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 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.

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).

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+.

scholarly journals Streptomyces lavendulaeProtease Inhibitor: Purification, Gene Overexpression, and 3-Dimensional Structure

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
D. E. El-Hadedy ◽  
El-Sayed E. Mostafa ◽  
Moataza M. Saad
Keyword(s):  
Protease Inhibitors ◽  
Gel Filtration ◽  
Dimensional Structure ◽  
Fulminant Myocarditis ◽  
Ammonium Sulfate Precipitation ◽  
3 Dimensional ◽  
Pcr Product ◽  
Sds Page ◽  
Cvb3 Infection ◽  
Causative Agents

Protease inhibitorstrypsin (STI1, Streptomyces trypsin inhibitor 1) has been identified, purified by ammonium sulfate precipitation and Sephadex G-100 gel filtration. SDS-PAGE of protease inhibitor showed molecular weight of approximately 10 KDa. PCR product (~1615 bp) ofsti1gene was cloned in expression vectorpACYC177/ET3dand transformed inEscherichia coliJM109.Protease inhibitorstrypsin was purified and used as antivirus against Coxsackievirus B3 (CVB3). CVB3 is one of the major causative agents of chronic, subacute, acute, and fulminant myocarditis as well as pancreatitis and aseptic meningitis. It has been reported that more than 50% of human myocarditis is associated with CVB3 infection.

Protease inhibitors of Acacia leucophloea gum extracts

2012 ◽  
Vol 1 (10) ◽  
pp. 91
Author(s):  
Balaji M Panchal* ◽  
Manvendra S Kachole
Keyword(s):  
Protease Inhibitors ◽  
Plant Pathogens ◽  
Mangifera Indica ◽  
Gel Filtration ◽  
Dot Blot ◽  
Plant Protease ◽  
Dot Blot Assay ◽  
Thermo Stability ◽  
Acacia Leucophloea ◽  
Chymotrypsin Inhibitors

Plant protease inhibitors (PIs) are very important for their defensive function against plant pathogens and predators. In present work trypsin and chymotrypsin inhibitors (PIs) from gum of one tree about seven species, Azadirachta indica, Acacia leucophloea, Acacia nilotica, Terminalia spp., Anogeissus latifolia, Mangifera indica and Moringa oleofera are studied. Protease inhibitory activity in gum extract was detected by dot blot assay. PI bands were      resolved on electrophoresis gel and detected by Gel X-ray film contact print technique (GXCP). PIs from gum extracts were purified by gel filtration (Sephadex G-75).  Among all   gum extracts studied, the gum extract from Acacia leucophloea showed highest number of trypsin and chymotrypsin inhibitors. One PI from the Acacia leucophloea of 97.00kDa was purified and characterized.  Purified PI was not destroyed by heat treatments up to 70ºC, but lost its activity when incubated at 80°C, showing moderate thermo stability.

Purification and Characterization of Superoxide Dismutase from Martianus Dermestoides Chevrola

2013 ◽  
Vol 773 ◽  
pp. 336-341 ◽  
Author(s):  
Wen Ge Yu ◽  
Bei Bei Zhang ◽  
Yong Jian Shen ◽  
Ying Li ◽  
Yong Bin Tian ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Ph Value ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Exchange Chromatography ◽  
Enzyme Activation ◽  
Purification And Characterization ◽  
Sds Page ◽  
Optimum Reaction

In this paper, the superoxide dismutase fromMartianus dermestoidesis purified by the following methods: heat treatment, polyethylene concentration, Sephadex G-75 gel filtration, and DEAE-Sepharose FF ion exchange chromatography. The result shows that the purification multiple is 3.86, the activation yield is 21.89% and the specific activation of the enzyme is 447.6 U/mg. The purified SOD appears to be a sole protein on SDS-PAGE and the molecular weight is estimated to be 40.58 kDa. H2O2can obviously inhibit the enzyme activation and CHCl3-CH3CH2OH only demonstrates basically no inhibitory effect. The type of the dermestoides SOD might be Cu/Zn-SOD. After purification, some enzymatic characterizations of the SOD are studied. The optimum reaction temperature of purified SOD is 50°C. The optimum reaction pH value of purification is 6. The dermestoide SOD has a preferable stability below 50°C and at pH values between 5-8.

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 Activation of phospholipase PLA2 actvity in Ricinus communis leaves in response to mechanical wounding

2007 ◽  
Vol 19 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Sarah J.S. Domingues ◽  
Thiago F. de Souza ◽  
Alexandra M.S. Soares ◽  
Tânia Jacinto ◽  
Olga L.T. Machado
Keyword(s):  
Time Course ◽  
Ricinus Communis ◽  
Gel Filtration ◽  
High Sensitivity ◽  
Pla2 Activity ◽  
Mechanical Wounding ◽  
Protein Activity ◽  
Leaf Extracts ◽  
Native Page ◽  
Sds Page

In order to investigate the defense response in castor bean (Ricinus communis) against predators, we analyzed the effect of mechanical wounding upon the phospholipase A2 (PLA2) activity of leaf extracts. Time course experiments revealed that the highest levels of increased PLA2 activity (ca. two fold) occurred 15 min and 60 min after injury. The induced activities demonstrated high sensitivity towards aristolochic acid (10 mM), a PLA2 inhibitor. Based on SDS-PAGE analysis, the PLA2 activity induced 15 min after wounding migrated with a molecular mass of 40 kDa and was denoted RcPLA2 I. The protein activity induced 60 min after wounding, RcPLA2 II, migrated with a molecular weight of 14 kDa. Furthermore its N-terminal sequence shared homology with PLA2 from elm and rice. The PLA2 enzymes were purified to near homogeneity by a combination of gel filtration and electro-elution of protein bands after native PAGE.

scholarly journals Production, Purification, and Characterization of Polygalacturonase from Mucor circinelloides ITCC 6025

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Akhilesh Thakur ◽  
Roma Pahwa ◽  
Smarika Singh ◽  
Reena Gupta
Keyword(s):  
Gel Filtration ◽  
Casein Hydrolysate ◽  
Inhibitory Effect ◽  
Mucor Circinelloides ◽  
Maximum Activity ◽  
Purification And Characterization ◽  
Production Parameters ◽  
Sds Page ◽  
S 100

Mucor circinelloides produced an extracellular polygalacturonase enzyme, the production of which was enhanced when various production parameters were optimized. Maximum polygalacturonase (PGase) activity was obtained in 48 h at 30∘C and pH 4.0 with pectin methyl ester (1% w/v) as carbon source and a combination of casein hydrolysate (0.1% w/v) and yeast extract (0.1% w/v) as nitrogen source. The enzyme was purified to homogeneity (13.3-fold) by Sephacryl S-100 gel-filtration chromatography. Its molecular weight was 66 kDa on SDS-PAGE. The enzyme was found to have Km and Vmax values of 2.2 mM and 4.81 IU/ml at 0.1% to 0.5% (w/v) concentration of the substrate. The addition of phenolic acids (0.05 mM), metal ions such as Mn+2, Co+2, Mg+2, Fe+3, Al+3, Hg+2, and Cu+2, and thiols had inhibitory effect on the enzyme. The enzyme showed maximum activity in the presence of polygalacturonic acid (0.1% w/v) at pH 5.5 and 42∘C.

scholarly journals Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Camel Liver

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Mahmoud A. Ibrahim ◽  
Abdel-Hady M. Ghazy ◽  
Ahmed M. H. Salem ◽  
Mohamed A. Ghazy ◽  
Mohamed M. Abdel-Monsef
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Native Form ◽  
Native Page ◽  
Sds Page ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Sepharose 4B

Glucose-6-phosphate dehydrogenase from camel liver was purified to homogeneity by ammonium sulfate precipitation and a combination of DEAE-cellulose, Sephacryl S-300 gel filtration, and 2′, 5′ ADP Sepharose 4B affinity chromatography columns. The specific activity of camel liver G6PD is increased to 1.80438 units/mg proteins with 63-fold purification. It turned out to be homogenous on both native PAGE and 12% SDS PAGE, with a molecular weight of 64 kDa. The molecular weight of the native form of camel liver G6PD was determined to be 194 kDa by gel filtration indicating a trimeric protein. The Km value was found to be 0.081 mM of NADP+. Camel liver G6PD displayed its optimum activity at pH 7.8 with an isoelectric point (pI) of pH 6.6–6.8. The divalent cations MgCl2, MnCl2, and CoCl2 act as activators; on the other hand, CaCl2 and NiCl2 act as moderate inhibitors, while FeCl2, CuCl2, and ZnCl2 are potent inhibitors of camel liver G6PD activity. NADPH inhibited camel liver G6PD competitively with Ki value of 0.035 mM. One binding site was deduced for NADPH on the enzyme molecule. This study presents a simple and reproducible purification procedure of G6PD from the camel liver.

Sign in / Sign up

Export Citation Format

Share Document