scholarly journals Purification and Characterization of Amyloglucosidase Enzyme from the Thermophilic Endomycopsis fibuligera Using Sago Starch as a Substrate

2018 ◽  
Vol 16 (3) ◽  
pp. 302
Author(s):  
Ahyar Ahmad ◽  
Harningsih Karim
Keyword(s):  
Ammonium Sulphate ◽  
Deae Cellulose ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Degree Of Saturation ◽  
Sago Starch ◽  
Purification And Characterization ◽  
Ammonium Sulphate Fractionation ◽  
Crude Extract Enzyme

An investigation on purification and characterization of amyloglucosidase enzyme from Endomycopsis fibuligera by fermentation of sago starch has been carried out. This enzyme is inductive and can be produced by fermenting sago starch in a medium containing E. fibuligera. Crude enzyme was obtained by centrifuging the medium cultures containing E. fibuligera at 6,000 rpm for 20 min and then adding with 0.15 M acetate buffer (pH 5.0). Enzyme activity was determined using Somogyi-Nelson method by quantifying the released glucose from amyloglucosidase catalysis of starch (0.2%) as substrate. Prepurification process was conducted by ammonium sulphate fractionation and it showed that the ammonium sulphate fractionation with the degree of saturation of 40-60% produced a maximum activity of enzyme. Purification by DEAE-Cellulose and Sephadex G-75 column chromatography produced three and one fractions with purifity 17.4 and 22.5 times, respectively, compared to the crude extract enzyme. Characterization of this enzyme showed the optimum condition at pH 5.0 and 55 °C with 0.2% starch as substrate. The amyloglucosidase activities was strongly increased by addition of Co2+ and Mn2+ ions, whereas the activities were weakly decreased by addition of K+, Mg2+, and Fe3+ ions.

scholarly journals Purification and characterization of amylase from local isolate Pseudomonas sp.SPH4

2012 ◽  
Vol 6 (1) ◽  
pp. 69-79
Author(s):  
Hala M. Ali ◽  
Ghazi M. Aziz
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Silver Ions ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Mercury Ions ◽  
Local Isolate ◽  
Optimum Ph

The amylase produced from local isolate Pseudomonas sp. SPH4 was purified by precipitation with 30% saturation ammonium sulphate, followed by ion-exchange chromotography using DEAE-cellulose column, and Gel filtration using Sephacryl S-300 column.The two iso-enzymes (a, b) were purified to (2.83, 3.47) times in the last step with an enzymes yields of (32.36, 76.34)% respectively. Enzyme characterization of the two iso-enzymes indicated that the optimum pH for the two iso-enzymes a and b were (7, 7.5) respectively, while the optimum pH for the iso-enzymes stability were (6.5, 7) respectively. The maximum activity for iso-enzymes (a, b) appeared at 45ºC and stable for 15 min at 30-50ºC and lost approximately 50% of it's activity at rang above 75ºC. Enzyme characterization results showed that the chlorides of silver and mercury had inhibitory effect on enzyme activity, the remaining enzyme activity for the iso-enzymes (a, b) were (46.66, 36.36)% for silver ions and (41.33, 33.63)% for mercury ions at 5 mM respectively, and (28, 28.18)% for silver ions and (25.33, 19.09)% for mercury ions at 10 mM respectively. The iso-enzymes a and b were affected by chelating agent ethylene diamine tetra acetic acid (EDTA) at concentration 2mM the remaining activity (45.33, 43.63)% respectively, and 5mM the remaining activity (28, 28.18)% respectivily, and these iso-enzymes (a, b) refered to metalloenzymes. The iso-enzymes (a, b) were kept their activity when treated by reducing agent (2-mercaptoethanol) at 2 mM the remaining activity (92, 92.72)% respectively, and 5 mM the remaining activity (85.3, 89.09)% respectivily. The iso-enzymes (a, b) were kept their activity when treated by phenyl methyl sulphonyl fluoride (PMSF) at concentration 1mM the remaining activity (93.33, 90.90)% respectivily,and 5 mM the remaining activity (90.66, 87.27)% respectivily, and these indicated that these iso-enzymes didnot referred to serineamylases group.

scholarly journals Purification and characterization of a heterogeneous glycosylated invertase from the rumen holotrich ciliate Isotricha prostoma

1989 ◽  
Vol 264 (3) ◽  
pp. 721-727 ◽  
Author(s):  
T Dauvrin ◽  
D Thinès-Sempoux
Keyword(s):  
Enzyme Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Product Inhibition ◽  
Maximum Activity ◽  
Kinetic Properties ◽  
Transferase Activity ◽  
Purification And Characterization ◽  
Sepharose 4B

The invertase (beta-fructofuranosidase, EC 3.2.1.26) of the rumen holotrich ciliate Isotricha prostoma has been purified. This is the first report of an enzyme purification from a known species of rumen protozoon. Cells were disrupted by ultrasonic treatment and the enzyme was purified from the cell-free extract by three successive liquid column chromatographies (Sepharose CL4B/octyl-Sepharose CL4B, DE52 DEAE-cellulose and concanavalin A-Sepharose 4B). This resulted in a 160-fold purification and a 15% yield. The major form of the purified enzyme was a tetramer with Mr about 350,000 that was readily dissociated by electrophoresis. The invertase was heterogeneous, as five types of monomers were shown by SDS/polyacrylamide-gel electrophoresis after denaturation. Part of this heterogeneity was due to different glycosylated forms of one of the polypeptides present in the purified enzyme. Isotricha prostoma invertase exhibited maximum activity at pH 5.5-6.0 and 50 degrees C. The kinetic properties of the purified enzyme were very similar to those of invertases from other sources such as yeast or plants (substrate and product inhibition, transferase activity).

scholarly journals Pemurnian dan Karakterisasi Enzim Lipase dari Aspergillus oryzae pada Kopra Berjamur

2012 ◽  
Vol 14 (1) ◽  
pp. 26
Author(s):  
Seniwati Dali Dali ◽  
Abdul Rauf Patong ◽  
Muhammad Noor Jalaluddin ◽  
Pirman Andi Parenrengi
Keyword(s):  
Ammonium Sulphate ◽  
Aspergillus Oryzae ◽  
Enzyme Purification ◽  
Maximum Activity ◽  
Crude Enzyme ◽  
Lipase Catalysis ◽  
Lipase Enzyme ◽  
Km Value ◽  
Ammonium Sulphate Fractionation

An investigation on purification and characterization of lipase enzyme production Aspergillus oryzae from copra by fermentation of oliveoil has been carried out. This enzyme can be produced by fermenting olive oil in a medium containing Aspergillus oryzae. Crude enzyme isobtained by centrifuging the medium cultures containing Aspergillus oryzae at 3500 rpm for 30 minutes and than adding 0.2 M borat buffer(pH 8.2). Enzyme activity was determined from paranitrophenol as product of lipase catalysis of paranitrophenylbutirat (0.2 M) assubstrate measured by the Vorderwulbecke method. Prepurification process was by ammonium sulphate fractionation. Precipitation60-80% ammonium sulphate produced maximum activity of enzyme. Purification by Q sepharosa FF and sephadex G-75 collumchromatography produced four and three fractions with purifity of 12.85 and 20.25 times than crude enzyme respectively. Characterizationof this enzyme showed optimum condition at pH 8.2, temperature at 350C, the Km value at 0.046 M, and Vmaks is 1.926 μmol/menit and themolecular weight at 40.7 kDa.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

Purification and Characterization of Two Proteins from Culture Filtrates of Mycobacterium tuberculosis H 37 Ra Strain

1970 ◽  
Vol 1 (2) ◽  
pp. 164-168
Author(s):  
Thomas M. Daniel ◽  
Lavenia E. Ferguson
Keyword(s):  
Molecular Weight ◽  
Mycobacterium Tuberculosis ◽  
Skin Testing ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Purification And Characterization ◽  
Culture Filtrates ◽  
Zone Electrophoresis

Two proteins have been purified from culture filtrates of Mycobacterium tuberculosis , H 37 Ra strain by a procedure combining gel filtration, diethylaminoethyl (DEAE)-cellulose chromatography, and zone electrophoresis. The two proteins are similar in molecular weight but differ slightly in charge. The faster migrating protein, designated a 1 , is not antigenic. The slower migrating protein, designated a 2 , is antigenic both with respect to antisera and as a skin-testing antigen.

scholarly journals Detection, purification and characterization of a human cancer-associated galactosyltransferase acceptor

1979 ◽  
Vol 178 (2) ◽  
pp. 279-287 ◽  
Author(s):  
D K Podolsky ◽  
M M Weiser
Keyword(s):  
Molecular Weight ◽  
Human Cancer ◽  
Deae Cellulose ◽  
Structural Data ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Purification And Characterization ◽  
Peptide Moiety ◽  
Cellulose Column

A low-molecular-weight acceptor of galactosyltransferase activity was detected in sera and effusions of patients with extensive maligant disease. This substance was purified to homogeneity from both human serum and effusion by using sequential charcoal/Celite and DEAE-cellulose column chromatography. The purified acceptor was shown to act as substrate for both purified normal and cancer-associated human galactosyltransferase (EC 2.4.1.22) isoenzymes, but had a higher affinity for the cancer-associated isoenzyme (Km = 20 microM) than for the normal isoenzyme (Km = 500 microM). The substrate was found to be a glycopeptide with mol.wt. approx. 3600 determined by polyacrylamide-gel chromatography. Carbohyydate analysis demonstrated only the presence of glucosamine and mannose. Amino acid analysis revealed that the peptide moiety consisted of eight different amino acids, including two residues of asparagine and one residue of serine, but no threonine. These structural data suggest that the acceptor is a fraction of an asparagine-glucosamine type of glycoprotein.

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 An inducible hydrolase from Aspergillus niger, acting on carbon–carbon bonds, for phlorrhizin and other C-acylated phenols

1970 ◽  
Vol 116 (5) ◽  
pp. 889-897 ◽  
Author(s):  
T. Minamikawa ◽  
N. P. Jayasankar ◽  
B. A. Bohm ◽  
I. E. P. Taylor ◽  
G. H. N. Towers
Keyword(s):  
Aspergillus Niger ◽  
Metal Ion ◽  
Deae Cellulose ◽  
Hydrolytic Activity ◽  
Maximum Activity ◽  
Protamine Sulphate ◽  
Carbon Carbon Bonds ◽  
Alkaline Conditions ◽  
Km Value ◽  
Ammonium Sulphate Fractionation

1. An inducible enzyme catalysing the hydrolysis of phloretin to form phloroglucinol and phloretic acid has been extracted from the acetone-dried powders of the mycelial felts of an Aspergillus niger strain grown in the presence of phlorrhizin. The enzyme was partially purified by treatment with protamine sulphate, ammonium sulphate fractionation, negative adsorption on tricalcium phosphate gel, and DEAE-cellulose column chromatography. 2. The hydrolytic activity on phloretin appeared to be maximal at about pH9.6. However, the characteristics of the enzyme were studied at pH7.2, because of the lability of the product, phloroglucinol, under alkaline conditions. 3. The apparent Km value at pH7.2 was about 0.3–0.4mm for phloretin and 0.15mm for 3′-methylphloracetophenone. 4. Maximum activity of the enzyme was obtained without the addition of any cofactor or metal ion. The involvement of thiol groups in the reaction was demonstrated by the potent inhibitory action of both heavy-metal ions and p-chloromercuribenzoate. 5. The enzyme showed a rather broad substrate specificity, and some other C-acylated phenols related to phloretin were hydrolysed. It was found that 3′-methylphloracetophenone, phloracetophenone and 2′,4,4′-trihydroxydihydrochalcone were attacked more efficiently than phloretin. We propose the systematic name C-acylphenol acylhydrolase for the enzyme. This enzyme belongs to EC group 3.7.1.

scholarly journals Purification and characterization of the heat-stable serine proteinase from Thermomonospora fusca YX

1987 ◽  
Vol 246 (2) ◽  
pp. 511-517 ◽  
Author(s):  
T W Gusek ◽  
J E Kinsella
Keyword(s):  
Thermal Stability ◽  
Ionic Strength ◽  
Serine Proteinase ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Purification And Characterization ◽  
Thermomonospora Fusca ◽  
Heat Stable

The proteinase secreted from Thermomonospora fusca YX grown on cellulose was purified by (NH4)2SO4 fractionation and cation-exchange chromatography. The isolated proteinase readily hydrolysed several proteins and demonstrated activity towards casein from 35 to 95 degrees C (at pH 8.0) with maximum activity at 80 degrees C. It exhibited broad pH and ionic-strength optima centered at pH 9.0 and 0.2 M-NaCl respectively, and it retained high activity in the presence of 2% (w/v) SDS, 20 mM-dithiothreitol and 1.0 M-NaCl. The proteinase, which was fully inhibited by phenylmethanesulphonyl fluoride, had an Mr of 14,500 and an isoelectric point at 9.21. A measurement of proteinase thermal stability demonstrated a T50% (15 min) of 85 degrees C at pH 4.5.

Purification and Characterization of Limit Dextrinase from Malted Barley

2012 ◽  
Vol 550-553 ◽  
pp. 1747-1754
Author(s):  
Ya Li Peng ◽  
Fei Hu
Keyword(s):  
Metal Ion ◽  
Ph Value ◽  
Maximum Activity ◽  
Effect Of Temperature ◽  
Purification And Characterization ◽  
Crude Extracts ◽  
Extraction And Purification ◽  
Sds Page ◽  
Limit Dextrinase

Limit dextrinase is one of three main amylases in malted barley, which plays a significant role during the mashing stage of brewing. Due to very low content and similar properties compared to other amylases in malted barley, limit dextrinase is hard to separate effectively. Our work had been directed towards the extraction and purification of limit dextrinase from malted barley. Final products were obtained through fraction precipitation with ammonium sulfate and column chromatography, and purified limit dextrinase acquired a high purity of 31.23 times as much as that of crude extracts. The previous results were also confirmed by sodiumdodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE) revealing a single band of protein (~97KDa). Effect of temperature, pH value, and metal ion on hydrolysis characterization of limit dextrinase was investigated. The results indicated that the maximum activity of purified samples changed significantly compared with that of crude extracts. The activity of purified limit dextrinase could be activated by lower concentration of Mg2+、Ca2+、Mn2+ and inhibited by the action of Zn2+、Fe2+. But this influence was not so obvious for K+.

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