Purification, immobilization and characterization of thermostable α-amylase from a thermophilic bacterium Geobacillus sp. TF14

2017 ◽  
Vol 42 (6) ◽  
Author(s):  
Şaban Keskin ◽  
Nagihan Saglam Ertunga
Keyword(s):  
Ion Exchange ◽  
Hydrophobic Interaction ◽  
Hydrophobic Interaction Chromatography ◽  
Maximal Activity ◽  
Thermophilic Bacterium ◽  
Single Band ◽  
Sds Page

AbstractObjective:In this study, α-amylase from a thermophilic bacterium Geobacillus sp. TF14 was purified and immobilized on two different supports.Methods:Ion exchange and hydrophobic interaction chromatography techniques were employed for the purification.Results:The enzyme was purified as 17.11 fold and determined as a single band of 54 kDa on SDS-PAGE. Purified enzyme showed two pH optimums of pH 5.00 and pH 9.00 and the enzyme is quite stable at these pHs over a period of 48 h. Purified enzyme showed maximal activity at 75°C and stability at this temperature over a period of 72 h. It was observed that CaConclusion:It can be concluded that the purified enzyme may find application in many fields of starch based industries.

scholarly journals ISOLASI DAN KARAKTERISASI ENZIM b-1,3-ENDOGLUKANASE DARI TANAMAN KUBIS (Brassica oleracea cv. Capitata L.)

2010 ◽  
Vol 15 (2) ◽  
pp. 99-105
Author(s):  
Y. Sri Manuhara
Keyword(s):  
Ion Exchange ◽  
Brassica Oleracea ◽  
Hydrophobic Interaction Chromatography ◽  
Inhibition Zone ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Optimum Ph

Isolation and characterization of β-1,3-endoglucanase from cabbage (Brassica oleracea var. capitata L.) have been done. It showed 40° C of optimum temperature, and optimum pH is 7. After the purification with hydrophobic interaction chromatography and ion exchange chromatography, it’s activity was increased. Based on SDS-PAGE analysis, β-1,3-endoglucanase have molecular weight around 48 kD. Antifungal activity of β-1,3-endoglukanase show that it has best inhibition zone on Fusarium solanii at extract from ion exchange chromatography.

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

Prediction of protein retention times in hydrophobic interaction chromatography by robust statistical characterization of their atomic-level surface properties

2016 ◽  
Vol 32 (2) ◽  
pp. 372-381 ◽  
Author(s):  
Alexander T. Hanke ◽  
Marieke E. Klijn ◽  
Peter D. E. M. Verhaert ◽  
Luuk A. M. van der Wielen ◽  
Marcel Ottens ◽  
...  
Keyword(s):  
Surface Properties ◽  
Hydrophobic Interaction ◽  
Hydrophobic Interaction Chromatography ◽  
Atomic Level ◽  
Level Surface ◽  
Statistical Characterization ◽  
Protein Retention

Preparation and Characterization of Cellulose Based Adsorbents for Large Scale Hydrophobic Interaction Chromatography

1994 ◽  
Vol 17 (4) ◽  
pp. 749-760 ◽  
Author(s):  
A. Šėrys ◽  
J. Liesienė ◽  
J. Urbonavičienė ◽  
A. Maruška ◽  
K. Radzevičius ◽  
...  
Keyword(s):  
Hydrophobic Interaction ◽  
Large Scale ◽  
Hydrophobic Interaction Chromatography

scholarly journals Biochemical Characterization of a Thiol-Activated, Oxidation Stable Keratinase from Bacillus pumilus KS12

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Rinky Rajput ◽  
Richa Sharma ◽  
Rani Gupta
Keyword(s):  
Ion Exchange ◽  
Bacillus Pumilus ◽  
Biochemical Characterization ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amidolytic Activity ◽  
Alkaline Serine Protease ◽  
Sds Page ◽  
Temperature Optima

An extracellular keratinase from Bacillus pumilus KS12 was purified by DEAE ion exchange chromatography. It was a 45 kDa monomer as determined by SDS PAGE analysis. It was found to be an alkaline, serine protease with pH and temperature optima of 10 and 60C, respectively. It was thiol activated with two- and eight-fold enhancement in presence of 10 mM DTT and β-mercaptoethanol, respectively. In addition, its activity was stimulated in the presence of various surfactants, detergents, and oxidizing agents where a nearly 2- to 3-fold enhancement was observed in presence of H2O2 and NaHClO3. It hydrolyzed broad range of complex substrates including feather keratin, haemoglobin, fibrin, casein,and α-keratin. Analysis of amidolytic activity revealed that it efficiently cleaved phenylalanine → leucine → alanine- p-nitroanilides. It also cleaved insulin B chain between Val2- Asn3, Leu6-Cys7 and His10-Leu11 residues.

Characterization of denatured proteins by hydrophobic interaction chromatography: A preliminary study

Biopolymers ◽  
2003 ◽  
Vol 69 (3) ◽  
pp. 293-300 ◽  
Author(s):  
Emilia Bramanti ◽  
Fabrizio Ferri ◽  
Chandra Sortino ◽  
Massimo Onor ◽  
Giorgio Raspi ◽  
...  
Keyword(s):  
Hydrophobic Interaction ◽  
Hydrophobic Interaction Chromatography ◽  
Denatured Proteins ◽  
Preliminary Study
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