scholarly journals Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site

1989 ◽  
Vol 261 (1) ◽  
pp. 49-55 ◽  
Author(s):  
S S Keskar ◽  
M C Srinivasan ◽  
V V Deshpande
Keyword(s):  
Chemical Modification ◽  
Active Site ◽  
Submerged Culture ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Cellulase Activity ◽  
Original Activity ◽  
Larch Wood ◽  
End Products ◽  
Temperature Optima

Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.

scholarly journals PREPARATION, PURIFICATION AND PROPERTIES OF LIPASE FROM HEPATOPANCREAS OF TRA (PANGASIUS) CATFISH

2011 ◽  
Vol 14 (3) ◽  
pp. 5-11
Author(s):  
Thy Bao Vuong ◽  
Lam Bich Tran ◽  
Duan Luu
Keyword(s):  
Heavy Metals ◽  
Molecular Weight ◽  
Enzyme Activity ◽  
Crude Extract ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Purification And Properties ◽  
Temperature Optima

Lipase from the hepatopancreas of Tra (Pangasius) catfish was purified by ammonium sulfate fractionation, followed by ion-exhange chromatography on DEAE Cellulose and gel filtration Sephadex G-75. The preparation was homogeneous on polyacrylamide disc gel electrophoresis. The specific activity of the purified enzyme was 37.95 times higher than that of the crude extract. The enzyme showed a molecular weight of 57000 Da. The pH and temperature optima of purified lipase were 8 and 500C respectively. Enzyme activity was enhanced by Ca2+ but inhibited by heavy metals Zn2+, Cd2+, Mg2+.

scholarly journals Prolinase and non-specific dipeptidase of human kidney

1985 ◽  
Vol 231 (3) ◽  
pp. 689-694 ◽  
Author(s):  
D A Priestman ◽  
J Butterworth
Keyword(s):  
Active Site ◽  
Enzyme Activities ◽  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Human Kidney ◽  
Competitive Inhibitor ◽  
Dipeptidase Activity ◽  
Single Enzyme

Human kidney prolinase, assayed with Pro-Ala, and non-specific dipeptidase, assayed with Gly-Leu, were purified by using DEAE-cellulose, gel-filtration, metal-ion-chelate, hydrophobic and adsorption chromatography and chromatofocusing. Both enzymes gave single peaks of activity that were congruent and the ratio of their activities was constant throughout the purification. Gel filtration indicated an Mr of 100 000 and chromatofocusing a pI of 5.4. Ni2+-chelate chromatography demonstrated the presence of exposed histidine residues on the enzyme and was an effective separative procedure. Polyacrylamide-gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. Both enzyme activities decayed at the same rate at 53 degrees C and were inhibited to the same extent by p-hydroxymercuribenzoate. Of six non-specific dipeptidase substrates tested Gly-Leu gave the highest activity, and of six prolinase substrates Pro-Leu had the highest activity. Gly-Leu was hydrolysed at double the rate of Pro-Leu. Pro-Ala was a competitive inhibitor of activity towards Gly-Leu, and Gly-Leu was a competitive inhibitor of activity towards Pro-Ala. Mixed-substrate studies strongly suggested that Gly-Leu and Pro-Ala were hydrolysed at a common active site. The data are consistent with prolinase and non-specific dipeptidase activity in human kidney being due to a single enzyme.

Production of pectin lyase from Geobacillus pallidus p26, purification, characterization and fruit juice application

2014 ◽  
Vol 7 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Safinur Yildirim Çelik ◽  
Nazan Demir ◽  
Yaşar Demir ◽  
Ahmet Adiguzel ◽  
Medine Gulluce
Keyword(s):  
Molecular Weight ◽  
Fruit Juice ◽  
Hot Spring ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Pectin Lyase ◽  
Anion Exchange Column ◽  
Sds Page ◽  
Temperature Optima ◽  
Geobacillus Pallidus

Abstract A bacterial strain was isolated from Pasinler hot spring, Erzurum, Turkey. The purified thermophilic isolate was identified as Geobacillus pallidus P26 and used to produce extracellular pectin lyase (EC 4.2.2.10). Pectin lyase enzyme was purified 34 fold by using DEAE-cellulose anion exchange column chromatography and characterized. Molecular weight of the enzyme was determined as 56 kDa by using Sephadex G-100 gel filtration chromatography. Purification of enzyme was verified by SDS-PAGE. The pH- and temperature optima of enzyme were determined (pH 9.0 and 60°C, respectively). Pectin lyase was mostly stable at 50 oC for 24 hours. Its’ activity decreased to 50 % for 24 h at 60°C. KM and Vmax were calculated as 24.8 mg/mL and 2.28 μmol/L min, respectively. Purified pectin lyase was inhibited by Fe3+, Zn2+, Cu2+, Ca2+, Co2+ and Hg2+ but not by Mg2+. The purified pectin lyase enzyme was used for getting fruits juices. It was found that yields of fruits juices increased when they were compared with control.

Purification and characterization of two xylanases from Chaetomium thermophile var. coprophile

1989 ◽  
Vol 35 (9) ◽  
pp. 836-842 ◽  
Author(s):  
Ramesh K. Ganju ◽  
Paul J. Vithayathil ◽  
S. K. Murthy
Keyword(s):  
Amino Acids ◽  
Enzyme Purification ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Larch Wood ◽  
Hydrolysis Products ◽  
Xylanase Enzyme ◽  
Temperature Optima

Two xylanases (I and II) out of several extracellular xylanases produced by the thermophilic fungus Chaetomium thermophile var. coprophile were purified to homogeneity by a combination of ion exchange and gel filtration chromatographic procedures. They had molecular weights of 26 000 (xylanase I) and 7000 (xylanase II). The temperature optima for xylanase I and II were 70 and 60 °C, and they were optimally active at pH 4.8–6.4 and 5.4–6.0, respectively. Xylanase I was found to be comparatively more stable than xylanase II at higher temperatures. Amino acid composition indicated that xylanase I contained high amounts of glycine, threonine, and low amounts of histidine and sulphur-containing amino acids. Each enzyme released different hydrolysis products from larch wood xylan. Xylanase I produced mainly xylobiose and xylotriose whereas xylanase II produced mainly xylobiose.Key words: Xylanase, enzyme purification, characterization, Chaetomium thermophile.

scholarly journals β-d-Glucosidases and related enzymic activities in pig kidney

1969 ◽  
Vol 111 (5) ◽  
pp. 749-755 ◽  
Author(s):  
Hope E. Abrahams ◽  
D. Robinson
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Active Site ◽  
Gel Electrophoresis ◽  
Small Difference ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Heat Denaturation ◽  
Pig Kidney ◽  
Approximate Molecular Weight

1. The β-glucosidase activity of pig kidney is located in the unsedimentable fraction of the cell and is not associated with the lysosomes. 2. The enzyme is active towards β-d-glucosides, β-d-galactosides, β-d-xylosides and α-l-arabinosides. 3. These activities could not be separated by gel electrophoresis, gel filtration or DEAE-cellulose chromatography. 4. Response to inhibitors, heat-denaturation and competitive substrates suggests that a single active site is responsible for all four activities. 5. Two forms of the enzyme were found to occur either separately or together in kidneys of pigs from several different breeds. 6. Electro-focusing experiments show these to have a small difference in isoelectric point (4·9 and 5·1), and gel filtration gives an approximate molecular weight of 50000 for both forms. 7. The characteristics of these two enzymes are compared.

Turnover of Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits

1981 ◽  
Vol 46 (03) ◽  
pp. 658-661 ◽  
Author(s):  
C Korninger ◽  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Intravenous Injection ◽  
Active Site ◽  
Half Life ◽  
Degradation Products ◽  
Gel Filtration ◽  
Tissue Type ◽  
Organ Distribution ◽  
Small Rise

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

Anticoagulants Produced by Thrombin from Fibrin, the Effect on Blood Coagulation, Some Physical Characteristics

1971 ◽  
Vol 26 (02) ◽  
pp. 211-223 ◽  
Author(s):  
Ch R. Muirhead ◽  
D. C Triantaphyllopoulos
Keyword(s):  
Blood Coagulation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fibrin Clot ◽  
Disc Electrophoresis ◽  
Physical Characteristics ◽  
Advanced Stage ◽  
Kallikrein Inhibitor ◽  
Shed Blood ◽  
Complete Lysis

SummaryChromatographed thrombin in the presence of both 50 Kallikrein inhibitor units of Trasylol per ml and 0.1 M E-ACA solubilized fibrin and the products of lysis possessed anticoagulant properties. The peak of the antithrombic activity coincided with the time of complete lysis of the fibrin clot, plasmin lysed fibrin exhibited the peak of its antithrombic activity much earlier. The effect of thrombin lysed fibrin on the prothrombin consumption of shed blood was found to be inhibitory.The products of the digestion of fibrin by thrombin and by plasmin, isolated at an advanced stage of proteolysis were compared by gel filtration, disc electrophoresis and DEAE cellulose chromatography. Differences in physical characteristics of these fibrin breakdown products offer evidence that they were produced by two different enzymes.

INHIBITION BY HUMAN SERUM OF THE ADIPOKINETIC EFFECT OF A HUMAN PITUITARY LIPID-MOBILIZING FACTOR

1972 ◽  
Vol 71 (3) ◽  
pp. 443-453 ◽  
Author(s):  
Olav Trygstad ◽  
Irene Foss
Keyword(s):  
Human Serum ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Disc Electrophoresis ◽  
Inhibitory Protein ◽  
Human Pituitary ◽  
Albumin Fraction ◽  
Pituitary Glands ◽  
Normal Human

ABSTRACT A lipid-mobilizing factor (LMF) with an adipotrophic effect in human and animal fat tissue has been prepared from human pituitary glands. The addition of normal human serum to LMF reduced its lipolytic effect, and it was completely abolished by serum from a group of obese patients, whereas the lipolysis was not influenced by serum from patients with generalized lipodystrophy. By DEAE-cellulose chromatography of human serum the inhibitory effect on LMF was found to be present in a protein fraction less acidic than the main serum albumin fraction. The inhibitory fraction was deprived of some contaminants by Sephadex gel filtration. Disc electrophoresis demonstrated the presence of three components in the inhibitory protein (IP), and they were identified as albumin, transferin, and haemopexin by immuno-electrophoresis. Precipitation of these proteins by their rabbit antisera demonstrated that the inhibitory effect was present in the albumin fraction. Insulin like activity was not observed in IP. A protein binding of LMF by IP could not be demonstrated. Incubation at 37°C for one hour of a mixture of LMF and IP eliminated the electrophoretic picture of LMF. It is concluded that the inhibitory effect of human serum may be due to proteolysis of LMF.

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

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