Immobilized enzymes optimization

Immobilization. Enzymes, as individual water-soluble molecules, are generally efficient catalysts. In biological systems they are predorninandy intracellular or associated with cell membranes, ie, in a type of immobilized state. This enables them to perform their activity in a specific environment, be stored and protected in stable form, take part in multi-enzyme reactions, acquire cofactors, etc. Unfortunately, this optimization of enzyme use and performance in nature may not be directiy transferable to the laboratory. 

When selecting a suitable feed symp, the main criteria are optimization of enzyme productivity and minimization of the formation of by-products. Typical feed symp specifications are shown in Table 5. Higher symp concentration and higher viscosity results in a reduced isomerization rate due to diffusion resistance in the pores of the immobilized enzyme. A deaeration step is desirable to remove dissolved oxygen that would otherwise iacrease the formation of by-products. The pH is adjusted to the optimum level for the productivity of the enzyme. 

An intriguing influence of a cosolvent immiscible with water on the enantioselec-tivity of the enzyme-catalyzed hydrolysis was observed. It was proven that enzyme enantioselectivity is directly correlated with the cosolvent hydrophobicity. In the best example, for ethyl ether as cosolvent, the reaction proceeded with E = 55, and the target compound was obtained in 33% yield with 92.7% ee. This finding may be of great practical importance, particularly in industrial processes [24], since it will enable better optimization of enzyme-catalyzed processes. It is clear that, in future, immobilized enzymes, as heterogeneous catalysts, wiU be widely used in most industrial transformations, especially in the preparation of pharmaceuticals [25]. 

It can be seen from Eq. (5) that the maximum possible concentration on the surface, c, influences significantly the transport rate. This parameter is a function of the available surface area as well as of the density of the reactive sites. Because of that, the matrix structure plays a very important role in such adsorp-tion/desorption processes. In the case of biological reactions, where the chemical conversion is performed by immobilized enzymes, the immobilization also plays an important role in order to achieve an optimal enzyme density on the reactive surface. 

Since the large-scale application of immobilized enzymes in the 1960s, substantial research efforts have aimed to optimize the structure of carrier materials for better catalytic efficiency. To date, nanoscale materials may provide the upper limits in... 

In the daily production routine, both enzyme costs and operating costs play decisive roles. In addition, the amount of production is likely to be adapted to demand the load for the product, here HFCS, so that at high demand the optimal production temperature is higher than at lower demand. From Figure 19.9 it becomes apparent how optimal operating times and temperatures can be estimated if the operating costs of a column as well as the costs for the immobilized enzyme... 

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. 

Compound 25 (Fig. 18.9), a prodrug of 9-P-D-arabinofuranosyl guanine (26), was developed for the potential treatment of leukemia. Compound 24 is poorly soluble in water and its synthesis by conventional techniques is difficult. An enzymatic demethoxylation process was developed using adenosine deaminase (Mahmoudian et al., 1999, 2001). Compound 25 was enzymatically prepared from 6-methoxyguanine (27) and ara-uracil (28) using uridine phosphorylase and purine nucleotide phosphorylase. Each protein was cloned and overexpressed in independent Escherichia coli strains. Fermentation conditions were optimized for production of both enzymes and a co-immobilized enzyme preparation was used in the biotransformation process at 200 g/L substrate input. Enzyme was recovered at the end of the reaction by filtration and reused in several cycles. A more water soluble 5 -acetate ester of compound 26 was subsequently prepared by an enzymatic acylation process using immobilized Candida antarctica lipase in 1,4-dioxane (100 g/L substrate) with vinyl acetate as the acyl donor (Krenitsky et al., 1992). 

A characteristic of immobilized enzymes that is often ignored is the potential partitioning of ions and substrates and/or products due to electrostatic potentials or hydrophobic moments. This factor could be used to advantage, for example, if the optimal conditions for enzyme activity do not match those of the process stream. To use the example cited earlier, a succinamidopropyl surface was shown by electrostatic partitioning of ions and independent chemical analysis to have 96 ymol charged groups/g dry beads (25). Attachment of 2 ymol trypsin/g did not significantly alter this characteristic. 

The activity of the immobilized enzyme bioreactor plays an important role in the quality of the spectra. Immobilized enzyme activity is dependent on pH, temperature, solvents and buffers used for the hydrolysis. Extremes in any one condition can irreversibly destroy the bioreactor activity. Hydrolysis at conditions far from optimal can lead to no-hydrolysis or partial hydrolysis of a peptide, providing limited information on the peptide. In order to successfully employ an immobilized enzyme column on-line with HPLC/thermospray MS, organic modifiers must be kept minimal (less than 30-50%), pH must be between 6.5 and 8.5 and buffer (ammonium acetate) concentration around 0.05-0.1M [12]. 

EIA often makes use of solid phases. The relative merits and disadvantages of such techniques, as well as ways to optimize them will be discussed. An important aspect, which has not yet been investigated in detail for EIA, is the influence immobilization of enzymes has on enzyme kinetics. The solid-phase may cause strong local differences in the microenvironment of the enzyme, the implications of which can only be inferred from studies on immobilized enzymes in solid-phase biochemistry (Chapter 9). Major or minor flaws in EIA design, which may discredit an otherwise perfectly valid EIA, will be discussed. 

With glucose sensors the different coupling variants of immobilized enzymes with signal transducers may be very well demonstrated. The variety of constructions result from the specific requirements and limits as well as the different optimal operating conditions of the sensor... 

Besides these conventional reactors with spherical immobilizates, urease has also been immobilized inside nylon tubing and pipette tips ( enzyme pipette , Sundaram and Jayonne, 1979), on nylon fibers ( enzyme brush , Raghavan et al., 1986), and on the surface of a magnetic stirrer (Guilbault and Starklov, 1975). The urease reaction was in each case carried out at optimal pH after removal of the immobilized enzyme NH3 was assayed electrochemically or photometrically according to Berthelot s method. 

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