scholarly journals Purifikasi dan Karakterisasi α-amilase Termostabil dari Bacillus stearothermophilus TII-12

2011 ◽  
Vol 7 (1) ◽  
pp. 56 ◽  
Author(s):  
Puji Lestari ◽  
Nur Richana ◽  
Abdul Aziz Darwis ◽  
Khaswar Syamsu ◽  
Untung Murdiyatmo
Keyword(s):  
Bacillus Stearothermophilus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Total Activity ◽  
Purification And Characterization ◽  
Food Industries ◽  
High Temperature Process ◽  
Hydrolysis Products ◽  
Starch Liquefaction

<p>Purification and Characterization of Thermostable<br />α-amylase from Bacillus stearothermophilus TII-12. Puji<br />Lestari, Nur Richana, Abdul A. Darwis, Khaswar Syamsu,<br />and Untung Murdiyatmo. Thermostable α-amylase is a<br />potential enzyme employed in the starch processing and<br />widely used in food industries, but this enzyme is still<br />imported. The local enzyme production would be more<br />economist and useful for its broad applications. Here we<br />report α-amylase from indigenous bacteria TII-12 which was<br />purified and characterized, as well as analyzed its hydrolysis<br />product on cassava starch. The enzyme of Bacillus<br />stearothermophilus TII-12 partially purified by ultrafiltration,<br />acetone precipitation and gel filtration (Sephadex G-100)<br />showed the reduced total activity, total protein and yield, but<br />increased the specific activity. The enzyme had a Km of 1,06<br />mg/ml and Vmax of 1,21 mol/min, with optimal activity at pH 7<br />and 90oC. An apparent molecular mass was of 192.932,8<br />Dalton, as estimated by Native-Polyacrylamide Agarose Gel<br />electrophoresis. Its activity was inhibited by the divalent<br />cation chelator such as EDTA and CuSO4 but activated by<br />calcium ion. Hydrolysis products of this enzyme on cassava<br />starch were glucose, dextrin, maltose and oligosaccharides.<br />After 24 hours of hydrolysis, the concentration of glucose<br />and maltose reached 51.970 and 10.090 ppm, respectively.<br />The thermostable α-amylase of TII-12 is an endo-α-amylase<br />and prospective to be applied on starch liquefaction with<br />high temperature process.</p>

scholarly journals The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus

1986 ◽  
Vol 237 (2) ◽  
pp. 415-420 ◽  
Author(s):  
C R Goward ◽  
R Hartwell ◽  
T Atkinson ◽  
M D Scawen
Keyword(s):  
Large Scale ◽  
Bacillus Stearothermophilus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Km Value ◽  
Extraneous Material ◽  
Sepharose 4B

Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.

scholarly journals Purification and Characterization of Endoglucanase from local isolate of Aspergillus flavus

2013 ◽  
Vol 10 (3) ◽  
pp. 844-853
Author(s):  
Baghdad Science Journal
Keyword(s):  
Aspergillus Flavus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Stability ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Original Activity ◽  
A Value ◽  
Optimum Ph

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.

scholarly journals Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase

1980 ◽  
Vol 191 (1) ◽  
pp. 117-124 ◽  
Author(s):  
R Zecher ◽  
H U Wolf
Keyword(s):  
Specific Activity ◽  
Total Activity ◽  
Partial Purification ◽  
Half Maximum ◽  
Purification And Characterization ◽  
Dimeric Molecule ◽  
Maximum Inhibition ◽  
Optimum Ph ◽  
Triton X

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5′-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.

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.

Studies on the Purification and Characterization of Human Urinary Plasminogen and Plasmin

1979 ◽  
Vol 42 (05) ◽  
pp. 1536-1547
Author(s):  
Hsin Fu Chen ◽  
Masao Nakabayashi ◽  
Kazuo Satoh ◽  
Shoichi Sakamoto
Keyword(s):  
Disseminated Intravascular Coagulation ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Gel Filtration ◽  
New Method ◽  
Chronic Glomerulonephritis ◽  
Purification And Characterization ◽  
Intravascular Coagulation ◽  
Human Plasminogen

SummaryA new method is described for the preparation of highly purified human plasminogen and plasmin with specific activity of 32 CTA units per mg of protein. With this method, the purification of the urinary plasminogen + plasmin antigenic materials from patients with chronic glomerulonephritis, disseminated intravascular coagulation syndrome and severe toxemia of pregnancy was performed, and the resulting highly purified proenzyme and enzyme were analyzed by immunoelectrophoresis, separative agar electrophoresis, gel filtration and SDS-gel electrophoresis.Our findings indicated that urinary plasmin reflects more closely the extent of intraglomerular fibrinolysis, while urinary plasminogen reflects non-selective proteinuria in patients with chronic glomerulonephritis or severe toxemia of pregnancy.

scholarly journals Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

1996 ◽  
Vol 316 (3) ◽  
pp. 841-846 ◽  
Author(s):  
Stuart M. PITSON ◽  
Robert J. SEVIOUR ◽  
Barbara M. McDOUGALL ◽  
Bruce A. STONE ◽  
Maruse SADEK
Keyword(s):  
Molecular Mass ◽  
Filamentous Fungus ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Eisenia Bicyclis ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Sds Page ◽  
Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

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.

Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

1982 ◽  
Vol 47 (4) ◽  
pp. 1139-1148 ◽  
Author(s):  
Karel Hauzer ◽  
Linda Servítová ◽  
Tomislav Barth ◽  
Karel Jošt
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

scholarly journals Purification and characterization of versatile peroxidase from Lentinus squarrosulus and its application in biodegradation of lignocellulosics

2019 ◽  
Vol 6 (6) ◽  
pp. 280-286
Author(s):  
Aarthi Ravichandran ◽  
Ramya G Rao ◽  
Maheswarappa Gopinath ◽  
Manpal Sridhar
Keyword(s):  
Finger Millet ◽  
Nutritive Value ◽  
Crop Residues ◽  
Specific Activity ◽  
Gel Filtration ◽  
Versatile Peroxidase ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Km Value

Versatile Peroxidases are high redox potential peroxidases capable of degrading lignin of lignocellulosic crop residues. Hence Versatile Peroxidases are prominent biocatalysts in upgrading lignocellulosic biomass for biotechnological applications. In the interest of exploiting the potential of Versatile Peroxidase in improving the digestibility of crop residues through delignification, a novel Versatile Peroxidase was purified and characterized from the immobilized cultures of native isolate Lentinus squarrosulus. The enzyme was purified with a specific activity of 62 U/mg through ion exchange and gel filtration chromatographic procedures. The enzyme possessed high affinity towards RB5 and manganese with a Km value of 6.84 µM for RB5 and 0.15 mM for manganese. The optimum temperature for oxidation was identified to be 30°C and optimum pH for manganese and RB5 oxidation was 5 and 3 respectively. Reactivity of the enzyme towards diverse substrates was investigated besides studying the effect of metal ions and inhibitors on RB5 oxidation. The enhanced potential of this purified Versatile Peroxidase in biodegradation of crop residues was demonstrated through augmentation of digestibility of finger millet and paddy straws by 20%.The results demonstrated that Versatile Peroxidase from Lentinus squarrosulus is capable of enhancing the nutritive value of crop residues through delignification

Purification and characterization of the extracellular proteinase ofPseudomonas fluorescensNCDO 2085

1986 ◽  
Vol 53 (3) ◽  
pp. 457-466 ◽  
Author(s):  
David J. Fairbairn ◽  
Barry A. Law
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Extracellular Proteinase ◽  
Original Activity ◽  
Heat Stable ◽  
D Values

SUMMAEYPseudomonas fluorescensNCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 °C and pH 7·0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3·5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5·40±0·05 and a mol. wt of 40200±2100. It is heat-stable having D-values at 74 and 140 °C of 1·6 and 1·0 min respectively; 40 and 70% of the original activity remained after HTST (74 °C/17 s) and ultra high temperature (140°C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from otherPseudomonasspp.

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