Enzymatic reactor

Enzymatic Reactors Adding free enzyme to a batch reactor is practical only when the value of the enzyme is relatively low. With expensive enzymes, reuse by retaining the enzyme with some type of support makes great economic sense. As some activity is usually lost in tethering the enzyme and the additional operations cost money, stabihty is very important. However, many enzymes are stabilized by immobilization thus, many reuses may be possible. 

The CCC instruments have even been used as enzymatic reactors to carry out enantioselective processes. Thus, the hydrolysis of 2-cyanocyclopropy 1-1,1-dicar-boxylic acid dimethylester including a bacterial esterase in the stationary phase was reported [131]. After 8 h, the procedure yielded the desired product automatically, without any extraction and with an 80 % e.e. 

For bio-transformation processes, immobilised enzymes are often used because their activity persists over a longer period of time than that of free enzymes. The reduction of enzyme activity in enzymatic reactors is a consequence of energy dissipation by sparging and stirring, which is required for instance for oxygen transport or realisation of constant reaction conditions as regards temperature and pH. In the other hand low and high pH-values leads also to a decrease of enzyme activity and increase the stress sensitivity. 

Lvov, Y. and Caruso F. (2001) Biocolloids with ordered urease multilayer shells as enzymatic reactors. Analytical Chemistry, TS, 4212-4217. 

Figure 9.2-2. Operating principle of dense-gases enzymatic reactor types a), extractive semibatch b), recirculating batch c), semicontinuous flow.

Figure 9.2-3. Design of continuous experimental apparatus for synthesis under supercritical conditions S, substrates 1, molecular sieves 2, saturation column 3, packed-bed enzymatic reactor 4,5, separators P, high-pressure pump PI, pressure indicator TI, temperature indicator H, heat exchanger [17].

Numerous postcolumn enzymatic reactors have been designed for LCEC. Enzymes can be used to produce an electroactive compound from the analyte of interest or, alternatively, to generate an electroactive species that is proportional to analyte concentration. An example of the latter is the detection of acetylcholine [46]. In this case, acetylcholinesterase is used to convert acetylcholine to choline. The resulting choline is reacted with choline oxidase to produce hydrogen peroxide. The amount of hydrogen peroxide produced is directly proportional to the initial concentration of acetylcholine. Detection limits are in the 100 femto-mole range. 

Separation of products from the reaction mixture In situ product removal from enzymatic reactor via a nanofiltration or ultrafiltration membrane Removal of selected enantiomer via a liquid membrane Removal of water in esterification reactions via a pervaporation membrane... 

Enzyme kinetics were evaluated in a PDMS-glass chip using a continuous-flow system. A biotinylated enzyme (HRP or (5-galactosidase) was coupled to streptavidin-coated beads via the amide coupling of an aminocaproyl spacer. These beads (15.5 pm) were retained by a weir in the chip. The channel wall was passivated by 1 mg/mL BSA. The apparent enzyme kinetic parameters were evaluated using the Lilly-Homby model, as developed for the packed-bed enzymatic reactor systems. It was found that the apparent Michaelis constant (Km) approached the tme Km value of the free enzyme at zero-flow rate of a homogeneous reaction [845]. 

Interestingly, DO has been utilized as a control parameter to establish steady state conditions during the operation of a continuous enzymatic reactor for dye decolorization [8]. An unexpected decrease in the level of DO was the warning signal of the loss of peroxidase activity or dye overload, whereas the sudden increase reflected an extra dosing of H202, which would probably imply higher enzyme deactivation rate. 

When looking for an economically feasible enzymatic system, retention and reuse of the biocatalyst should be taken into account as potential alternatives [98, 99]. Enzymatic membrane reactors (EMR) result from the coupling of a membrane separation process with an enzymatic reactor. They can be considered as reactors where separation of the enzyme from the reactants and products is performed by means of a semipermeable membrane that acts as a selective barrier [98]. A difference in chemical potential, pressure, or electric field is usually responsible from the movement of solutes across the membrane, by diffusion, convection, or electrophoretic migration. The selective membrane should ensure the complete retention of the enzyme in order to maintain the full activity inside the system. Furthermore, the technique may include the integration of a purification step in the process, as products can be easily separated from the reaction mixture by means of the selective membrane. 

Enzymatic reactors with enzyme immobilized into the membrane... 

The enzymatic reactor consisted of a 500 mL-continuous tank reactor BIOSTAT Q (Braun-Biotech International) coupled to the ultrafiltration membrane. The additional volume of the ultrafiltration unit and piping was around 150 mL. Three solutions were added to the reactor by means of three variable speed peristaltic pumps, containing (a) Orange II, Mn2+ and oxalic acid (b) H2O2 and (c) MnP. Another peristaltic pump was used in order to feed the effluent into the ultrafiltration unit (Fig. 10.3). The recycling feed flow ratio was maintained at 12 1, as this flow rate allowed an adequate homogeneity of the enzymatic mixture but prevented the polarization or fouling of the membrane. 

The decolorization of the azo dye Orange II by Mn3+-chelate was performed in batch with the complex that had been previously produced in the enzymatic reactor. The capability of the complex to degrade Orange II was evaluated under different initial concentrations of the dye, from 10 to 22 mg/L, whereas the initial concentration of the complex was 165 pM (Fig. 10.10). In all these cases, the complex was able to degrade the dye reaching the same percentage of degradation, 80-85%. 

Fig. 10.12 Profile of production of Mn3+-malonate and degradation of Orange II-.(closed square) Mn3+-malonate produced in the enzymatic reactor (closed triangle) Mn3+-malonate introduced in the oxidation reactor (open circle) decolorization of Orange II...

This high value resulted due to the high conversion efficiencies of the enzymatic reactor. 

Figure 2. Design of continuously operating experimental apparatus for the synthesis of oleyl oleate under supercritical conditions 1-substrates, 2-saturation column, 3-enzymatic reactor, 4, 5-separators, P-high pressure pump, Pi-presssure indicator, T-temperature indicator,...

Kr enkova, J. and Foret, F. (2004). Immobilized microfluidic enzymatic reactors. Electrophoresis 25 3550-3563. 

Subsequently, a much simplified FIA system has been reported in which the co-immobilization of GOD and HPR in an enzymatic reactor permitted the use of luminol as a single reagent for monitoring blood glucose during a glucose load experiment in which the sample was obtained from a microdialysis probe in the jugular vein of a rabbit [136]. 

In the field of food analysis, milk was the real matrix in which several metabolites have been analysed by the use of the microdialysis sampling technique. Acetylcholine, glucose and glutamate were analysed by Yao et al. [189], coupling a microdialysis probe with an on-hne enzymatic reactor and a Pt electrode coated with a 1,2-diaminobenzene polymer. 

Due to high biocompability and large surface are of cobalt oxide nanoparticles it can be used for immobilization of other biomolecules. Flavin adenine FAD is a flavoprotein coenzyme that plays an important biological role in many oxidoreductase processes and biochemical reactions. The immobilized FAD onto different electrode surfaces provides a basis for fabrication of sensors, biosensors, enzymatic reactors and biomedical devices. The electrocatalytic oxidation of NADH on the surface of graphite electrode modified with immobilization of FAD was investigated [276], Recently we used cyclic voltammetry as simple technique for cobalt-oxide nanoparticles formation and immobilization flavin adenine dinucleotide (FAD) [277], Repeated cyclic voltammograms of GC/ CoOx nanoparticles modified electrode in buffer solution containing FAD is shown in Fig.37A. 

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