ (mL•min), respectively. Due to this enzymatic characterization, this pectinase is an attractive candidate for applications in degradation of pectin.

scholarly journals A highly inducible β-galactosidase from Enterobacter sp.

2020 ◽  
Vol 85 (5) ◽  
pp. 609-622
Author(s):  
Bestoon Shaikhan ◽  
Kemal Güven ◽  
Fatma Bekler ◽  
Ömer Acer ◽  
Reyhan Güven
Keyword(s):  
Gel Filtration ◽  
Gel Permeation Chromatography ◽  
Inhibitory Effect ◽  
Native Page ◽  
Environment And Health ◽  
Low Concentrations ◽  
Sds Page ◽  
Gel Permeation ◽  
Optimum Ph ◽  
Strong Inhibitory Effect

Enterobacter sp. 3TP2A isolated from a petroleum station was found to produce a novel, highly inducible mesophilic intracellular ?-galactosidase in the presence of lactose up to 76.5 U mg-1. The enzyme was purified to 17.3- -fold after gel permeation chromatography with a yield of approximately 11 %. The optimum pH and temperature values of the purified enzyme were found to be 8.0?9.0 and 35 ?C, respectively. The molecular weight of the enzyme was approx. 60 kDa with a single band by both SDS-PAGE and native-PAGE, and estimated by gel filtration chromatography. The enzyme was inhibited by Zn2+ and EDTA, while Cu2+ had strong inhibitory effect even at low concentrations. Activation by Mg2+ and inhibition by EDTA show that the enzyme is metal- -dependent or a metalloenzyme. The enzyme was slightly activated by 2-mercaptoethanol, while slightly inhibited by iodoacetamide. On the other hand, PCMB inhibited the enzymatic activity to a great extent, whereas it was completely inhibited by N-ethylmaleimide. The Vmax and Km values were calculated as 0.701 ?mol min-1 and 0.104 mM, respectively. The results indicated that the ?-galactosidase Enterobacter sp. 3TP2A might well be a good candidate for use in biotechnology, particularly in the area of environment and health.

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

Purification of sorbitol dehydrogenase from diapause eggs of Dysdercus cingulatus (fabricus 1775)

2017 ◽  
Vol 51 (03) ◽  
Author(s):  
A. S. Shahjahan ◽  
Enciy R. Martin
Keyword(s):  
Low Temperature ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Total Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sorbitol Dehydrogenase ◽  
Purification Technique ◽  
Dysdercus Cingulatus ◽  
Sds Page

<span>Sorbitol Dehydrogenase (SDH) is important enzymes responsible for the protection of eggs from harsh condition as it convert fructose to sorbitol which serves as polyol and stabilize the soluble proteins and lipid bilayer. The enzyme SDH (d-Idiol NAD oxidoreductase EC 1.1.1.14) is purified from the eggs of red cotton bug, Dysdercus cingulatus exposed to low temperature. The SDH from eggs were purified by using different protein purification technique viz, ion exchange chromatography, gel filtration and SDS–PAGE. The ion exchange chromatography showed 0.185U total activity, with a purification fold of 1.48 and yield 3.36%. Gel filtration chromatography showed an increase in purification of SDH activity by 2.73 with 0.095 U total activity and yield of 1.73%.  SDS-PAGE revealed that SDH molecular weight of 38 KDa. The Km value of Fructose: NADH is 0.4:0.01 and the Vmax was 0.6 and 0.03. Thus low temperature increases the activity of SDH which convert fructose to sorbitol which protects the cell during diapause condition.</span>

Sign in / Sign up

Export Citation Format

Share Document