scholarly journals Purification of papain from fresh latex of Carica papaya

2000 ◽  
Vol 43 (5) ◽  
pp. 501-507 ◽  
Author(s):  
Rubens Monti ◽  
Carmelita A. Basilio ◽  
Henrique C. Trevisan ◽  
Jonas Contiero
Keyword(s):  
Carica Papaya ◽  
Specific Activity ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
Enzymatic Assays ◽  
Papaya Latex ◽  
High Concentrations ◽  
Natural Inhibitors

In the present study we wish to report a method of crystallizing papain from fresh papaya latex which gave higher yields than previously reported. This method does not involve the use of sulphydryl reagents. The papain thus obtained is practically pure and shows a single band when submitted to electrophoresis on polyacrylamide gel, and is identical to the papain obtained by other methods. In routine enzymatic assays, specific activity was measured using Z-gly-pNP and BAEE as substrates. Papain crystallized by this method, without the use of high concentrations of salts or thiol-containing substances such as cysteine and dithiothreitol, is obtained in the form of a complex with natural inhibitors existent in latex which can be removed by dialysis.

scholarly journals Isolation of natural inhibitors of papain obtained from Carica papaya latex

2004 ◽  
Vol 47 (5) ◽  
pp. 747-754 ◽  
Author(s):  
Rubens Monti ◽  
Jonas Contiero ◽  
Antonio José Goulart
Keyword(s):  
Positive Reaction ◽  
Carica Papaya ◽  
Competitive Inhibition ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Negative Reaction ◽  
Ionic Exchange ◽  
Papaya Latex ◽  
Carica Papaya Latex ◽  
Natural Inhibitors

Studies were carried out to natural papain inhibitor from papaya latex. Fresh latex from green fruits of Carica papaya was collected and immediately transported in ice bath to the lab, from which three fractions with inhibitor effect of esterase papain activity were isolated by latex dialysis, Sephadex G-25 gel filtration and ionic exchange chromatography in SP-Sephadex C-25. The isolated fractions, identified as inhibitors I and II, showed a negative reaction with ninhydrin; however, the fraction identified as P-III showed positive reaction with ninhydrin. Kinetics data showed non-competitive inhibition (inhibitor I) and uncompetitive (inhibitors II and P-III).

scholarly journals PEMURNIAN PARSIAL DAN KRISTALISASI PAPAIN DARI GETAH Carica papaya

2019 ◽  
Vol 4 (2) ◽  
pp. 152
Author(s):  
Dwi Putri Mashfufatur Rohmah ◽  
Sofijan Hadi ◽  
Afaf Baktir
Keyword(s):  
Ammonium Sulphate ◽  
Carica Papaya ◽  
Specific Activity ◽  
Total Activity ◽  
Partial Purification ◽  
Purification Factor ◽  
Papaya Latex ◽  
Carica Papaya Latex

AbstractThis research has done partial purification by fractionation and optimization crystallization of papain from Carica papaya latex with the addition of ammonium sulphate. The enhancement of purification factor was determined by its specific activity. The fractionation results show that papain fraction of Carica papaya can be obtained by adding 40-80% saturated ammonium sulphate, with the highest specific activity, i.e. 2,0819 U/µg and the factor purification increase of 6,27 fold than papain extract. Meanwhile, the highest total activity, i.e. 10,7780 U of papain crystals can be obtained by presipitant agent addition of ammonium sulphate in the level / concentration 80% saturated at 15 °C. Microscopycally papain crystals profile in this condition have cube and tetragonal shape.Key words: crystallization, fractionation, ammonium sulphate, papain

scholarly journals Heparin-sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase

1979 ◽  
Vol 183 (3) ◽  
pp. 531-537 ◽  
Author(s):  
P N Srivastava ◽  
A A Farooqui
Keyword(s):  
Hyaluronic Acid ◽  
Affinity Chromatography ◽  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
National Formulary ◽  
Turnover Number

Bull seminal-plasma hyaluronidase was purified 180-fold by chromatography on concanvalin A-Sepharose, heparin Sepharose, Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 mumol/min per mg (104000 National Formulary units/mg of protein) and 214 min-1 (mol of product formed/mol of enzyme per min) respectively. Polyacrylamide-gel electrophoresis indicated that the purified enzyme migrated as a single band on 7.5 and 10% (w/v) gels at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenylhydrazine and semicarbazide. Purified hyaluronidase (1.25 munits; 1 unit = 1 mumol of N-acetylglucosamine liberated/min at 37 degrees C) dispersed the cumulus clot of rabbit ova in 1 h at 22 degrees C.

Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

2005 ◽  
Vol 32 (9) ◽  
pp. 839
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Enzyme Activity ◽  
Inorganic Phosphate ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

scholarly journals Ribonucleic acid stimulation of mammalian liver nuclear-envelope nucleoside triphosphatase. A possible enzymic marker for the nuclear envelope

1977 ◽  
Vol 162 (3) ◽  
pp. 671-679 ◽  
Author(s):  
P S Agutter ◽  
J R Harris ◽  
I Stevenson
Keyword(s):  
Nuclear Envelope ◽  
Specific Activity ◽  
Assay Medium ◽  
Exogenous Rna ◽  
Mammalian Liver ◽  
High Concentrations ◽  
Enzymic Marker ◽  
Stimulation Of ◽  
Nucleoside Triphosphatase

1. The specific activity of rat and pig liver nuclear-envelope nucleoside triphosphatase (EC 3.6.1.3) decreases when the system is depleted of RNA. The activity can be restored by adding high concentrations of yeast RNA to the assay medium. 2. Exogenous RNA also increases the activity of the enzyme in control envelopes (not RNA-depleted). The effect appears to be largely specific for poly(A) and poly(G); it is not stimulated by rRNA or tRNA preparations, ribonuclease-hydrolysed RNA, AMP, or double- or single-stranded DNA. 3. Inhibitors of the enzyme, in concentrations at which half-maximal inhibition of the enzyme is achieved, do not affect the percentage stimulation of the enzyme by yeast RNA. 4. The simulation is abolished by the inclusion of 150 mM-KCl or -NaCl in the assay medium, but not by increasing the assay pH to 8.5. 5. The results are discussed in the light of the possible role of the nucleoside triphosphatase in vivo in nucleo-cytoplasmic ribonucleoprotein translocation. 6. It is proposed that poly(G)-stimulated Mg2+-activated adenosine triphosphatase activity should be adopted as an enzymic marker for the nuclear envelope.

Rates of de novo Synthesis of Malate Synthase and Albumins during the Very Early Phase of Germination

1979 ◽  
Vol 34 (12) ◽  
pp. 1237-1242 ◽  
Author(s):  
Wolfram Köller ◽  
Helmut Kindl
Keyword(s):  
Early Phase ◽  
De Novo ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Malate Synthase ◽  
Polyacrylamide Gel ◽  
De Novo Synthesis ◽  
Albumin Fraction ◽  
Average Value ◽  
Large Pool

Abstract Malate synthase is synthesized de novo in the very early phase of germination. Its molecular and immunological properties do not differ from those of malate synthase from fully developed cotyledons. Radioactive leucine was administered to dry seeds of cucumber, and its incorporation into proteins of cotyledons was examined after 2 days of germination. The specific radioactivity of malate synthase, purified by immunoprecipitation and electrophoresis on polyacrylamide gel, was only 1/20 the average value of the total albumin fraction. The minimal incorporation documented by the comparatively low specific activity of isolated malate synthase is discussed in relation to the large pool of malate synthase already present in dry seeds.

scholarly journals Purification, Characterization, and Potential of Saline Waste Water Remediation of a Polyextremophilicα-Amylase from an Obligate HalophilicAspergillus gracilis

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Imran Ali ◽  
Ali Akbar ◽  
Benjawan Yanwisetpakdee ◽  
Sehanat Prasongsuk ◽  
Pongtharin Lotrakul ◽  
...  
Keyword(s):  
Waste Water ◽  
Water Activity ◽  
Column Chromatography ◽  
Specific Activity ◽  
Single Band ◽  
Water Remediation ◽  
Saline Waste ◽  
Sds Page ◽  
Nacl Concentration ◽  
Burk Plot

An obligate halophilicAspergillus graciliswhich was isolated from a hypersaline man-made saltern from Thailand was screened for its potential of producing extracellularα-amylase in the previous studies. In this study theα-amylase was extracted and purified by the help of column chromatography using Sephadex G-100 column. Presence of amylase was verified by SDS-PAGE analysis, showing a single band of approximately 35 kDa. The specific activity of the enzyme was found to be 131.02 U/mg. The Lineweaver-Burk plot showed theVmaxandKmvalues of 8.36 U/mg and 6.33 mg/mL, respectively. The enzyme was found to have the best activity at 5 pH, 60°C, and 30% of NaCl concentration, showing its polyextremophilic nature. The use of various additives did not show much variation in the activity of enzyme, showing its resilience against inhibitors. The enzyme, when tested for its use for synthetic waste water remediation by comparing its activity with commercial amylase in different salt concentrations showed that theα-amylase fromA. graciliswas having better performance at increasing salt concentrations than the commercial one. This shows its potential to be applied in saline waste water and other low water activity effluents for bioremediation.

scholarly journals PURIFICATION AND PHYSICAL PROPERTIES OF GROUP C STREPTOCOCCAL PHAGE-ASSOCIATED LYSIN

1971 ◽  
Vol 133 (5) ◽  
pp. 1105-1117 ◽  
Author(s):  
Vincent A. Fischetti ◽  
Emil C. Gotschlich ◽  
Alan W. Bernheimer
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Physical Properties ◽  
Total Protein ◽  
Sedimentation Coefficient ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
Purification Procedure ◽  
Sh Group ◽  
Stokes Radius

A purification procedure for the Group C phage-associated lysin is described utilizing tetrathionate to protect the enzyme's -SH group(s) from thiol-inactivating agents. A 652-fold purification has been accomplished yielding a solution in which the enzyme activity corresponds to essentially a single band on polyacrylamide gel which accounts for 70% of the total protein in the preparation. A molecular weight of 101,000 and frictional ratio of 1.526 was determined for the lysin utilizing experimentally determined values for its Stokes radius and sedimentation coefficient.

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