Enzyme by adsorption

In general, immobilization of the enzyme by adsorption or bioaffinity immobilization can be accomplished rapidly. Consequendy, being able to constimte (or reconstitute) the catalyst just prior to use is an advantage. 

The immobilization of invertase on aluminium hydroxide (2) was one of the earliest reports of adsorption technology. The use of aminoacylase adsorbed on DEAE-Sephadex for producing L-amino acids from a racemic mixture of their corresponding ethyl esters (4) was the first industrial application of an immobilized enzyme system. The basic disadvantage of this convenient technique is that binding is weak and the enzyme slowly leaches out. However, for many purposes, this slow leakage is not an important handicap. Immobilizing enzymes by adsorption has been extensively reviewed (5, 6, 27). Some special approaches are described (1, 28-30). 

Researchers turned their attention to applications of silica gel as a new electrode material. Silica gel, which has a three-dimensional structure with high specific surface area and is electroinactive in an aqueous medimn can be used as a support for electroactive species during their formation and/or enzymes by adsorption or entrapment [92,93]. Patel et al. recently reported application of poljwinyl ferrocene immobilized on silica gel particles to construct glucose sensors. Efficiency of carbon paste electrodes prepared with these polymeric electron mediators and GOx was comparable to electrodes constructed with other ferrocene based polymeric electron transfer systems. The fact that 70% of initial anodic current was retained after a month when electrodes were kept in the buffer at room temperature shows that polymerization of monomer vinylferrocene in the pores of silica gel and entrapping GOx in the matrix of poljwinyl ferrocene appears to have added stability to the sensors [94]. 

The adsorption method is the simplest one and is often used in bioelectrocatalysis research. Essentially it involves the incubation of protein in the carrier suspension with the subsequent washing of the nonadsorbed protein. Adsorption of proteins on different types of surfaces is effected due to electrostatic, hydrophobic, and dispersion interactions. The most popular carriers are carbon, soot, clays, aluminum oxide, silica gel, and glass. The optimal inert carrier is glass. It has recently been shown that porous glass with calibrated pore size can be used for immobilization of enzymes by adsorption. An interesting method of immobilization by adsorption has been proposed in which lipid is first adsorbed on carbon or silica gel and then the enzyme is adsorbed on the so-called soft surface of the lipid. 

Hurst (19) discusses the similarity in action of the pyrethrins and of DDT as indicated by a dispersant action on the lipids of insect cuticle and internal tissue. He has developed an elaborate theory of contact insecticidal action but provides no experimental data. Hurst believes that the susceptibility to insecticides depends partially on the cuticular permeability, but more fundamentally on the effects on internal tissue receptors which control oxidative metabolism or oxidative enzyme systems. The access of pyrethrins to insects, for example, is facilitated by adsorption and storage in the lipophilic layers of the epicuticle. The epicuticle is to be regarded as a lipoprotein mosaic consisting of alternating patches of lipid and protein receptors which are sites of oxidase activity. Such a condition exists in both the hydrophilic type of cuticle found in larvae of Calliphora and Phormia and in the waxy cuticle of Tenebrio larvae. Hurst explains pyrethrinization as a preliminary narcosis or knockdown phase in which oxidase action is blocked by adsorption of the insecticide on the lipoprotein tissue components, followed by death when further dispersant action of the insecticide results in an irreversible increase in the phenoloxidase activity as a result of the displacement of protective lipids. This increase in phenoloxidase activity is accompanied by the accumulation of toxic quinoid metabolites in the blood and tissues—for example, O-quinones which would block substrate access to normal enzyme systems. The varying degrees of susceptibility shown by different insect species to an insecticide may be explainable not only in terms of differences in cuticle make-up but also as internal factors associated with the stability of oxidase systems. 

In biological systems, the enzymes are homogeneons catalysts. For their use in heterogeneons electrochemical reactions, they mnst be immobilized on a carrier suitable for fashioning an electrode. This is most often achieved by adsorption of the enzyme on a carbon material (carbon black, graphite, etc.). This immobilization usually leads to some decrease in activity of the enzymes, bnt on the other hand, raises their stability. 

In order to overcome some limitations of the adsorption process due to surface accessibility or diffusional hindering, immobilization of enzymes by direct in situ encapsulation has been investigated. When inorganic supports can be prepared in mild conditions compatible with the enzyme stability, then such processes allow... 

In this communication a study of the catalytic behavior of the immobilized Rhizomucor miehei lipase in the transesterification reaction to biodiesel production has been reported. The main drawbacks associated to the current biodiesel production by basic homogeneous catalysis could be overcome by using immobilized lipases. Immobilization by adsorption and entrapment have been used as methods to prepare the heterogeneous biocatalyst. Zeolites and related materials have been used as inorganic lipase supports. To promote the enzyme adsorption, the surface of the supports have been functionalized by synthesis procedures or by post-treatments. While, the enzyme entrapping procedure has been carried out by sol-gel method in order to obtain the biocatalyst protected by a mesoporous matrix and to reduce its leaching after several catalytic uses. 

Table 1 shows the enzyme immobilization results. By adsorption procedure, it is possible to observe that no enzyme was retained on the N-ITQ-6 material after... 

In view of the conductive and electrocatalytic features of carbon nanotubes (CNTs), AChE and choline oxidases (COx) have been covalently coimmobilized on multiwall carbon nanotubes (MWNTs) for the preparation of an organophosphorus pesticide (OP) biosensor [40, 41], Another OP biosensor has also been constructed by adsorption of AChE on MWNTs modified thick film [8], More recently AChE has been covalently linked with MWNTs doped glutaraldehyde cross-linked chitosan composite film [11], in which biopolymer chitosan provides biocompatible nature to the enzyme and MWNTs improve the conductive nature of chitosan. Even though these enzyme immobilization techniques have been reported in the last three decades, no method can be commonly used for all the enzymes by retaining their complete activity. 

Mineral colloid-enzyme interactions have been documented (e.g., Theng 1979 Bums 1986 Naidja et al. 2000 Bums and Dick 2002). Besides cation-exchange reactions, adsorption of enzymes by mineral colloids may proceed through ionic, covalent, hydrophobic, hydrogen bonding, and van der Waals forces. When enzymes are adsorbed on mineral colloids, changes in the tertiary structures (i.e., the folding of the helix or... 

Ferrocene-mediated enzyme sensors have usually been obtained by adsorption of the mediator onto the electrode surface because of this insolubility in aqueous solution. Due to the good solubility of ferrecinium cations in aqueous solution complications arise from leakage of the mediator from the electrode surface. 

Immobilized enzymes are attached to a solid support by adsorption or chemical binding or mechanical entrapment in the pores of a gel structure but retain their catalytic power. Their merit is ease of separation from the finished reaction product. 

An enzyme is immobilized by adsorption on porous pellets of a carrier. The differential equation for the concentration of a reactant in a porous spherical pellet is derived in problem P7.03.01 and integrated for a first order reaction, rc = kC, in problem P7.03.06. An expression is derived for the effectiveness of the adsorbed enzyme for first order reaction as... 

Selected entries from Methods in Enzymology [vol, page(s)] Activation of lipolytic enzymes by interfaces, 64, 341 model for lipase action on insoluble lipids, 64, 345 interfacial enzyme inactivation, 64, 347 reversibility of the adsorption step, 64, 347 monolayer substrates, 64, 349 kinetic models applicable to partly soluble amphiphilic lipids, 64, 353 surface dilution model, 64, 355 and 364 practical aspects, 64, 357. 

Biotin is present in natnre predominantly bonded to protein and is relatively stable therefore it can be extracted under hard conditions, snch as autoclaving in sulfuric acid. Enzyme digestion is also applied to break the protein-biotin bond. A following purification is usually performed by adsorption on charcoal or by lEC. 

Some of the high cost enzymes are concentrated by adsorption chromatography. After adsorption, the adsorbent with bound enzyme is centrifuged and washed in order to remove unbound protein and solutions. Elution is performed by manipulation of pH and/or ionic strength. 

Immobilization can be accomplished by several methods. Adsorption is the easiest and least expensive. The bond is weak, however, and loss of enzyme by washout is inevitable. Covalent bonding can be accomplished by activating the... 

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