Kinetic behavior, effect immobilized enzymes

Another case of heterogeneous systems refers to immobilized enzymes. The kinetic behavior of a bound enzyme can differ significantly from that of the same enzyme in free solution. The properties of an enzyme can be modified by suitable choice of the immobilization protocol, whereas the same method may have appreciably different effects on different enzymes. These changes may be due to conformational alterations within the enzyme, immobilization procedure and the presence and nature of the immobifization support. The advantages of immobifized enzymes are for instance in reusabifity and possibility to use continuous mode. 

Considerable progress has been made within the last decade in elucidating the effects of the microenvironment (such as electric charge, dielectric constant and lipophilic or hydrophilic nature) and of external and internal diffusion on the kinetics of immobilized enzymes (7). Taking these factors into consideration, quantitative expressions have been derived for the kinetic behavior of relatively simple enzyme systems. In all of these derivations the immobilized enzymes were treated as simple heterogeneous catalysts. 

Several thermodynamic and kinetic behaviors of enzyme-catalyzed reactions performed in ILs, with respect to enzymatic reactions carried out in conventional solvents, could lead to an improvement in the process performance [34—37]. ILs showed an over-stabilization effect on biocatalysts [38] on the basis of the double role played by these neoteric solvents ILs could provide an adequate microenvironment for the catalytic action of the enzyme (mass transfer phenomena and active catalytic conformation) and if they act as a solvent, ILs may be regarded as liquid immobilization supports, since multipoint enzyme-1L interactions (hydrogen. Van der Waals, ionic, etc.) may occur, resulting in a flexible supramolecular not able to maintain the active protein conformation [39]. Their polar and non-coordinating properties hold considerable potential for enantioselective reactions since profound effects on reactivities and selectivities are expected [40]. In recent years attention has been focused on the appUcation of ILs as reaction media for enantioselective processes [41—43]. 

The attachment of catalytlcally active sites to materials which may be easily recovered from a reaction mixture has been the sine qua non of most useful examples of catalysis outside of enzymology, and this latter area has begun to follow suit (see for example, the six Enzvme Engineering Conferences,as well as several summary texts ). In a pleasantly exhaustive review of the kinetics of immobilized enzyme systems, Goldstein several years ago assigned "the effects of Immobilization on the kinetic behavior of an enzyme" to four situations ... 

Intraparticle diffusion can have a significant effect on the kinetic behavior of enzymes immobilized on solid carriers or entrapped in gels. In their basic analysis of this problem. Moo-Young and Kobayashi (1972) derived a general modulus and effectiveness factor. The results also predicted possible multiple steady-states as well as unstable situations for certain systems. While these results are very interesting it should be remembered that they are primarily mathematical and await extensive experimental support data. 

Intrinsic kinetics of the immobilized enzyme represents its proper behavior and corresponds to that observed in the absence of partition and mass transfer limitations of the reacting species. This kinetic behavior and the corresponding kinetic parameters are not directly measurable for an immobilized enzyme, except in special conditions where these effects are purposely avoided. Even if the intrinsic behavior could be revealed, it may differ from that of the free enzyme counterpart because of conformational changes. 

Effective (or apparent) kinetics of the immobilized enzyme is that directly determined from the observed behavior. Effective and apparent seem opposite concepts, but this is not so, since it is effective from the standpoint of the enzyme user (it is what one gets), but apparent from the standpoint of the enzyme since it does not reflect its actual catalytic potential which is obscured by the heterogeneous nature of the system. 

When using immobilization onto supports, one potential problem is that the matrix strurture can slow diffusion rates, an effect that depends critically on pore size and microenvironment. It is therefore essential to consider both the chemical and physical properties of the matrix since these can alter thermal stability, kinetic behaviors, and the optimal pH and temperature of the enzyme for a given reaction [30],... 

Several studies have demonstrated the improved stability of peroxidases when they were subjected to immobilization. Akhtar and Husain observed that bitter gourd peroxidase (BGP) was able to remove higher percentage of phenols over a wider range of pH when immobilized on a bioaffinity support [37]. Sasaki et al. highlighted an improvement of thermal stability of MnP immobilized on FSM-16 mesoporous material [59]. Furthermore, some other studies demonstrated a protective effect of peroxidase immobilization against inactivation by H202 [7, 20]. The different behavior of immobilized peroxidases with respect to soluble ones points out the necessity of an optimization of the process conditions when immobilized enzyme is used. Nevertheless, the possible improvement in stability should balance the usual decrease in kinetic rates, due to substrate transfer limitations to reach the enzyme inside the support. 

Free enzyme versus immobilized enzyme can influence the yield of lOS, additionally an immobilized system would be favorable economically as the biocatalyst can be reused, enables continuous production and the end product is free of contamination. Kim et al. [275] intended to make a comparison between the reaction kinetics of free and immobilized endo-inulinases in a batch reactor however significant differences were observed in the reaction behavior and product composition due to the form of enzyme used and the initial concentration of substrate. Yun et al. [276] investigated the effect of inulin concentration on the production of lOS by free and immobilized endo-inulinase from Pseudomonas sp. Their findings corroborate those of Kim et al. [275] whereby different products are formed depending on the form of enzyme a soluble enzyme yielded inulobiose and DP3 products, whereby the immobilized form predominantly produced inulobiose. As the concentration of inulin increased the yield of lOS did not increase in the soluble system and in the immobilized the yield remained the same. Although the enzyme was derived from Pseudomonas the immobilized form required a differ-... 

If kinetic rate data from an immobilized enzyme are collected directly, as presented in section 3.2.2, only effective (apparent) parameters are obtained that do not reflect the actual behavior of the enzyme. This information, though useful, is valid only at the precise conditions at which the experiment was performed. Eor design... 

The products of the reaction they catalyze may inhibit many enzymes through Michaelis-Menten kinetic retroaction. Protons, which are involved as products or reactants in a number of cases, may also influence the enzymatic kinetics. The course of the reaction may therefore be altered by the attending production or depletion of protons. It is thus interesting to examine whether these phenomena may be revealed by the effect they might have on the electrochemical responses of immobilized enzyme films under appropriate conditions [92]. A first clue of the existence of such inhibition effects is the observation of hysteresis behaviors of the type shown in Fig. 18(a) where data obtained with 10 glucose oxidase monolayers with ferrocene methanol as cosubstrate have been taken as example. In the absence of inhibition, the forward and reverse traces should be exactly superimposed. Hysteresis increases to the point of making a peak appear on the forward trace as the scan rate decreases and as the concentration of the buffer decreases, as illustrated in Fig. 18c, c , c , c by comparison with Fig. 18(a). 

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