scholarly journals Canine submandibular-gland hyaluronidase. Purification and properties

1968 ◽  
Vol 110 (1) ◽  
pp. 19-25 ◽  
Author(s):  
Y. H. Tan ◽  
J. M. Bowness
Keyword(s):  
Submandibular Gland ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
Gel Filtration ◽  
Acid Precipitation ◽  
Ammonium Sulphate Precipitation ◽  
Purification And Properties ◽  
Freeze Dried ◽  
Specific Activities

1. Methods for the purification of dog submandibular-gland hyaluronidase from sedimentable and non-sedimentable portions of a homogenate and from the whole homogenate are presented. The method consists of three main steps: removal of mucin by acid precipitation or gel filtration on Sephadex G-200, ammonium sulphate precipitation and CM-cellulose chromatography. By this method specific activities of up to 1·28 and 0·78μmoles of N-acetylglucosamine/min./mg. of protein were obtained for the purified freeze-dried non-sedimentable hyaluronidase and for the sedimentable hyaluronidase respectively. 2. A comparison of some of the properties of the non-sedimentable and the sedimentable hyaluronidase preparation indicated that there was little difference between the two and that they both resembled lysosomal hyaluronidase from rat liver.

scholarly journals Production and optimization of Aspergillus niger glucoamylase using amylopectin from guinea corn starch as the sole carbon source

2017 ◽  
Vol 52 (4) ◽  
pp. 263-272
Author(s):  
PC Okwuenu ◽  
AL Ezugwu ◽  
FC Chilaka
Keyword(s):  
Carbon Source ◽  
Ammonium Sulphate ◽  
Sole Carbon Source ◽  
Corn Starch ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Glucoamylase Activity ◽  
Ammonium Sulphate Precipitation ◽  
Specific Activities ◽  
Two Peaks

A Fourteen day experimental study was carried out to determine the day of highest glucoamylase activity using amylopectin from guinea corn starch as the sole carbon source. Two peaks of high activity were observed on the fifth and twelveth days, and were thus mass produced. Specific activities for crude enzymes were found to be 729.45 U/mg and 1046.82 U/mg for day five and twelve harvested enzymes respectively. Ammonium sulphate saturations, 70% and 20%, were found suitable to precipitate proteins with highest glucoamylase activity for day five and twelve harvested enzymes respectively. After ammonium sulphate precipitation and gel filtration, specific activities were found to be 65.98 U/mg and 180.52 U/mg respectively for day five harvested enzyme and 61.51 U/mg and 272.81 U/mg for day twelve harvested enzyme. The pH optimum for day five harvested enzyme were found to be 7.5, 7.5 and 6.0 using tiger nut, cassava and guinea corn starches as substrates respectively, also, the pH optimum for day twelve harvested enzyme were found to be 5.0, 8.5 and 7.0 using tiger nut, cassava and guinea corn starches as substrate, respectively. Optimum temperatures were found to be 50˚C and 45˚C for day five and twelve harvested enzymes, respectively. Km and Vmax, of day five harvested enzyme were found to be 770.75 mg/ml and 2500 μmol/min, 158.55 mg/ml and 500 μmol/min and 46.23 mg/ml and 454.53 μmol/min using cassava, guinea corn and tiger nut starches as substrate respectively. Km and Vmax of day twelve harvested enzyme were found to be 87.1 mg/ml and 384.61 μmol/min, 29.51 mg/ml and 243.90 μmol/min, and 2364 mg/ml and 2500 μmol/min, using cassava, guinea corn and tiger nut starches as substrate respectively.Bangladesh J. Sci. Ind. Res. 52(4), 263-272, 2017

Anreicherung und Charakterisierung einer „Transhydrogenase-Aktivität“ und der 17 β-Hydroxysteroid-Oxidoreduktase aus der Cytoplasma-Fraktion der Leber von weiblichen Ratten / The Enrichment and Characterisation of Cytoplasmatic “Transhydrogenase-activity” and 17 β-hydroxysteroid-oxidoreductase from Rat Liver

1972 ◽  
Vol 27 (8) ◽  
pp. 981-988
Author(s):  
Kunhard Pollow ◽  
Barbara Pollow
Keyword(s):  
Molecular Weight ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
Isoelectric Focusing ◽  
Isoelectric Point ◽  
Column Chromatography ◽  
Enzymatic Activities ◽  
Kinetic Constants ◽  
Gel Chromatography ◽  
Ammonium Sulphate Precipitation

The cytoplasmatic fraction of rat liver contains both 17 β-hydroxysteroid-oxidoreductase and a “transhydrogenase-activity”, which catalyses the transfer of hydrogen from the 17 β-position of estradiol-17 β to the 17-position of 4-androstene-3,17-dione. The 17 β-hydroxysteroid-oxidoreductase was purified 718-fold and the “transhydrogenase-activity” 264-fold by ammonium sulphate precipitation, gel chromatography with Sephadex G-200, column chromatography on DEAE-Sephadex and isoelectric focusing. The two enzymic activities could not be separated. The characteristics of the two enzymatic activities give some evidence that the “transhydrogenase-activity” is identical with the already known 17 β-hydroxysteroid-oxidoreductase.Isoelectric focusing of the chromatographycally enriched 17 β-enzyme gave an isoelectric point at 5,2. The 17 β-enzyme has a molecular weight of 62 — 65 000 as determined by mobility on Sephadex G-200 superfine.The kinetic constants for both the 17 β-enzyme and the “transhydrogenase-activity” were determined.

Fermentative Production, Purification and Characterization of Nisin

2008 ◽  
Vol 4 (5) ◽  
Author(s):  
Swapnali S Gujarathi ◽  
Sandip B. Bankar ◽  
Laxmi A. Ananthanarayan
Keyword(s):  
Ammonium Sulphate ◽  
Hydrophobic Interaction ◽  
Orthogonal Array ◽  
Gel Filtration ◽  
Statistical Design ◽  
Temperature Stability ◽  
Gel Chromatography ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation

Bacteriocins are bactericidal or bacteriostatic in action and active against closely related species. Nisin is the bacteriocin produced by the lactic acid bacteria (LAB). Nisin is a small (3353 Da), cationic, hydrophobic, and 34-amino acid peptide. It is used in products such as pasteurized processed cheese, salad dressing, and liquid whole eggs to inhibit the growth of Gram-positive microorganisms including Listeria monocytogenes. The objective of the present work was to study production, purification and characterization of nisin. The production of nisin was carried out using Lactococcus lactis subsp lactis MTCC 440 by one-factor-at-a-time method and statistical design (Orthogonal array). Purification was carried out using ammonium sulphate precipitation followed by hydrophobic interaction and gel chromatography. Characterization was done for pH and temperature stability. The activity of nisin after one factor at a time optimization was found to be 5120 AU/ml. The activity of the nisin increased to 6800 AU/ml after optimization by Orthogonal Array design. Ammonium sulphate precipitation gives good yield of nisin with 60 to 80% saturation with 2.52 fold purity. The overall purification by hydrophobic interaction and gel filtration chromatography was 10.87 fold with 50.84% yield and 8.8 fold with 49.65% yield as compared to crude broth respectively.

scholarly journals Purification and properties of a new glutathione-dependent thiol:disulphide oxidoreductase from rat liver

1982 ◽  
Vol 207 (1) ◽  
pp. 133-138 ◽  
Author(s):  
M G Battelli ◽  
E Lorenzoni
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Xanthine Dehydrogenase ◽  
Enzymic Activity ◽  
Oxidized Glutathione ◽  
Adult Rats ◽  
Purification And Properties ◽  
Rat Liver Cytosol

A new GSSG-dependent thiol:disulphide oxidoreductase was extensively purified from rat liver cytosol. The enzymic protein shows molecular weight 40 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and 43 000 as determined by thin-layer gel filtration on Bio-Gel P-100. The pI is 8.1. This enzyme converts rat liver xanthine dehydrogenase into an oxidase, in the presence of oxidized glutathione. Other disulphide compounds are either inactive or far less active than oxidized glutathione in the enzymic oxidation of rat liver xanthine dehydrogenase. The enzyme also catalyses the reduction of the disulphide bond of ricin and acts as a thioltransferase and as a GSH:insulin transhydrogenase. The enzymic activity was measured in various organs of newborn and adult rats.

scholarly journals Characterization of Bromelain from Morinda citrifolia (Noni)

2012 ◽  
Vol 4 (2) ◽  
pp. 445 ◽  
Author(s):  
K. D. Golden ◽  
J. Smith-Marshall
Keyword(s):  
Gel Filtration ◽  
Exchange Chromatography ◽  
Morinda Citrifolia ◽  
Mercury Chloride ◽  
Ammonium Sulphate Precipitation ◽  
Step Procedure ◽  
Fresh Plant Material ◽  
Freeze Dried ◽  
Inhibition Studies ◽  
Protein Enzyme

Morinda citrifolia from the Rubiaceae family, collected in parish of St. Andrew, Jamaica, West Indies, was investigated for the presence of the  Bromelain-like protein enzyme.  In the process Bromelain from Morinda citrifolia was partially purified and characterized. Fresh plant material was extracted in buffer (sodium acetate-acetic acid (10mM), L-cysteine (1mM), and sodium chloride (0.1M), freeze dried and analyzed using column chromatography and assayed using spectrophotometry in a five step procedure.  The major purification steps involved were ammonium sulphate precipitation, gel-filtration (Sephadex G200), ion exchange chromatography (CM sephadex C25) and (DEAE sephadex A25). The protein content was determined using  the Bradford method. The enzyme displayed an optimum activity at pH 7.1 and a temperature optimum at 35 ºC. A purification fold of 70.9 and percentage recovery of 3.3 was obtained. Inhibition studies using several different inhibitors of the enzyme revealed that the enzyme was susceptible to copper sulphate (0.1mM), mercury chloride (0.1mM), cobalt sulphate (0.1mM), zinc sulphate (0.1mM) and phenylmercury acetate (0.1mM). Both casein and p-Nitrophenylbenzyloxycarbonyl-L-lysinate (CLN) were used as substrates for the enzyme, with the enzyme displaying greater activity when using casein as substrate. Bromelain-like protein enzyme showed high affinity for the substrate casein with a Km of 48.5 µM. Key words: Morinda citrifolia; Bbromelain; Proteolytic enzyme. © 2012 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. doi: http://dx.doi.org/10.3329/jsr.v4i2.8125 J. Sci. Res. 4 (2), 445-456 (2012)

scholarly journals Biobleaching of Industrial Important Dyes with Peroxidase Partially Purified from Garlic

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Akudo Chigozirim Osuji ◽  
Sabinus Oscar O. Eze ◽  
Emmanuel Emeka Osayi ◽  
Ferdinand Chiemeka Chilaka
Keyword(s):  
Ammonium Sulphate ◽  
Specific Activity ◽  
Low Cost ◽  
Gel Filtration ◽  
Enzyme Concentration ◽  
Gel Filtration Chromatography ◽  
Vat Dyes ◽  
Ammonium Sulphate Precipitation ◽  
Ph Range ◽  
Better Than

An acidic peroxidase was extracted from garlic (Allium sativum) and was partially purified threefold by ammonium sulphate precipitation, dialysis, and gel filtration chromatography using sephadex G-200. The specific activity of the enzyme increased from 4.89 U/mg after ammonium sulphate precipitation to 25.26 U/mg after gel filtration chromatography. The optimum temperature and pH of the enzyme were 50°C and 5.0, respectively. The Km andVmaxfor H2O2and o-dianisidine were 0.026 mM and 0.8 U/min, and 25 mM and 0.75 U/min, respectively. Peroxidase from garlic was effective in decolourizing Vat Yellow 2, Vat Orange 11, and Vat Black 27 better than Vat Green 9 dye. For all the parameters monitored, the decolourization was more effective at a pH range, temperature, H2O2concentration, and enzyme concentration of 4.5–5.0, 50°C, 0.6 mM, and 0.20 U/mL, respectively. The observed properties of the enzyme together with its low cost of extraction (from local sources) show the potential of this enzyme for practical application in industrial wastewater treatment especially with hydrogen peroxide. These Vat dyes also exhibited potentials of acting as peroxidase inhibitors at alkaline pH range.

scholarly journals Purification and Characterization of Peroxidase From Anthracnose Disease Infected Papaya (Carica papaya L.)

2014 ◽  
Vol 6 (2) ◽  
pp. 49-57 ◽  
Author(s):  
L Bari ◽  
P Hassan ◽  
N Absar ◽  
S Khatun ◽  
MI Hossain
Keyword(s):  
Ammonium Sulphate ◽  
Gel Filtration ◽  
Potassium Cyanide ◽  
Ammonium Sulphate Precipitation ◽  
Reducing Conditions ◽  
Sds Page ◽  
Peroxidase Enzyme ◽  
Km Value ◽  
Temperature Optima

Peroxidase enzyme was isolated and purified from the pulp of disease infected ripen papaya of local variety by 90% ammonium sulphate precipitation, chromatography on DEAEcellulose followed by hydrophobic chromatography on Phenyl Sepharose CL-4B and the purifications achieved was about 7.2 fold with 2.5% recovery. The purified enzyme was homogeneous as judged by polyacrylamide slab gel electrophoresis. The purified enzyme had a Mr of about 55,000 and 50 000 as determined by gel filtration on Sephadex G-100 and SDS-PAGE, respectively. The molecular mass of the enzyme was found to be very similar under both reducing and non-reducing conditions indicating that the enzyme contains no subunit. The enzyme has the following characteristics: pH optima at 6.0, temperature optima around 38°C, enzyme activity was found to be strongly inhibited in the presence of potassium cyanide and Fe+2 while the activity was found to be remarkably increased in the presence of ammonium sulphate. The Km value for the peroxidase obtained with pyrogallol as substrate was 0.027 mM. DOI: http://dx.doi.org/10.3329/bjmb.v6i2.17643 Bangladesh J Med Biochem 2013; 6(2): 49-57

scholarly journals Purification of recombinant α-Synuclein: a comparison of commonly used protocols

2020 ◽  
Author(s):  
Amberley D. Stephens ◽  
Dijana Matak-Vinkovic ◽  
Gabriele S. Kaminski Schierle
Keyword(s):  
Degradation Products ◽  
Gel Filtration ◽  
Acid Precipitation ◽  
Alpha Synuclein ◽  
Exchange Chromatography ◽  
Great Effort ◽  
List Type ◽  
Initial State ◽  
E Coli ◽  
Ammonium Sulphate Precipitation

AbstractThe insoluble aggregated form of the protein alpha-synuclein (aSyn) is associated with synucleinopathies, such as Parkinson’s Disease, therefore great effort is put into understanding why and how this initially soluble protein misfolds. The initial state of aSyn, e.g. presence of contaminants, adducts, oligomers or degradation products, can greatly influence the outcome of an assay, such as determining its aggregation kinetics. Here, we compare four commonly used protocols for the isolation of recombinant aSyn from E. coli by boiling, acid precipitation, ammonium sulphate precipitation and periplasmic lysis followed by ion exchange chromatography and gel filtration. We identified, using non-denaturing electrospray ionisation mass spectrometry of the differently extracted aSyn samples, that aSyn isolated by acid precipitation and periplasmic lysis yielded the highest percentage of monomer, 100% and 96.5% respectively. aSyn purity was again highest in samples isolated by acid precipitation and periplasmic lysis, yet aggregation assays displayed differences in the aggregation rate of aSyn isolated by all four methods.HighlightsA rapid protocol; expression day one, two step purification day two.The periplasmic lysis-based protocol yielded 95% pure aSyn.Acid precipitation and periplasmic lysis-based protocols yielded the highest proportion of monomeric aSyn at 100% and 96.5%, respectively.

Inhibitory activity of salivary glycoproteins on phytohemagglutins (PHA): Possible molecules to enhance nutritional quality of red kidney beans

2018 ◽  
Author(s):  
Vishwanath B. Chachadi ◽  
Tejashwini R. Nayanegali ◽  
Bharamappa G. Pujari ◽  
Lakshmi V. Umarji ◽  
Vasundhara Budyhalamath ◽  
...  
Keyword(s):  
Ammonium Sulphate ◽  
Nutritional Quality ◽  
Gel Filtration ◽  
Human Saliva ◽  
Health Concern ◽  
Ammonium Sulphate Precipitation ◽  
Kidney Beans ◽  
Red Kidney Beans ◽  
Inhibitory Molecules

Food allergy caused by red kidney bean (Phaseolus vulgaris L.) is of serious health concern and is mainly due to its phytohemagglutinins (PHA) content. PHA can enter the circulation after oral uptake and cause IgE mediated allergy. However, studies describing enhancement of nutritional quality of red kidney beans by targeting PHA are not reported. This study was carried out to identify, PHA-inhibitory molecules present in saliva secretions. Results describe that PHA can be effectively inhibited by salivary glycoproteins. Fractionation of salivary proteins by ammonium sulphate precipitation revealed that, PHA-inhibitory proteins can be specifically precipitated at 30-60% of ammonium sulphate saturation. Gel filtration chromatography and lectin-blot analysis of 30-60% ammonium sulphate fraction suggest that only high molecular weight glycoproteins can act as potent inhibitors of PHA. In conclusion, human saliva secretions contain inhibitory glycoproteins which can be used to inhibit PHA effectively. If these glycoproteins are purified to homogeneity, can be used as potent food supplements in order to neutralize allergic potential of PHA, thus increasing the nutritional value of red kidney beans.

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