Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
1.
Electron. j. biotechnol ; 27: 84-90, May. 2017. graf, ilus, tab
Article in English | LILACS | ID: biblio-1010412

ABSTRACT

Background: Iron magnetic nanoparticles have attracted much attention. They have been used in enzyme immobilization because of their properties such as product is easily separated from the medium by magnetic separation. The present work was designed to immobilize horseradish peroxidase on Fe3O4 magnetic nanopraticles without modification. Results: In the present study, horseradish peroxidase (HRP) was immobilized on non-modified Fe3O4 magnetic nanoparticles. The immobilized HRP was characterized by FT-IR spectroscopy, scanning electron microscopy, and energy dispersive X-ray. In addition, it retained 55% of its initial activity after 10 reuses. The optimal pH shifted from 7.0 for soluble HRP to 7.5 for the immobilized HRP, and the optimal temperature shifted from 40°C to 50°C. The immobilized HRP is more thermostable than soluble HRP. Various substrates were oxidized by the immobilized HRP with higher efficiencies than by soluble HRP. Km values of the soluble and immobilized HRP were 31 and 45 mM for guaiacol and 5.0 and 7.0 mM for H2O2, respectively. The effect of metals on soluble and immobilized HRP was studied. Moreover, the immobilized HRP was more stable against high concentrations of urea, Triton X-100, and isopropanol. Conclusions: Physical immobilization of HRP on iron magnetic nanoparticles improved the stability toward the denaturation induced by pH, heat, metal ions, urea, detergent, and water-miscible organic solvent.


Subject(s)
Enzymes, Immobilized/chemistry , Ferrosoferric Oxide/chemistry , Horseradish Peroxidase/chemistry , Solubility , Spectrometry, X-Ray Emission , Temperature , Microscopy, Electron, Scanning , Spectroscopy, Fourier Transform Infrared , Enzymes, Immobilized/metabolism , Nanoparticles/chemistry , Horseradish Peroxidase/metabolism , Hydrogen-Ion Concentration
2.
Indian J Biochem Biophys ; 2014 Aug; 51(4): 314-320
Article in English | IMSEAR | ID: sea-154255

ABSTRACT

Immobilization of cellulase from Aspergillus niger on TiO2 nanoparticles was studied by two different approaches — physical adsorption and covalent coupling. A. niger was selected, as it is generally non-pathogenic, is found in nature in the broad range of habitats and produces cellulase extracellulary. For covalent method, TiO2 nanoparticles were modified with aminopropyltriethoxysilane (APTS). The adsorbed and covalently immobilized enzymes showed 76% and 93% activity, respectively, as compared to the free enzyme. The catalytic efficiency Vmax/Km increased from 0.4 to 4.0 after covalent attachment, whereas in adsorption method, it increased slightly from 0.4 to 1.2. The covalently-immobilized and adsorbed cellulase lost only 25% and 50% of their activity, respectively after 60 min of incubation at 75°C. The reusability and operational stability data also showed that covalent coupling increased the stability of the enzyme. The presence of enzyme on TiO2 nanoparticles was confirmed by Fourier-transform infrared spectroscopy. The high-resolution transmission electron microscopy (HR-TEM) and atomic force microscopy (AFM) studies indicated aggregation of enzyme when adsorbed on TiO2 surface and a monolayer of enzyme in covalent attachment. In conclusion, covalently attached cellulase retained good activity and thermal stability, as compared to physically adsorbed enzyme. The lower amount of enzyme activity and thermal stability in case of physically adsorbed immobilized enzyme was due to aggregation of the enzyme after adsorption on TiO2 nanoparticles, as revealed by HR-TEM and AFM. Thus, TiO2 nanoparticles could be suitable candidates for immobilization of cellulase for industrial applications like paper, textile, detergent and food industries.


Subject(s)
Aspergillus niger/enzymology , Cellulase/chemistry , Enzymes, Immobilized/chemistry , Kinetics , Microscopy, Atomic Force , Microscopy, Electron, Transmission , Spectroscopy, Fourier Transform Infrared , Titanium/chemistry
3.
Indian J Biochem Biophys ; 2011 Feb; 48(1): 29-34
Article in English | IMSEAR | ID: sea-135297

ABSTRACT

Bacterial organophosphate hydrolases (OPH) have been shown to hydrolyze structurally diverse group of organophosphate (OP) compounds and nerve agents. Due to broad substrate range and unusual catalytic properties, the OPH has successfully been used to develop eco-friendly strategies for detection and decontamination of OP compounds. However, their usage has failed to gain necessary acceptance, due to short half-life of the enzyme and loss of activity during process development. In the present study, we report a simple procedure for immobilization of OPH on biocompatible gelatin pads. The covalent coupling of OPH using glutaraldehyde spacer has been found to dramatically improve the enzyme stability. There is no apparent loss of OPH activity in OPH-gelatin pads stored at room temperature for more than six months. As revealed by a number of kinetic parameters, the catalytic properties of immobilized enzyme are found to be comparable to the free enzyme. Further, the OPH‑gelatin pads effectively eliminate OP insecticide methyl parathion and nerve agent sarin.


Subject(s)
Enzyme Stability , Enzymes, Immobilized/chemistry , Escherichia coli/enzymology , Escherichia coli/genetics , Gelatin/chemistry , Hydrolysis , Insecticides/poisoning , Methyl Parathion/chemistry , Organophosphorus Compounds/chemistry , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/isolation & purification , Phosphoric Monoester Hydrolases/metabolism , Sarin/chemistry , Substrate Specificity
4.
Indian J Biochem Biophys ; 2009 Aug; 46(4): 294-298
Article in English | IMSEAR | ID: sea-135208

ABSTRACT

ALP2 gene encoding alkaline protease cloned from Aureobasidium pullulans HN2-3 was ligated into the surface display plasmid and expressed in the cells of the yeast Yarrowia lipolytica. The expressed alkaline protease was immobilized on the yeast cells. The activity of the immobilized enzyme with 6 His tag was found to be significantly higher than that of without 6 His tag. The immobilized enzyme showed lower optimal temperature and a lower affinity for azocasein than the free enzyme purified from A. pullulans HN2-3. The thermal stability of the immobilized enzyme enhanced and the pH stability decreased, compared to that of the free enzyme.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Caseins/chemistry , Cations , Cell Membrane/metabolism , Cloning, Molecular , Endopeptidases/chemistry , Endopeptidases/genetics , Enzymes, Immobilized/chemistry , Fungi/enzymology , Gene Expression Regulation, Fungal , Genetic Engineering/methods , Hydrogen-Ion Concentration , Ions , Kinetics , Models, Biological , Temperature , Yarrowia/enzymology , Yarrowia/genetics
5.
Electron. j. biotechnol ; 10(4): 508-513, oct. 2007. ilus, tab
Article in English | LILACS | ID: lil-504124

ABSTRACT

The esterification of phenylpropanoid and hydrophenylpropanoid acids, catalyzed by candida antarctica lipase B (CAL-B), with several alcohols has demonstrated that the substitution pattern on the aromatic ring has a very significant influence on the reactivity of the carboxyl group due, mainly, to electronic effects, when compared to the unsaturated acids with the hydrogenated acids. It is also clear that in the saturated acids there still remain some unclear effects related to the aromatic substituents.


Subject(s)
Esterification , Phenylpropionates/metabolism , Lipase/metabolism , Catalysis , Chromatography, High Pressure Liquid , Candida/enzymology , Enzymes, Immobilized/metabolism , Enzymes, Immobilized/chemistry , Phenylpropionates/chemistry , Lipase/chemistry , Solvents
6.
Indian J Biochem Biophys ; 2007 Apr; 44(2): 82-7
Article in English | IMSEAR | ID: sea-27187

ABSTRACT

Glutathione (L-gamma-glutamyl-L-cysteinyl-L-glycine; GSH) forms a surface monolayer on gold nanoparticles by tethering via sulfur bonds (Au:GSH). In the present study, glucose oxidase (GOx; EC 1.1.3.4) was immobilized by covalent chemical coupling reactions on to Au:GSH nanoparticles and the enzyme coupled nanoparticles formed a stable colloid (stable for several weeks) in water. The immobilized enzyme was investigated for electrochemical characteristics to monitor the FAD (prosthetic group of the GOx) redox potentials. Various concentrations of substrate (glucose) were added to check the oxidation characteristics. It was observed that with increase in substrate concentrations, the oxidation rate increased proportionally with the current. The present study demonstrated that GOx was effectively coupled to the gold nanoparticle (Au:GSH). The coupled nanoparticle system could be used in a potential biosensor application. Similarly, other enzymes (e.g., horseradish peroxidase) could be immobilized to the Au:GSH nanoparticles via the peptide arm (GSH) to achieve the desired characteristics needed for a specific application in biosensor.


Subject(s)
Biosensing Techniques , Electrochemistry , Enzymes, Immobilized/chemistry , Glucose/chemistry , Glucose Oxidase/chemistry , Glutathione/chemistry , Gold , Metal Nanoparticles , Oxidation-Reduction
7.
Indian J Biochem Biophys ; 2007 Feb; 44(1): 38-43
Article in English | IMSEAR | ID: sea-28315

ABSTRACT

The covalent immobilization of bovine liver catalase (CAT) on to florisil via glutaraldehyde was investigated. Optimum immobilization pH and temperature were determined as pH 6.0, 10 degrees C respectively, while the amount of initial CAT per g of carrier and immobilization time was determined as 5 mg g(-1) and 120 min, respectively. The Vmax values for free and immobilized CAT were found to be 1.7 x 10(5) and 2.0 x 10(4) micromol H2O2 min(-1) mg protein(-1), respectively, whereas KM values were 33.3 mM and 1722.0 mM respectively. Operational stability was determined by using a stirred batch-type column reactor. Immobilized CAT retained about 40% of its initial activity after 50 uses. It showed higher storage stability than free CAT at 4 degrees C and 25 degrees C. Its storage stability increased with increasing relative humidity (RH) from 0 to 20% of the medium. The highest storage stability was obtained in 20% RH, however, further increase in RH from 40 to 100% significantly decreased the storage stability.


Subject(s)
Animals , Buffers , Catalase/chemistry , Cattle , Drug Storage , Enzyme Stability , Enzymes, Immobilized/chemistry , Hydrogen-Ion Concentration , Kinetics , Liver/enzymology , Magnesium Silicates
8.
Indian J Exp Biol ; 2006 May; 44(5): 392-8
Article in English | IMSEAR | ID: sea-61313

ABSTRACT

A monosodium glutamate (MSG) biosensor with immobilized L-glutamate oxidase (L-GLOD) has been developed and studied for analysis of MSG in sauces, soup etc. The immobilized enzymatic membrane was attached with oxygen electrode with a push cap system. The detection limit of the sensor was 1 mg/dl and the standard curve was found to be linear upto 20 mg/dl. Response time of the sensor was 2 min. Cross-linking with glutaraldehyde in presence of Bovine Serum Albumin (BSA) as a spacer molecule has been used for immobilization. Optimization of the sensor was done with an increase in L-GLOD concentration (6.3-31.5 IU) and also with increase in loading volume of enzyme solution (5-20 microl). Optimization of pH and temperature was also studied. The permeability of O2 through different membrane was studied with and without immobilized L-GLOD. The enzymatic membrane was used for over 20 measurements and stability of the membrane was observed.


Subject(s)
Amino Acid Oxidoreductases/chemistry , Biosensing Techniques , Enzymes, Immobilized/chemistry , Food Analysis , Sodium Glutamate/analysis
9.
Indian J Biochem Biophys ; 1999 Dec; 36(6): 449-52
Article in English | IMSEAR | ID: sea-27598

ABSTRACT

A membrane bound oxalate oxidase from leaves of Amaranthus spionsus has been partially purified and immobilized on alkylamine glass with a yield of 9.2 mg protein/g support. The enzyme retained 99.4% of initial activity of free enzyme after immobilization. There was no change in the optimum pH (3.5) and Vmax but the temperature for maximum activity was slightly decreased (35 degrees C) and energy of activation (Ea) and Km for oxalate were increased after immobilization. The immobilized enzyme preparation was stable for 6 months, when stored in distilled water at 4 degrees C. Presence of Cl- did not affect the activity of immobilized enzyme.


Subject(s)
Adult , Amines/chemistry , Enzymes, Immobilized/chemistry , Humans , Male , Oxidoreductases/chemistry , Plant Leaves/enzymology
10.
Hindustan Antibiot Bull ; 1998 Feb-Nov; 40(1-4): 1-4
Article in English | IMSEAR | ID: sea-2560

ABSTRACT

The role of sugars, polyhydroxy compounds, phenylacetic acid and 6-aminopenicillanic acid in stabilization of immobilized penicillin G acylase (IMPGA) was studied. The loss in the activity of IMPGA at 50 degrees C, 2 h, after incorporation of sucrose and mannitol at 0.1 M concentration was 16 and 18% respectively; the loss in the activity of the enzyme under these conditions in the absence of stabilizing agents was 40%.


Subject(s)
Carbohydrates/pharmacology , Enzyme Stability , Enzymes, Immobilized/chemistry , Hot Temperature , Penicillin Amidase/chemistry , Polymers/pharmacology
11.
Hindustan Antibiot Bull ; 1997 Feb-Nov; 39(1-4): 1-10
Article in English | IMSEAR | ID: sea-2619

ABSTRACT

Penicillin G-Acylase is produced by submerged cultivation of E. Coli (NCIM-2400) and extracted from the harvested fermented broth, purified (affinity chromatography) and immobilised on Eupergit C (Synthetic polymer in bead form). The immobilised penicillin G acylase properties are studied and compared with soluble penicillin G-acylase. The control parameters for conversion of penicillin G-K to 6 APA are optimised [e.g. substrate (Pen G-K) concentration ratio to immobilised penicillin G-acylase, temperature, pH etc.] in a stirred tank reactor. Our findings suggest that immobilised penicillin G-acylase can be used commercially and the productivity of 1 kg. of immobilised enzyme is around 400 kg of 6 APA under given desired stipulated conditions.


Subject(s)
Enzymes, Immobilized/chemistry , Escherichia coli/enzymology , Hydrogen-Ion Concentration , Penicillanic Acid/analogs & derivatives , Penicillin Amidase/isolation & purification , Polymers
12.
Braz. j. med. biol. res ; 28(3): 291-5, Mar. 1995. ilus, tab
Article in English | LILACS | ID: lil-154693

ABSTRACT

Xanthine oxidase was covalently immbolized on polyacrylamide gel beads, polyamide- 11 and dacron. Hypoxanthine (15 ml of 200 µM), prepared in 0.1 M phosphate buffer, pH 8.0, was circulated through a column containing 1.0g derivatized enzyme at a flow rate of 1.0 ml/min at 28§C. Specific activities of 0.660, 0.072 and 0.016 Units/mg of protein were demonstrable for the polyacrylamide gel beads, dacron and polyamide-11 derivatives, respectively. The action of these water insoluble enzyme derivatives on 6 mercaptopurine (15 ml of 660 µM) was also investigated, under the same experimental conditions, showing specific activites of 0.063 Units/mg, 0.574 µUnits/mg and 0.118 µUnitis/mg, respectively. The 6-mercaptopurine oxidative pathway catalyzed by immobilized xanthine oxidase on dacron stopped at the intermediate compound 6-mercaptopurine oxidative on dracon stopped at the intermediate compound, 6-mercapto-8-hydroxypurine, so that no 6-thiouric acid was produced, whereas the immobilized preparations using polyacrylamide gel beads and polyamide-11 behaved like the soluble enzyme, namely, 6-thiouric acid was the final product. The behavior of dracon-xanthine oxidase immobilized on these three supports was similar to the soluble enzyme. However, although its oxidation is stoichiometric for polyacrylamide gel beads and polyamide- 11 derivatives, and no xanthine formation is observed (steady-state equilibrium), under the action of the enzymedacron derivative the xanthine formation rate (0.164 µUnits/mg) is higher than the uric acid formation rate (0.017 µUnits/mg) compared to the hypoxanthine consumption (0.072 µUnits/mg). These findings suggest again that xanthine oxidase-dacron derivative is limited to the catalysis of oxidation of hypoxanthine carbon atom number 2 as in 6-mercaptopurine


Subject(s)
Mercaptopurine/chemistry , Enzymes, Immobilized/chemistry , Xanthine Oxidase/chemistry , Enzymes, Immobilized/metabolism , Enzymes, Immobilized/pharmacology , Xanthine Oxidase/metabolism , Xanthine Oxidase/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL