Enzymes immobilized, characterization

The reactivation of enzymes (after their partial inactivation in an acid medium) upon passing into a medium of pH 8 is also of great importance for oral use (Fig. 25). Enzymes immobilized in crosslinked polyelectrolytes are characterized by a structural memory even after considerable inactivation. Under changed conditions, this leads to a considerable or almost complete reactivation of the enzyme, whereas in the reactivation of a free enzyme in solution under similar conditions the enzymatic activity is restored on a lower level. 

For farther improvement of hydrogen enzyme electrode the commercial carbon filament materials were used as an electrode matrix. Such type of materials are accessible and well characterized, that provides the reproducibility of the results. A procedure for hydrogen enzyme electrode preparation included the pretreatment of electrode support with sulfuric acid followed by enzyme immobilization. This procedure is a critical step, since initially carbon filament material is completely hydrophobic [9]. 

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

Zhang, X., Guan, R. F., Wu, D. Q. and Chan, K. Y. Enzyme immobilization on amino-functionalized mesostmetured cellular foam surfaces, characterization and catalytic properties, J. Mol. Catal., B, 2005, 33, 43-50. 

A new development in the field of potentiometric enzyme sensors came in the 1980s from the work of Caras and Janata (72). They describe a penicillin-responsive device which consists of a pH-sensitive, ion-selective field effect transistor (ISFET) and an enzyme-immobilized ISFET (ENFET). Determining urea with ISFETs covered with immobilized urease is also possible (73). Current research is focused on the construction and characterization of ENFETs (27,73). Although ISFETs have several interesting features, the need to compensate for variations in the pH and buffering capacity of the sample is a serious hurdle for the rapid development of ENFETs. For detailed information on the principles and applications of ENFETs, the reader is referred to several recent reviews (27, 74) and Chapter 8. 

Packed-bed enzyme reactors, those employing enzymes immobilized onto a particulate phase that is subsequently packed into a column, may be characterized by their column capacity, C, and the degree of reaction P. The parameter C is defined in Eq. 4.23,... 

Akertek E, Tarhan L (1995) Characterization of immobilized catalases and their application in pasteurization of milk with H202. Appl Biochem Biotech 50 291-303 Alonso A, Almendral MJ, Baez MD, Porras MJ, Alonso C (1995) Enzyme immobilization on an epoxy matrix. Determination of L-Arginine by flow-injection analysis. Anal Chim Acta 308 164-169... 

Flow microcalorimetry, which makes many rapid and accurate measurements of the activity of immobilized biocatalysts, provides a tool for researchers that can be used to discriminate between different preparatives of immobilized biocatalysts. Table 3 shows previous experiments where the characterization of kinetic properties by flow micro calorimetry was used to compare different techniques of purified enzyme immobilization [27, 30, 31, 35] as well as the immobilization of enzymes fixed in cells [28,29,40]. More details can be found in our recent review article [41]. 

Enzymes are characterized by unusual specific activities and remarkably high selectivities. They are effective catalysts at relatively low temperatures and ambient pressure. The primary driving force for efforts to develop immobilized forms of these biocatalysts is cost, especially when one is comparing process alternatives involving either conventional inorganic catalysts or soluble enzymes. Immobilization can permit conversion of labile enzymes into forms appropriate for use as catalysts in industrial processes—production of sweeteners, pharmaceutical intermediates, and fine chemicals—or as biosensors in analytical applications. Because of their high specificities, immobilized versions of enzymes are potentially useful in situations where it is necessary to obtain high yields of the desired product... 

We here report on the design, synthesis, characterization, and applications of template-synthesized nanotube membranes. We will first briefly review the synthesis of the template-synthesized nanotube membranes. Some details of differential-surface chemistry on nanotnbes and nanotubes for bioextraction and biocatalysis will be presented. We discuss in details the drug detoxification nsing functionalized nanotnbes and apoenzyme-immobilized, enzyme-immobilized, and antibody and DNA-immobilized nanotubes for enantiomeric and DNA separations, biocatalysis, and bioextractions. ... 

A. Nomura, S. Shin, O. O. Mehdi, J-M. Kauffmann, Preparation, characterization and application of an enzyme-immobilized magnetic... 

Airoldi C., Monteiro Jr O.A.C. Chitosan-organosilane hybrids-syntheses, characterization, copper adsorption, and enzyme immobilization. J. Appl. Polym. Sci. 2000 77 797-804 Al-Saraj M., El-Nahal L, Baraka R. Bioaccumulation ofsome hazardous metals by sol-gel entrapped microorganisms. J. Non-Cryst. Solids 1999 248 137-140 Audebert P., Demaille C., Sanchez C. Electrochemical probing of the activity of glucose oxidase embedded in sol-gel matrices. Chem. Mater. 1993 5 911-913 Avnir D., Kaufman V.R. Alcohol is an unnecessary additive in the silicon alkoxide sol-gel process. J. Non-Cryst. Solids 1987 192 180-182... 

Fluorescence is an excellent tool to probe chemical microenvironments. For example, in 2010, Martin et al. used a specific shift in fluorescence emission spectra of polar sensitive fluorophores, for example, acrylodan, to characterize the polarity immediately surrounding immobilized enzymes [34]. Cytoplasmic malate dehydrogenase (cMDH) and mitochondrial malate dehydrogenase (mMDH), for example, were tagged with fluorescent probes and then entrapped within macroporous three-dimensional chitosan scaffolds. A blue shift in the acrylodan emission maximum was taken to indicate a polar shift in the chemical microenvironment directly surrounding enzymes immobilized within the scaffold (Figme 12.5). The emission... 

Cooney MJ, Petermarm J, CaroUn L, Minteer SD. Characterization and evaluation of hydrophohically modified chitosan scaffolds towards design of enzyme immobilized flowthrough electrodes. Carbohydr Polym 2009 75 428-435. 

Mohy Eldin MS, Seuror E, Nasr M, El-Aassar M, Tieama H. (2011c). Affinity covalent immobilization of glucoamylase onto p-benzoquinone activated alginate beads II. Enzyme immobilization and characterization. AppI Biochem Biotechnol, 64,45-57. 

The Michaehs-Menten equation and other similar nonhnear expressions characterize immobihzed enzyme kinetics. Therefore, for a spherical porous carrier particle with enzyme molecules immobilized on its external as well as internal surfaces, material balance of the substrate will result in the following ... 

Enzyme structure may be studied by fluorescence spectroscopy [238-244]. Excitation in the 280-310 nm absorption bands of proteins, usually results in fluorescence from tryptophan (Trp) residues in the 310-390 nm region. The fluorescence from the Trp residues is a convenient marker for protein denaturation and large decreases or red-shifts in fluorescence are observed when proteins are denatured. These changes are most often due to the exposure of the Trp residues that are buried in the protein and may be due to the changes in the proximities of specific residues that may act as fluorescence quenchers. Fluorescence emission characterization of the immobilized... 

Vertically aligned CNT-modified electrodes are based on a more elaborated technique than other methods, and microscopic images are used to characterize the integrity of this type of electrode. The technique has been applied for the immobilization of enzymes and DNA, and the sensors based on this technique have shown a lower detection limit than those based on other methods. More research activities using this technique, particularly with low density CNT arrays, are expected in the near future because of its sensitivity and versatility. 

Hublik G, Schinnera F (2000) Characterization and immobilization of the laccase from Pleurotus ostreatus and its use for the continuous elimination of phenolic pollutants. Enzyme Microb Technol 27 330-336... 

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