Purification and characterization of the protease inhibitor "monastatin" from a marine Alteromonas sp. with reference to inhibition of the protease produced by a bacterium pathogenic to fish

1985 ◽  
Vol 31 (12) ◽  
pp. 1089-1094 ◽  
Author(s):  
Chiaki Imada ◽  
Masachika Maeda ◽  
Nobuo Taga
Keyword(s):  
Protease Inhibitor ◽  
Inhibitory Activity ◽  
Marine Bacterium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization ◽  
Wide Range ◽  
Thiol Proteases

A novel protease inhibitor, which we named "monastatin," produced by a marine bacterium was purified using ammonium sulfate precipitation, DEAE-cellulofine column chromatography, and gel filtration through Sephadex G-100. Monastatin was purified to homogeneity, as a single staining band in polyacrylamide gel electrophoresis. Monastatin was a glycoprotein with a molecular weight of about 20 000. It was stable up to 100 °C for a 30-min incubation with loss of 20% of inhibitory activity and also showed stability over a wide range of pH from 2 to 12. Metal ions such as Cu2+ and Fe2+ repressed the inhibitory activity to 0%. Monastatin had an inhibitory activity against thiol proteases such as papain and ficin.

Purification and Characterization of a 3,17 β-Hydroxysteroid Dehydrogenase from Streptomyces hydrogenans

1979 ◽  
Vol 34 (7-8) ◽  
pp. 533-540 ◽  
Author(s):  
Helmut Duchmann ◽  
Lothar Träger
Keyword(s):  
Gel Electrophoresis ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Sedimentation Coefficient ◽  
Hydroxysteroid Dehydrogenase ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization

3,17 β-Hydroxysteroid dehydrogenase has been enriched and purified from cytosol of Streptomyces hydrogenans. After ammonium sulfate precipitation and filtration on Sephadex G-100 the enzyme was finally purified by preparative gel electrophoresis and DEAE-Sephadex A-50 chro­matography. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate gave a single band of mobility corresponding to molecular weight of 70 200 ± 2 500. 3 β-. 17 β- as well as 20 β-hydroxy steroids were dehydrogenated by the enzyme in the presence of NAD+. The dehydrogenation proceeded faster than the reduction of the corresponding ketosteroids in the presence of NADH. The enzyme does not accent NADP+ or NADPH as co-substrates. The apparent Km values were calculated to be 11 μᴍ for 5 α-dihydrotestosterone, 20 μᴍ for testosterone ana 68 μᴍ for epiandrosterone in the NAD+-driven reaction, 1.8 x 10-4 m for NADH+ and 1.9 x 10-4 ᴍ for NADH. The catalytic activity was influenced by the ratio of NAD+/ATP. The inhibition by ATP appears to be of a competitive type with respect to NAD+ (Ki 1.15 x 10-3 ᴍ).After sucrose gradient centrifugation in a preparative ultracentrifuge the enzyme sediments with 4.1 ± 0.1 S as estimated in comparison to other proteins of known sedimentation coefficient. The isoelectric point was determined to be 3.9 with the LKB preparative isoelectric focusing col­umn (pH 2-11) and 4.1 with the analytical flat bed polyacrylamide isofocusing (pH 3 - 5). The number of SH groups was determined to be 2 mol/mol enzyme. In the presence of 6 M urea the fig­ure inceases to 3 mol SH/mol enzyme. In the presence of an excess of p-chloromercuribenzoate the enzyme activity decreases only partially.

Purification and Characterization of an Acetyl Xylan Esterase from Bacillus pumilus

1998 ◽  
Vol 64 (2) ◽  
pp. 789-792 ◽  
Author(s):  
Giuliano Degrassi ◽  
Benedict C. Okeke ◽  
Carlo V. Bruschi ◽  
Vittorio Venturi
Keyword(s):  
Gel Electrophoresis ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Acetyl Xylan Esterase ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Naphthyl Acetate

ABSTRACT Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (Km ) andV max for α-naphthyl acetate were 1.54 mM and 360 μmol min−1 mg of protein−1, respectively.

Purification and Characterization of Kallikrein from Plasma of Patients with Acute Pancreatitis

1981 ◽  
Vol 60 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Naotika Toki ◽  
Hiroyuki Sumi ◽  
Sumiyoshi Takasugi
Keyword(s):  
Acute Pancreatitis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Purification And Characterization ◽  
Α2 Macroglobulin ◽  
Sepharose 4B

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with α2-macroglobulin only in the presence of trypsin.

scholarly journals Purification and characterization of histidine decarboxylase from mouse kidney

1986 ◽  
Vol 234 (2) ◽  
pp. 349-354 ◽  
Author(s):  
S A M Martin ◽  
J O Bishop
Keyword(s):  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Histidine Decarboxylase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Mouse Kidney ◽  
Purification Procedure ◽  
Purification And Characterization ◽  
A Subunit

Histidine decarboxylase was purified 800-fold from the kidneys of thyroxine-treated mice. The purification procedure included precipitation of protein from a crude supernatant after heating it to 55 degrees C at pH 5.5, fractionation with (NH4)2SO4, phosphocellulose column chromatography, chromatofocusing, DEAE-Sepharose column chromatography, gel filtration on Sephacryl S-300 and preparative polyacrylamide-gel electrophoresis. The native enzyme had an estimated Mr of 113 000. The protein was analysed in SDS/10%-polyacrylamide gels and formed a single band corresponding to a subunit Mr of 55 000, indicating that it is a dimer. Three forms of the enzyme were resolved on isoelectrofocusing gels, with pI 5.3, 5.5 and 5.7.

scholarly journals Innate Immunity in Lobsters: Partial Purification and Characterization of a Panulirus cygnus Anti-A Lectin

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Robert L. P. Flower
Keyword(s):  
Innate Immunity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Spiny Lobster ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Rat Erythrocytes ◽  
Group A ◽  
Panulirus Cygnus

A lectin detected in haemolymph from the Australian spiny lobster Panulirus cygnus agglutinated human ABO Group A cells to a higher titre than Group O or B. The lectin also agglutinated rat and sheep erythrocytes, with reactivity with rat erythrocytes strongly enhanced by treatment with the proteolytic enzyme papain, an observation consistent with reactivity via a glycolipid. The lectin, purified by affinity chromatography on fixed rat-erythrocyte stroma, was inhibited equally by N-acetylglucosamine and N-acetylgalactosamine. Comparison of data from gel filtration of haemolymph (behaving as a 1,800,000 Da macromolecule), and polyacrylamide gel electrophoresis of purified lectin (a single 67,000 Da band), suggested that in haemolymph the lecin was a multimer. The purified anti-A lectin autoprecipitated unless the storage solution contained chaotropic inhibitors (125 mmol/L sucrose: 500 mmol/L urea). The properties of this anti-A lectin and other similar lectins are consistent with a role in innate immunity in these invertebrates.

scholarly journals Purification and Characterization of a Polyextremophilicα-Amylase from an Obligate HalophilicAspergillus penicillioidesIsolate and Its Potential for Souse with Detergents

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Imran Ali ◽  
Ali Akbar ◽  
Mohammad Anwar ◽  
Sehanat Prasongsuk ◽  
Pongtharin Lotrakul ◽  
...  
Keyword(s):  
Specific Activity ◽  
Amylase Activity ◽  
Gel Filtration ◽  
Soluble Starch ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Good Potential

An extracellularα-amylase from the obligate halophilicAspergillus penicillioidesTISTR3639 strain was produced and enriched to apparent homogeneity by ammonium sulfate precipitation and Sephadex G100 gel filtration column chromatography. The mass of the purified amylase was estimated to be 42 kDa by SDS-PAGE. With soluble starch as the substrate it had a specific activity of 118.42 U·mg−1andVmax⁡andKmvalues of 1.05 µmol·min−1·mg−1and 5.41 mg·mL−1, respectively. The enzyme was found to have certain polyextremophilic characteristics, with an optimum activity at pH 9, 80°C, and 300 g·L−1NaCl. The addition of CaCl2at 2 mM was found to slightly enhance the amylase activity, while ZnCl2, FeCl2, or EDTA at 2 mM was strongly or moderately inhibitory, respectively, suggesting the requirement for a (non-Fe2+or Zn2+) divalent cation. The enzyme retained more than 80% of its activity when incubated with three different laundry detergents and had a better performance compared to a commercial amylase and three detergents in the presence of increasing NaCl concentrations up to 300 g·L−1. Accordingly, it has a good potential for use as anα-amylase in a low water activity (high salt concentration) and at high pH and temperatures.

scholarly journals Purification and Characterization of a Dimethoate-Degrading Enzyme of Aspergillus niger ZHY256, Isolated from Sewage

2001 ◽  
Vol 67 (8) ◽  
pp. 3746-3749 ◽  
Author(s):  
Yu-Huan Liu ◽  
Ying-Cheng Chung ◽  
Ya Xiong
Keyword(s):  
Aspergillus Niger ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Degrading Enzyme

ABSTRACT A dimethoate-degrading enzyme from Aspergillus nigerZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50°C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu2+. The Michaelis constant (K m ) andV max for dimethoate were 1.25 mM and 292 μmol min−1 mg of protein−1, respectively.

Purification and Characterization of 5'-Deoxy-5'-methylthioadenosine Nucleoside from Avocado Fruits

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 520f-521
Author(s):  
Chunlin Xiao ◽  
Mosbah M. Kushad
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Ethylene Biosynthesis ◽  
Plant Tissues ◽  
Salvage Pathway ◽  
Purification And Characterization ◽  
Insufficient Amount ◽  
Optimum Ph ◽  
Isozyme Activity

The polyamine and ethylene biosynthetic pathways utilize methionine as substrate for their biosynthesis. However, methionine pool in fruits and other plant tissues is very limited and is not sufficient to accommodate the amount of ethylene or polyamines that are synthesized during development and senescence. To compensate for the insufficient amount of methionine, plant tissues have evolved a mechanism to salvage 5'-deoxy-5'-methylthioadenosine (MTA), a by-product of polyamine and ethylene biosynthesis, back into methionine. The first enzyme involved in this salvage pathway is MTA nucleosidase. Purification of MTA nucleosidase from mature avocado fruits showed that there are two isozymes of MTA nucleosidase. In this study we will report on the purification of one of the isozymes, MTA nucleosidase I. Using ammonium sulfate fractionation, DEAE-Sepharose, Sephadex G-100 gel filtration, and hydroxyapatite chromatography, the isozyme was purified 3712-fold yielding about 11% protein. The isozyme migrated as a single band of 40 kDa molecular weight on SDS-polyacrylamide gel electrophoresis. MTA nucleosidase I exhibited an optimum pH of 7.2 and optimum temperature of 55 °C. The Km value of the isozyme for its substrate MTA is 7.69 μM and the Vmax is 58.82 × 10–6 μmol·min-1. Incubation of MTA nucleosidase I with analogs of MTA, 2.5 mM ethylthioadenosine and 2.5 mM 5'-S-isobutyl-5'-deoxyadenosine, completely blocked its activity. The isozyme activity was also inhibited by putrescine, spermidine, and spermine.

scholarly journals Purification and characterization of a second form of acid lipase in human liver

1987 ◽  
Vol 248 (1) ◽  
pp. 139-144 ◽  
Author(s):  
E R Sjoberg ◽  
J D Hatton ◽  
J S O'Brien
Keyword(s):  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Immunoblot Analysis ◽  
Polyacrylamide Gel ◽  
Acid Lipase ◽  
Purification And Characterization ◽  
Cellulose Acetate Electrophoresis

We describe here the purification and characterization of a form of acid lipase from human liver (designated ALII), which differed from the more abundant Mr-29000 form (ALI). ALII was solubilized from frozen human liver with Triton X-100 and purified 8500-fold by chromatography over concanavalin A-sepharose, CM-cellulose and finally h.p.l.c. over a Mono S column. ALII migrated as a single band on polyacrylamide-gel electrophoresis in both the presence and the absence of SDS. The Mr of ALII was estimated to be 58,500 by SDS/polyacrylamide-gel electrophoresis. Gel filtration on Sephacryl S-200 gave an apparent Mr of 69,000. 4-Methylumbelliferyl (4MU) palmitate, cholesterol oleate and triolein were substrates for ALII, with apparent Vmax values of 5000, 1100 and 2500 nmol/min per mg respectively and Km values of 1.0, 1.5 and 1.8 mM respectively. Cholesterol oleate and triolein were hydrolysed optimally by ALII at pH 4.5, whereas 4MU palmitate was hydrolysed optimally at pH 5.3. Antisera were raised against ALI and ALII and, on immunoblot analysis, no antigenic similarity was observed between ALI and ALII. Cellulose acetate electrophoresis followed by reaction with 4MU palmitate revealed two forms of lipase, corresponding to ALI and ALII. The two enzymes were also separated by hydrophobic chromatography. The activity of ALII was stimulated by several proteins and was partially inhibited by millimolar concentrations of NaCl, CaCl2 and MgSO4.

Purification and Characterization of an Alkaline Protease from Bacillus alcalophilus

2012 ◽  
Vol 192 ◽  
pp. 285-288 ◽  
Author(s):  
Shan Shan Liu ◽  
Li Li Wang ◽  
Lin Yuan
Keyword(s):  
Alkaline Protease ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization ◽  
Highly Active ◽  
Wide Range ◽  
Commercial Applications ◽  
Ph Range

The purification and characterization of an alkaline protease produced by Bacillus alcalophilus were investigated. The enzyme was purified in two steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by cation-exchange chromatography. The purified protease had a molecular mass of approximately 28 kDa, was highly active over an alkaline pH range of 10.0 to 11.0, and remained stable over a pH range of 7.0 to 12.0. The optimum temperature for the enzyme activity was found to be 40~60°C, while the thermotolerance of the enzyme was poor. Therefore, these characteristics of the protease indicate its potential for a wide range of commercial applications.

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