Purification and thermodynamic characterization of glucose oxidase from a newly isolated strain of Aspergillus niger

2006 ◽  
Vol 52 (6) ◽  
pp. 519-524 ◽  
Author(s):  
H N Bhatti ◽  
M Madeeha ◽  
M Asgher ◽  
N Batool
Keyword(s):  
Aspergillus Niger ◽  
Glucose Oxidase ◽  
Specific Activity ◽  
Gel Filtration ◽  
High Specificity ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Analytical Reagent ◽  
Intracellular Glucose

An intracellular glucose oxidase (GOD) was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger NFCCP. The enzyme was partially purified to a yield of 28.43% and specific activity of 135 U mg–1 through ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The enzyme showed high specificity for D-glucose, with a Km value of 25 mmol L–1. The enzyme exhibited optimum catalytic activity at pH 5.5. Optimum temperature for GOD-catalyzed D-glucose oxidation was 40 °C. The enzyme displayed a high thermostability having a half-life (t1/2) of 30 min, enthalpy of denaturation (H*) of 99.66 kJ mol–1, and free energy of denaturation (G*) of 103.63 kJ mol–1. These characteristics suggest that GOD from A. niger NFCCP can be used as an analytical reagent and in the design of biosensors for clinical, biochemical, and diagnostic assays.Key words: glucose oxidase, Aspergillus niger, kinetics, thermodynamics, thermal stability.

scholarly journals Pemurnian Parsial dan Karakterisasi Enzim Xilanase dari Bakteri Laut Bacillus safencis strain LBF P20 Asal Pulau Pari Jakarta

2017 ◽  
Vol 37 (1) ◽  
pp. 31
Author(s):  
Fitria Fitria ◽  
Nanik Rahmani ◽  
Sri Pujiyanto ◽  
Budi Raharjo ◽  
Yopi Yopi
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Centrifugal Filter ◽  
Sds Page ◽  
Enzyme Specific Activity ◽  
Hydrolysis Of

Enzyme xylanase (EC 3.2.1.8) is widely used in various industrial  fields for the hydrolysis of xylan (hemicellulose) into xylooligosaccharide and xylose. The aims of this study were to  conduct partial purification and characterization of xylanase from marine Bacillus safencis strain LBF P20 and to obtain the  xylooligosaccharide types from xylan hydrolysis by this enzyme.  Based on this research, the optimum time for enzyme production  occurred at 96 hours with the enzyme activity of 6.275 U/mL and  enzyme specific activity of 5.093 U/mg. The specific activities were  obtained from precipitation by amicon® ultra-15 centrifugal filter devices, gel filtration chromatography and anion exchange chromatography that were increased by 15.07, 34.7, and 96.0  U/mg. The results showed that the highest activity at pH 7, temperature of 60 °C, and stable at 4 °C. Type of  xylooligosaccharide produced by this study were xylohexoses, xylotriose, and xylobiose. SDS-PAGE analysis and zimogram  showed that the molecular weight of xylanase protein were about  25 kDa. ABSTRAKEnzim xilanase (EC 3.2.1.8) digunakan dalam hidrolisis xilan  (hemiselulosa) menjadi xilooligosakarida dan xilosa. Penelitian  ini bertujuan untuk melakukan purifikasi parsial dan karakterisasi xilanase dari bakteri laut Bacillus safencis strain LBF P20 serta uji  hidrolisis untuk mengetahui jenis xilooligosakarida yang  dihasilkan oleh enzim tersebut. Berdasarkan hasil penelitian, waktu optimum untuk produksi enzim terjadi pada jam ke 96  dengan aktivitas enzim sebesar 6,275 U/mL dan aktivitas spesifik enzim sebesar 5,093 (U/mg). Aktivitas spesifik enzim hasil  pemekatan dengan amicon® ultra-15 centrifugal filter devices,  kromatografi filtrasi gel dan kromatografi penukar anion  mengalami peningkatan berturut-turut sebesar 15,1; 34,7 dan96,0 U/mg. Hasil karakterisasi menunjukkan aktivitas  tertinggi pada pH 7, suhu 60 °C dan stabil pada suhu 4 °C. Analisis SDS-PAGE dan zimogram menunjukkan berat molekul protein xilanase berkisar 25 kDa. Jenis gula reduksi yang  dihasilkan yaitu xiloheksosa, xilotriosa, dan xilobiosa.

Kinetic and Thermodynamic Characterization of Lignin Peroxidase Isolated from Agaricus bitorqus A66

2012 ◽  
Vol 10 (1) ◽  
Author(s):  
Ismat Bibi ◽  
Haq Nawaz Bhatti
Keyword(s):  
Lignin Peroxidase ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Gel Filtration ◽  
Veratryl Alcohol ◽  
Exchange Chromatography ◽  
Different Temperatures ◽  
Scale Experiment ◽  
Menten Kinetic

This study deals with purification and characterization of lignin peroxidase (LiP) isolated from Agaricus bitorqus A66 during decolorization of NOVASOL Direct Black dye. A laboratory scale experiment was conducted for maximum LiP production under optimal conditions. Purification & fractionation of LiP was performed on DEAE-Sepharose ion exchange chromatography followed by Sephadex G-50 gel filtration. The purified LiP has a specific activity of 519 U/mg with 6.73% activity recover. The optimum pH and temperature of purified LiP for the oxidation of veratryl alcohol were 6.8 and 45 °C, respectively. Michaelis-Menten kinetic constants (Vmax and Km) were determined using different concentrations of veratryl alcohol (1-35 mM). The Km and Vmax were 16.67 mM and 179.2 U/mL respectively, for veratryl alcohol oxidation as determined from the Lineweaver-Burk plot. Thermal inactivation studies were carried out at different temperatures to check the thermal stability of the enzyme. Enthalpy of activation decreased where Free energy of activation for thermal denaturation increased at higher temperatures. A possible explanation for the thermal inactivation of LiP at higher temperatures is also discussed.

scholarly journals PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

2017 ◽  
Vol 18 (2) ◽  
pp. 1-10 ◽  
Author(s):  
Dzun Noraini Jimat ◽  
Intan Baizura Firda Mohamed ◽  
Azlin Suhaida Azmi ◽  
Parveen Jamal
Keyword(s):  
Specific Activity ◽  
Hot Spring ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Bacillus Sp ◽  
Sds Page ◽  
Fast Flow ◽  
Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60°C.

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 Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants (Camellia sinensis (L.) O. Kuntze)

2005 ◽  
Vol 37 (6) ◽  
pp. 363-370 ◽  
Author(s):  
Ye-Yun Li ◽  
Chang-Jun Jiang ◽  
Xiao-Chun Wan ◽  
Zheng-Zhu Zhang ◽  
Da-Xiang Li
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Optimum Activity ◽  
Development Of Resistance ◽  
Sds Page ◽  
C 50 ◽  
Tea Plants

Abstractβ-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity, with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50 °C and was stable at temperatures lower than 40 °C. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.

scholarly journals Hyaluronidase in ram semen. Quantitative determination, and isolation of multiple forms

1988 ◽  
Vol 252 (3) ◽  
pp. 865-874 ◽  
Author(s):  
R A Harrison
Keyword(s):  
Ionic Strength ◽  
Quantitative Determination ◽  
Specific Activity ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
High Yield ◽  
Poly Vinyl Alcohol ◽  
The Media ◽  
Low Ionic Strength

A study was made of hyaluronidase in ram semen. The end-group assay conditions used to determine activity quantitatively were chosen to ensure reliability as well as sensitivity [Gacesa, Savitsky, Dodgson & Olavesen (1981) Anal. Biochem. 118, 76-84]; they led to 1 W.H.O. Standard International Hyaluronidase Unit displaying 0.1263 EC munit (1 EC unit of activity releases 1 mumol equivalent of N-acetylglucosamine end groups/min at 37 degrees C). All the activity in the semen was shown to be sperm-derived, and intact spermatozoa were estimated to contain 1.23 EC units per 10(9) cells. In a low-ionic-strength medium, only some 20% of the hyaluronidase was extractable, although up to 80% of the activity could be extracted as the ionic strength was increased; further addition of detergent extracted the remainder. During purification of the enzyme, it was found that inclusion of poly(vinyl alcohol) in the media stabilized the activity; detergent inclusion also improved the yield, especially during early stages. As a consequence both of reliable quantitative determination and of stabilization, a number of forms of hyaluronidase could be isolated in high yield, by using anion-exchange chromatography, cation-exchange chromatography, affinity chromatography and gel filtration. The existence of all these forms was confirmed by electrophoresis and immunoblotting with the use of a monoclonal anti-(ram hyaluronidase) antibody, and their presence in very freshly prepared sperm extracts was demonstrated. The specific activity of the isolated major hyaluronidase form was 15.0 EC units/mg; this was equivalent to 119,000 W.H.O. units/mg, higher than any other previously reported values.

Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation

1994 ◽  
Vol 40 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Andreas Prokop ◽  
Peter Rapp ◽  
Fritz Wagner
Keyword(s):  
Molecular Mass ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glucanase Activity ◽  
Sds Page ◽  
S 100 ◽  
Reduced Activity

Production of extracellular β-1, 3-glucanase activity by a monokaryotic Schizophyllum commune strain was monitored and results indicated that the β-glucanase activity consisted of an endo- β-1, 3-glucanase activity, besides a negligible amount of β-1, 6-glucanase and β-glucosidase activity. Unlike the β-1, 3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the β-1, 3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo- β-1, 3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing β-1, 3-linkages, including lichenan, a β-1, 3-1, 4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular β-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo- β-1, 3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass β-1, 3-/β-1, 6-glucan. The Km of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50 °C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50 °C. The enzyme was completely inhibited by 1 mM Hg2+. Growth of the monokaryotic S. commune strain was not affected by its constitutive endo- β-1, 3-glucanase formation.Key words: endo- β-1, 3-glucanase, Schizophyllum commune, monokaryon, constitutive endo- β-1, 3-glucanase formation.

scholarly journals PURIFICATION AND CHARACTERIZATION OF SALIVARY AMYLASE INHIBITOR EXTRACTED FROM BARLEY

2016 ◽  
Vol 47 (4) ◽  
Author(s):  
Abood & Hakeem
Keyword(s):  
Ammonium Sulfate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Alpha Amylase ◽  
Ionic Exchange ◽  
Inhibition Activity ◽  
Amylase Inhibitors

Amylase inhibitors were purified by many sequential steps included concentration by gradual addition of ammonium sulfate at  saturation ratios. ranged from 0 to 90% . The best ratio of saturation was found to be 70% as the specific activity and inhibition activity toward Human alpha-amylase(HAS)  were the highest ( 8 U/mg and 6 U/ml respectively as compared to those of the rest ratios, the ratio of saturation with ammonium sulfate 60 % and then 50%, (5.8 ,5.5  )U/ml and( 7.7 ،7 )U/mg respectively for inhibition activity and specific activity and for  40% ,30%20%  saturation  the inhibition activity and specific activity were(5 ،4.8 ،4 ) u/ml (6.6 ،6 ،5.8) u/mg respectively .The precepitation step was followed by ionic exchange chromatography technique by DEAE-cellulose column( 3×11 )cm and the results showed that there was one peak with inhibition activity toward (HAS). Further  purification steps were conducted using gel filtration on Sephacryl S-200 column    (1.5  ×  60)cm; the purification folds was5.59 times with outcome of 46.5%.The results of alpha-amylase inhibitors characterization showed that the molecular weight was about 23.44 and 22.9  kDa  as determined by electrophoresis and gel filteration respectively.                                         

Purification and Partial Characterization of Cellulase Produced by Aspergillus niger Cultured on Vitellaria paradoxa shells

2017 ◽  
Vol 6 (2) ◽  
Author(s):  
Abdulhakeem Olarewaju Sulyman ◽  
Yusuf A Iyanda ◽  
Afolabi Olaniyi Opasola ◽  
OtunOla Adedayo ◽  
Raliat Abimbola Aladodo
Keyword(s):  
Aspergillus Niger ◽  
Specific Activity ◽  
Gel Filtration ◽  
Inoculum Size ◽  
Partial Characterization ◽  
Effect Of Temperature ◽  
Vitellaria Paradoxa ◽  
Endoglucanase Activity ◽  
Ammonium Sulphate Precipitation

This research investigated the purification and partial characterization of cellulase produced by Aspergillus niger cultured on Vitellaria paradoxa shells. Cellulase (endoglucanase) from A. niger was produced under optimum fermentation conditions at 35 °C, pH 4.7, V. paradoxa, 4 g/L, inoculum size of 10 mm and the fermentation media incubated for 120 hours. The crude endoglucanase obtained were partially purified by subjecting to ammonium sulphate precipitation, dialysis and gel filtration chromatography for further purification. The effect of temperature and pH on the activity of purified endoglucanase was determined. Cellulase was purified to 734.33 folds by Sephadex G-100 column chromatography with a specific activity and yield of 4.406 U/mg and 63.03% respectively. Fractions 4 and 7 contained the highest endoglucanase activity out of 18 fractions collected and the two fractions were pooled for further analysis. The activity of purified endoglucanase was optimum at a temperature of 40 °C and pH 5. Therefore, the purified endoglucanase produced may be explored in detergent industry.

scholarly journals Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

2002 ◽  
Vol 68 (4) ◽  
pp. 1980-1987 ◽  
Author(s):  
Yolanda Sanz ◽  
Fidel Toldrá
Keyword(s):  
Specific Activity ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Sulfhydryl Group ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Lactobacillus Sakei ◽  
C Terminus ◽  
Arginine Aminopeptidase

ABSTRACT An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu2+, Hg2+, and Zn2+) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The Km values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.

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