Enzyme using adsorption technique

Control of Juice Bitterness. A number of advances have been reported in this field since it was last reviewed (3). A commercial application of the cellulose acetate adsorption technique for the removal of limonin from citrus juices was undertaken (49). New sorbent gel forms of cellulose esters for adsorption of limonin were developed (50). Knowledge was gained that limonoids are biosynthesized in citrus leaves and translocated to the fruit (12) and that specific bioregulators can inhibit accumulation of XIV in citrus leaves (15). Additional studies were carried out on the use of neodiosmin to suppress limonin and other types of bitterness (30,51). The influence of extractor and finisher pressures on the level of limonin and naringin in grapefruit juice was reported (34). Also, further studies were conducted on the microbial sources and properties of limonoate dehydrogenase (52), the enzyme that converts XIV to XV and can be used to prevent limonin from forming in freshly expressed citrus juices (53). 

Methods for Immobilization of enzymes on Insoluble supports can be generally classified as using adsorption, entrapment or covalent attachment techniques (Table II). In a few cases, enzymes have also been rendered Insoluble by covalent crosslinking however, more often, enzymes are crossllnked within entrapped cells. These methods have been reviewed extensively in the past [for example, see refs. 

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). 

Figure 2. Comparison of current output for three sensors in a 100 mg/dL glucose solution in vitro. The sensors were identical in electrode area and meUiod of fabrication, except for the enzyme deposition. One of the sensors was coated using the Ikariyama adsorption technique (16) and the odier two were coated using the method described here utilizing two different current values. The sensors were not covered with an outer membrane. (Reproduced with permission from ref. 19. Copyright 1991 Elsevier Sequoia.)...

Figure 10.9 Immobilization of enzyme using the adsorption technique.

The number of publications dealing with this topic is increasing rapidly. For studying the interaction of dyes with an enzyme the adsorption on solid surfaces with different binding characteristics may serve as a model. In this case the dyes are used in their classical role as indicators. Dyes are well suited for this purpose because of their strong tendency towards chemi- and physisorption. When the UV-visible properties of the adsorbed species change sufficiently the immediate comparison with dye molecules dissolved in a homogeneous solution is possible. Also, dynamic aspects of the adsorption of molecules on these surfaces can be examined by electronic absorption techniques. 

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. 

A potentially more sensitive and selective approach involves reaction of formic acid with a reagent to form a chromophore or fluorophore, followed by chromatographic analysis. A wide variety of alkylating and silylating reagents have been used for this purpose. Two serious drawbacks to this approach are that inorganic salts and/or water interfere with the derivatisation reaction, and these reactions are generally not specific for formic acid or other carboxylic acids. These techniques are prone to errors from adsorption losses, contamination, and decomposition of the components of interest. Enzymic techniques, in contrast, are ideal for the analysis of non-saline water samples, since they are compatible with aqueous media and involve little or no chemical or physical alterations of the sample (e.g., pH, temperature). 

Preparation of enzyme-DNA films one layer at a time provides excellent control over the thickness of films designed to the specifications of the builder. Films containing two layers each of enzyme and DNA that are 20-40 nm thick are easily made. Alternate adsorption of layers of biomolecules and polyions is a general method that has been developed over the past decade by Lvov et al.[17 201 The technique has been used to make ultrathin films of a wide variety of proteins and oppositely charged... 

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