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 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 A study of production, purification and characterization of amylases from Escherichia coli for medical uses

2018 ◽  
Vol 9 (SPL1) ◽  
Author(s):  
Baydaa Abood Hassan
Keyword(s):  
Escherichia Coli ◽  
Incubation Temperature ◽  
High Titer ◽  
Synthetic Medium ◽  
Maximum Activity ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation ◽  
Enhanced Production ◽  
Sds Page

This study was conducted in the laboratories of Biology Department,facultyofScience,which deal with isolation,purification and characterization ofof amylase by Escherichia coli which carried out for enhanced production of amylase using starch (1%) asthe substrate of enzyme, the production was carried out by submerged fermentation, the best conditions were the isolated ofamylase in synthetic medium, it gave high titer of amylase activity, the ammonium sulfate as nitrogen source, incubation period 48 h, the starch as carbon source,, incubation temperature 30 °C and pH = 7, The enzyme was purified using ammonium sulphate precipitation(60%) anddialysis, the purified amylase had a maximum activity at pH =7,the amylase was stable with pH values ranging between (7 - 8) and in temperature 30 °C also amylase was stable in (30- 40 ) °C analyses of the amylase for molecular weight was carried out by SDS-PAGE electrophoresiswhich revealed 52 KDa.

Activation of Purified Prothrombin in Ammonium Sulfate Solutions: Purification of Autoprothrombin C

1966 ◽  
Vol 15 (01/02) ◽  
pp. 001-011 ◽  
Author(s):  
W. H Seegers ◽  
D. L Heene ◽  
Ewa Marciniak
Keyword(s):  
Ammonium Sulfate ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Sulfate Solution ◽  
Intermediate Stage ◽  
Freezing And Thawing ◽  
Bovine Plasma ◽  
The Stability ◽  
Autoprothrombin C ◽  
Better Than

SummaryFor the generation of autoprothrombin C activity from prothrombin preparations in concentrated ammonium sulfate solution the optimum conditions were found to be near 2 M at pH 7. In addition to thrombin and autoprothrombin C an inhibitor was obtained. Methods were developed to obtain the autoprothrombin C consistently with a specific activity of about 4,700 units per mg protein, and a yield of one mg per liter of bovine plasma. At an intermediate stage of purification the stability of autoprothrombin C was far better than in the final product which lost activity even by simple freezing and thawing. Preservation of activity in 50% glycerol solutions at pH 7.2 was found to be the most convenient procedure. The alterations produced in purified prothrombin with DEAE cellulose chromatography are discussed with respect to possible significance for thrombin and autoprothrombin C.

Isolation, purification and characterization of chymosin from riverine buffalo (Bubalos bubalis)

2003 ◽  
Vol 70 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Ashok K Mohanty ◽  
Utpal K Mukhopadhyay ◽  
Jai K Kaushik ◽  
Sunita Grover ◽  
Virender K Batish
Keyword(s):  
Amino Acid ◽  
Proteolytic Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Amino Acid Sequences ◽  
Milk Clotting ◽  
Sds Page ◽  
Terminal Amino ◽  
The Stability

Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29–fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35·6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 °C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7·0. The ratio of milk clotting to proteolytic activity was 3·03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.

Cytosolic and membrane-bound chitinases of two mucoraceous fungi: a comparative study

1992 ◽  
Vol 38 (4) ◽  
pp. 331-338 ◽  
Author(s):  
R. Balasubramanian ◽  
M. S. Manocha
Keyword(s):  
Chitinase Activity ◽  
Lysozyme Activity ◽  
Acid Protease ◽  
Glucanase Activity ◽  
Chromatographic Techniques ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Choanephora Cucurbitarum ◽  
Membrane Bound ◽  
Temperature Optima

Chitinases isolated from membrane and cytosolic fractions of two mucoraceous fungi, Choanephora cucurbitarum and Phascolomyces articulosus, were investigated. The membrane-bound chitinase was isolated by Bio-Gel P-100 and DEAE Bio-Gel A chromatographic techniques. On SDS–PAGE the chitinase from both fungi migrated as a single band of Mr 66 kDa. The cytosolic chitinase from the mycelial extracts of these fungi was separated by heat treatment, ammonium sulphate precipitation, and by affinity chromatography with regenerated chitin. SDS–PAGE showed two bands for each fungus with Mr of 69.5 and 55 kDa in C. cucurbitarum and Mr 69.5 and 53 kDa in Ph. articulosus. Chitinases, membrane bound or cytosolic, hydrolyzed regenerated chitin, colloidal chitin, glycol chitin, N,N′-diacetylchitobiose, and N,N′,N′ ′-triacetylchitotriose. Heavy metals, inhibitors, and N-acetylglucosamine inhibited chitinase activity, whereas trypsin and an acid protease enhanced its activity. Chitinase preparations showed lysozyme activity that was inhibited by histamine but not by N-acetylglucosamine. There was no N-acetylglucosamanidase activity, but β-1,3 glucanase activity was found in cytosolic preparations only. Despite slight differences in their molecular mass, both the membrane-bound and cytosolic chitinases showed similarities in substrate utilization, response to inhibitors, and activation by trypsin and acid protease; pH and temperature optima also were similar. Key words: chitinase, membrane-bound chitinase, cytosolic chitinase, Choanephora cucurbitarum, Phascolomyces articulosus.

scholarly journals Purification and Characterization of Carboxymethyl Cellulase from Bacillus sp. Isolated from a Paddy Field

2012 ◽  
Vol 61 (1) ◽  
pp. 51-55 ◽  
Author(s):  
PONNUSWAMY VIJAYARAGHAVAN ◽  
S.G. PRAKASH VINCENT
Keyword(s):  
Paddy Field ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Relative Molecular Mass ◽  
Bacillus Sp ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Ph Range

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.

Antimicrobial activity of bacteriocin isolated and purified from rumen liquor collected from slaughtered goats

2015 ◽  
Vol 49 (6) ◽  
Author(s):  
S. Barathiraja ◽  
J. Thanislass ◽  
P. X. Antony ◽  
S. Venkatesaperumal
Keyword(s):  
Molecular Weight ◽  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Diffusion Method ◽  
Chromatographic Techniques ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Overlay Method

Bacteriocin like substance with antimicrobial activity was purified from freshly collected rumen liquor using 60% ammonium sulphate precipitation followed by ion exchange(SP-Sepharose) and gel filtration (Sephadex G25) chromatographic techniques. Purity of the product was checked on SDS-PAGE, having molecular weight of 6.5 kDa. Anti-microbial activity was demonstrated using <italic>Bacillus subtilis</italic> by gel overlay method and agar cut well diffusion method. Proteomic analysis confirmed the substance as bacteriocin. The purified sample was resistant to the action of protease. The substance was active at pH 4, 7 and 10. It was also active at autoclave temperature. The peptide purified was found to inhibit the growth of <italic>Staphylococcus aureus</italic> (MTCC87), <italic>Listeria monocytogenes</italic> (MTCC 657) and <italic>Pseudomonas aeurginosa</italic> (MTCC 424).

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.

scholarly journals Biochemical Isolation and Characterization of Hyaluronidase Enzyme from Venom of Egyptian Honey Bee Apis Mellifera Lamarckii

2020 ◽  
Vol 64 (1) ◽  
pp. 153-164
Author(s):  
Mohammed M. Abdel-Monsef ◽  
Hind A. Zidan ◽  
Doaa A. Darwish ◽  
Hassan M. Masoud ◽  
Mohamed S. Helmy ◽  
...  
Keyword(s):  
Specific Activity ◽  
Deae Cellulose ◽  
Bee Venom ◽  
Native Form ◽  
Native Page ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Wide Range ◽  
First Time

AbstractThe hyaluronidase enzyme has been used in many such fields of medicine as ophthalmology, orthopaedia, internal medicine, gynecology, surgery, oncology and dermatology. In this study, the hyaluronidase enzyme was purified and characterized for the first time from Egyptian bee venom homogeneously using DEAE-cellulose and Sephacryl S-300 columns. Bee venom hyaluronidase specific activity was 411.7 units/mg protein with 49.9% yield and 3.23-fold purification. The molecular weight of the purified bee venom hyaluronidase native form was 37 kDa. The purified enzyme was found homogeneous on native PAGE and SDS-PAGE, with two congruent subunits of 18.4 kDa and isoelectric point (pI) of 8.6–8.8. The enzyme was found to be stable over a wide range of temperature (20–60°C) and pH (4.5–6.5), and its optimum activity at 37°C, pH 5.4 and 0.15 M NaCl. Km for bee venom hyaluronidase was 0.029 mg/ml hyaluronic acid and its activity was elevated in presence of MgCl2 and ZnCl2 and lowered in presence of FeCl2. Heparin inhibited the hyaluronidase enzyme noncompetitively with a Ki value of 2.9 units heparin and one binding site on the enzyme molecule.

Methods for the Concentration of Bovine Ac-Globulin (Factor V)

1961 ◽  
Vol 06 (03) ◽  
pp. 435-444 ◽  
Author(s):  
Ricardo H. Landaburu ◽  
Walter H. Seegers
Keyword(s):  
Room Temperature ◽  
Barium Carbonate ◽  
Deae Cellulose ◽  
Reducing Agents ◽  
Factor V ◽  
Isoelectric Precipitation ◽  
Stabilizing Agent ◽  
Bovine Plasma ◽  
The Stability

SummaryAn attempt was made to obtain Ac-globulin from bovine plasma. The concentrates contain mostly protein, and phosphorus is also present. The stability characteristics vary from one preparation to another, but in general there was no loss before 1 month in a deep freeze or before 1 week in an icebox, or before 5 hours at room temperature. Reducing agents destroy the activity rapidly. S-acetylmercaptosuccinic anhydride is an effective stabilizing agent. Greatest stability was at pH 6.0.In the purification bovine plasma is adsorbed with barium carbonate and diluted 6-fold with water. Protein is removed at pH 6.0 and the Ac-globulin is precipitated at pH 5.0. Rivanol and alcohol fractionation is followed by chromatography on Amberlite IRC-50 or DEAE-cellulose. The final product is obtained by isoelectric precipitation.

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