Continuous-flow enzyme reactors

Third, a poly[bis(phenoxy)phosphazene] has been coated on porous alumina particles, surface nitrated, reduced to the amino-derivative, and then coupled to the enzyme glucose-6-phosphate dehydrogenase or trypsin by means of glutaric dialdehyde. The immobilized enzymes were more stable than their counterparts in solution, and they could be used in continuous flow enzyme reactor equipment (25). 

Once a polymer is fuUy saturated, the physical tests described above can be conducted with confidence. Naturally, minimizing the evaporation of water should be considered. The one exception in this new category of testing is flow of water through the foam. This is not covered in the standard but will be very important for some applications, particularly in environmental remediation. If the intent is to build a biofilter or a continuous flow enzyme reactor, we must know the hydrodynamic properties of the materials we produce. Since polyurethanes are rarely used in these environments, the flow of water even through a reticulated foam is not described by the manufacturers. Furthermore, if we are to make composites of reticulated foams, the amount of polymer grafted to the surface will have a dominating effect on the flow of water. In a later chapter, we will describe our work in this area. 

Table 9.3 Stereoselective biotransformations in continuous-flow enzyme reactors (ranges between brackets indicate the interval of an investigated property).

Although enzymes have the advantage of selectivity and efficiency, they are, as previously mentioned, sometimes not very fast. To increase the total reaction time in microspace, Muller et al. [428] constructed a circulating continuous flow enzyme membrane reactor and investigated the reduction of acetophenone to (S)-phenylethanol in the presence of the enzyme alcohol dehydrogenase (ADH)... 

Giordano, R.L.C., Giordano, R.C. and Cooney, C.L. (2000). Performance of a continuous Taylor-Couette-Poiseuille vortex flow enzymic reactor with suspended particles. Process Biochemistry, Vol. 35, No. 10, pp. 1093-1101. 

Active immobilize enzyme process engineering studies in a packed colunm and a continuous flow stirred reactor... 

When the substrate concentration S is much greater than Km, Eqs. (12) and (13) reduce to the same form. In this case, the continuous flow stirred reactor and the plug flow device achieve similar conversion values in a given time. In contrast, when S Km, the reaction rate becomes first order in the substrate concentration (see Eq. (9)), and the plug flow reactor provides higher conversion values in comparison with the well-mixed continuous flow device. In the latter bioreactor, all the enzyme would be exposed to the same low concentration of the substrate which is not useful except when the reaction is inhibited by the substrate. 

Synthesis of 2-amino-1,3,4-butanetriol by a transketolase/ transaminase enzyme cascade via continuous flow dual reactor. The enzymes were immobilized inside the silica microreactors separately. 

The alternative to batch mode operation is continuous operation. In the continuous mode there is a continuous flow of medium into the fermentor and of product stream out of the fermentor. Continuous bioprocesses often use homogenously mixed whole cell suspensions. However, immobilised cell or enzyme processes generally operate in continuous plug flow reactors, without mixing (see Figure 2.1, packed-bed reactors). 

The flow diagram of the enzyme reactor for continuous production of the L-amino add is given in Figure A85. The acetyl amino add is continuously charged into the enzyme column through a filter and a heat exchanger. The effluent is concentrated and the L-amino add is crystallised. The acyl-D-amino add contained in the mother liquor is racemised by heating in a racemisation tank, and reused. 

The use of glutaric dialdehyde as a coupling agent bound the enzymes trypsin or glucose-6-phosphate dehydrogenase to the surface. A large part of the enzymic activity was retained (Fig. 4), and the activity was such that the particle-enzyme conjugate could be used in laboratory scale continuous-flow reactors. 

The decolorization of Orange II by immobilized P. chrysosporium in a continuous packed-bed reactor (PBR) was investigated [50]. Nearly complete decolorization (95%) with immobilized fungus on PuF was obtained when working at optimal conditions [dye load rate of 0.2 g/l/d, temperature of 37°C, a hydraulic retention time (HRT) of 24 h], and also oxygen gas in a pulsed flow was applied. A correlation between residual MnP activity and decolorization was observed, but no laccase and LiP enzyme activities were detected. 

An immobilized-enzyme continuous-flow reactor incorporating a continuous direct electrochemical regeneration of NAD + has been proposed. To retain the low molecular weight cofactor NADH/NAD+ within the reaction system, special hollow fibers (Dow ultrafilter UFb/HFU-1) with a molecular weight cut-off of 200 has been used [32],... 

Enzymes can be used in several ways in chromatographic applications to improve selectivity or to enhance the detector response. Applications may involve enzymes with either a broad specificity toward a group of related compounds or a high specificity toward a particular compound. In the field of drug residue analysis, most current applications concern enzymatic reactions taking place in separate reactors incorporated in LC systems before or after the analytical column. Reactors with immobilized enzymes have proven to be suitable in such continuous flow systems. 

Batch-, stirred-tank-, extractive semibatch-, recirculating batch-, semicontinuous flow-, continuous packed-bed-, and continuous-membrane reactors have been used as enzyme reactors, with dense gases used as solvents. 

Cooking extruders have been studied for the liquefaction of starch, but the high temperature inactivation of the enzymes in the extruder demands doses 5—10 times higher than under conditions in a jet cooker (69). For example, continuous nonpressure cooking of wheat for the production of ethanol is carried out at 85°C in two continuous stirred tank reactors (CSTR) connected in series plug-flow tube reactors may be included if only one CSTR is used (70). 

A main adimntage of immobilized enzyme is that it can be reused since it can be easily separated from the reaction solution and can be easily retained in a continuous-flow reactor. Furthermore, immobilized enzyme may show selectively altered chemical or physical properties and it may simulate the realistic natural environment where the enzyme came from, the cell. 

In the following we attempt to describe the acetylcholinesterase/choline acetyltransferase enzyme system inside the neural synaptic cleft in a simple fashion see Figure 4.49. The complete neurocycle of the acetylcholine as a neurotransmitter is simulated in our model as a simple two-enzymes/two-compartments model. Each compartment is described as a constant-flow, constant-volume, isothermal, continuous stirred tank reactor (CSTR). The two compartments (I) and (II) are separated by a nonselective permeable membrane as shown in Figure 4.50. 

A large part of the enzymic activity was retained after immobilization, as illustrated in Figure 3.20. Moreover, packing of the conjugate particles into a chromatography-type column allowed the construction of a continuous flow reactor. Figure 3.20 illustrates that the enzyme molecules continued to catalyze their reactions for long... 

J. Y. Lee, A. Veiayudhan, and M. R. Iadisch, Maintaining constant enzyme activity in a continuous flow reactor, Chem. Eng. J. 

Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon. The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of >90% were observed. 

Fig. 7. Effect of enzyme loading on conversion in 50-mm PDMS-E continuous-flow reactors.

Fig. 9. Effect of time on conversions for 1000-mm PDMS-E continuous-flow reactor with various enzyme loadings.

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]. 

Having confirmed that the immobilization strategy was robust enough to be used in conjunction with a continuous flow reactor, the authors evaluated the immobilization of a second Hisg-tag enzyme, BsubpNBE, and employed the synthesis of 6-O-acetyl-D-glucal 162 (Scheme 44) as a model reaction. 

Csajagi et al. (2008) recently demonstrated the enantioselective acylation of racemic alcohols in a continuous flow bioreactor, using Candida antarctica lipase B (CaLB) 167. Employing a packed-bed reactor, containing 0.40 g of enzyme 167, and pumping a solution of rac-phenyl-1-ethanol 119 (10 mg ml-1) in hexane THF vinyl acetate 168 (2 1 1) at a flow rate of 100 gl min-1 (at 25 °C), the authors found the reactor reached steady state after 30 min of operation. Under the aforementioned conditions, the... 

To demonstrate the synthetic application of this methodology, the authors subsequently demonstrated its use for the preparative kinetic resolutions of a series of 2° alcohols, Table 24, whereby 20 ml solutions of each racemic alcohol were passed through the bioreactor (3.3 h) and found to afford analogous results to those obtained during the initial optimization experiments. The authors successfully demonstrated the use of immobilized and lyophilized enzymes within a continuous flow reactor, presenting a synthetically viable approach to the kinetic resolution of racemic alcohols. 

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