Multistep enzyme systems immobilized

The results of one of the first multistep enzyme systems to be immobilized were presented in 1970 by Mosbach and Mattiasson (3). They covalently bonded hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6-PDH) to individual polymer particles via the cyanogen bromide reaction. Using a solution containing glucose, ATP and NADP" ", they demonstrated that the coimmobilized enzymes formed product... 

The quantitative data and observations discussed above appear to be consistent with the qualitative concepts put forward by Mosbach and others to explain the behavior of multistep enzyme systems. Mathematical modelling of gel entrapped, multistep, immobilized enzyme systems, using the collocation technique, is quite straightforward and provides the opportunity not only to compare experiment with theory but also to explore the effect of parameters of the system which are experimentally inaccessible. 

The development of analytically useful enzyme electrodes is limited by the availability of purified and stable enzyme preparations. In an effort to extend the range of measurable species using ISE devices further, Rechnitz and co-workers (Rl) recently introduced bacterial- and tissue-based bio-selective electrode systems. These sensors are prepared in much the same manner as the enzyme probes except that whole intact cells are utilized as the immobilized reagents. There are several potential advantages to this novel approach, including (1) no need to extract and purify the enzymes involved, i. e., low cost (2) enzymes which are unstable when extracted from the cell may be used in situ to maximize and preserve their activity (3) if desired enzyme reactions require cofactors, these co ctors need not be added to the assay mixture because they are already present in the intact cell and (4) analytical reactions involving multistep enzyme sequences already present in the cells may be used to detect given analytes. 

The immobilization of whole cells provides a means for the entrapment of multistep and cooperative enzyme system present in the intact cell, repetitive use and improved stabihty. This technique is also advantageous in the separation of bioproducts from cell mass in a continuous bioconversion process [114,115]. The other advantages of immobilized growing cells include (1) protection of cells against unfavourable environmental factors (2) changes in the permeability of the cells (3) reduced inhibition by substrate and product (4) reusability and (5) faster removal of end product. 

The immobilization concept was later extended and applied to living cells41 . Immobilization of whole cells rather than purified enzymes reduced the expense of separation, isolation and purification of the enzyme. Furthermore, in multistep reactions, in which several enzymes are involved, the application of immobilized cells is advantageous. Since the enzymes are in their native state their stability is enhanced. Such systems may widely be applied, which is not possible with isolated pure enzymes, and are less expensive than processes based on free intact cells 42). 

Polyelectrolyte multilayers are able to very effectively immobilize various molecules such as enzymes, for example. These multilayers have been shown to exhibit useful biocatalytic (i.e., enzymatic) [220,221] activities hybrid multilayers containing polyoxometalate are promising for catalytic applications [165], ESA multilayers have also been used for molecular recognition [199,344] or, more specifically, as biosensors [76,167,253-255,345,346], Electrocatalytic [196,256,259] and electrosensing [257] capabilities of multilayers deposited on electrodes have also been demonstrated. As already mentioned, the possibility of putting different enzymes at specific locations in the multilayer has been employed to design systems for multistep catalysis [6] (Fig. 16). 

In all of the multistep immobilized enzyme work done to date, theoretical or experimental, for modelling purposes or for applications, there exists one common factor the chemical reactions are affected by the diffusive processes so that the macroscopically observed kinetics are strongly perturbed by the incorporation of the enzymes into a gel. This perturbation is caused by the development of localized concentrations and concentration gradients within the gel which are quite different from that found in free solution. Only one instance appears to have been reported where exact modelling of real experimental data has been attempted. All other work has been either purely theoretical or qualitative interpretations of limited experimental data. There is still much to be learned of the role played by the gel matrix in affecting the overall kinetic performance of gel entrapped multienzyme systems before they can be well designed for applications or used with any confidence in a quantitative way as models for living systems. 

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