Enzymes reactors

Enzymes are frequently used as catalysts to promote specific reactions in free solution. They are typically required in small amounts and are attractive in that they obviate both the need to provide the nutritional support which would be required for micro-organisms to perform the same conversion, and the possible subsequent removal of those microbes. Furthermore, the enzyme need not necessarily be of microbial origin so that a wider choice of operating conditions and characteristics may be available. 

In a batch reactor where substrate is being converted to product by the enzyme, a material balance for the substrate gives  

If the substrate concentration is S, the volume of the reactor is V and t is the time then this becomes  

As discussed before, the rate of an enzyme-catalysed reaction is dependent, not only on the substrate concentration at any instant, but also on the temperature, pH and the degree of decay of that enzyme. For given values of pH and temperature, the specific rate of reaction v is given, in the simplest case, by the Michaelis-Menten equation  

These design equations for a free-enzyme batch reaction may be formulated in a similar manner for kinetic schemes other than the case used above. If, for example, the back reaction is significant then the procedure would make use of equation A5.4.13 to define 9t0, and at infinite time an equilibrium mixture of substrate and product would be obtained. 

Enzyme Reactors. We turn now to new developments, not fully commercialized, which utilize UF membranes in the processing of pharmaceuticals and biologicals. The first of these is the enzyme reactor. 

Enzymes facilitate many biochemical reactions, serving as a catalyst-i.e., the enzyme itself is not consumed in the reaction. In fact, it must be separated from the products of the reaction. In the past, the enzyme was discarded and fresh enzyme was charged to the reactor with substrate. Obviously, the economics of such a reactor can be improved dramatically if the enzyme can be recovered and reused. One way of doing this is to immobilize the enzyme on a column of glass beads substrate is fed into one end of the column and products are withdrawn from the other end. Because the enzyme is affixed to the stationary phase, it does not contaminate the product and may be used over and over again. 

Enzymes may also be immobilized on membranes in the same fashion. Three types of enzyme reactors using UF membranes are introduced here. The equa- 

Method of Operation Recirculating Time Conversion. Time Conversion, 

Fibers used 1n this study were 20 mil diameter XM50 and 45 mil diameter XM50. These fibers have a molecular weight cutoff of 50,000. Fibers with molecular weight cutoffs of 10,000 and 5,000 are also available. 

The biologically active enzymes can be used as catalysts either in a soluble, dispersed form or in a carrier-bound form. Because of the need for the isolation step and losses of enzyme activity, enzyme processes are sparingly used at the present time. Immobilized enzymes show promise for minimizing these activity losses and for facilitating enzyme recovery (Pitcher, 1978). 

The problems of working with carrier-bound enzymes and cells in biofilm reactors and the biofloc reactors are compared in Table 3.1. 

More than 80% of the commercial value of enzymes is linked to their applications as process catalysts. Hydrolytic reactions conducted mainly with the enzyme dissolved in the aqueous medium has been the traditional way of using enzymes, this technology still representing a major share of enzyme processes. However, in recent decades the use of enzymes in organic synthesis has widened its scope of application to unprecedented levels. 

Immobilized enzymes, despite the complexities introduced by the heterogeneous nature of the catalytic process, are usually much more stable than their soluble 

School of Biochemical Engineering, Pontificia Universidad CatoUca de Valparaiso, Avenida Brasil 2147, Valparaiso, Chile 

Several reactor configurations have been proposed and used for conducting enzyme-catalyzed processes, as shown in Fig. 5.1. Batch operations with soluble enzymes are conducted mainly in stirred tank reactors (BSTR) provided with mixing 

Decristoforo and Danielsson (1984) designed an enzyme thermistor for penicillin determination based on (3-lactamase immobilized on porous 

Base sensor (J-Lactamase Amidase Linear range (mmol/1) Lifetime (d) Sample frequency Of CV(%) 

Olsson (1988) obtained a pH signal which was linearly dependent on penicillin concentration by using a penicillinase reactor in an FIA manifold. The linearity was due to complete substrate hydrolysis in the reactor and an almost constant buffer capacity of the carrier stream. To exclude disturbances from varying sample pH the penicillin concentration was calculated as the difference between the response with and without hydrolysis. 

In the case of utilizing isolated enzymes for generation and modification of flavour substances from precursors, enzyme reactors may be applied. 

To put it simple, the enzyme reactor is an agitated tank reactor in which the substrate is placed and stirred together with the enzyme for a certain period of time at a certain temperature in the liquid phase. The enzyme is added preferably in a concentration of 0.1 to 1% and is lost after completion of the reaction. 

A great savings in enzyme consumption can be achieved by immobilizing the enzyme in the reactor (Fig. 12). In addition to the smaller amount of enzyme required, immobilization often increases the stability of the enzyme. Several designs of immobiliz-ed-enzyme reactors (lERs) have been reported, with open-tubular and packed-bed being the most popular. Open-tubular reactors offer low dispersion but have a relatively small surface area for enzyme attachment. Packed-bed reactors provide extremely high surface areas and improved mass transport at the cost of more dispersion. 

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Here we will focus on the biochemical aspects. The techniques of isolating enzymes, the process of enzyme immobilisation and the behaviour of immobilised enzyme reactors are discussed in detail in the BIOTOL text Technological Applications of Biocatalysts", so will not deal with these aspects in detail here. In outline, however, once the desired enzyme is isolated, it is attached to a carrier material. In order to ascertain sufficient accessibility of the enzyme, a bifunctional spacer molecule is attached to the carrier ... 

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. 

Solution Most enzyme reactors use such high concentrations of water that the fluid density is constant. Applying Michaelis-Menten kinetics to the component balance for a steady-state CSTR gives... 

By incorporating the entire analytical scheme (enzyme reaction and electrochemical detection) into the flow system a great improvement in precision can be realized. Sample manipulation is minimized because only a single injection into the flow system is required versus sampling of aliquots for the off-line method. Precision is also improved because the timing of the enzyme reaction and detection are much better controlled in the flow system. Finally, less of both enzyme and sample are needed with on-line enzyme reactor methods. 

The simplest design for an enzyme reactor is to merely have the substrate and enzyme in two merging buffer streams followed by a reaction delay coil (Fig. 12). The delay... 

Fig. 12. On-line enzyme reactor system designs, merging stream system (Top) and immobilized-enzyme reactor system (Bottom). A = mobile phase, B = enzyme solution...

Numata, M., Funazaki, N., Ito, S., Asano, Y., and Yano, Y., Flow injection analysis for hypoxanthine in meat with dissolved oxygen detector and enzyme reactor, Talanta, 43,2053,1996. 

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). 

Fig. 2.2 Enzyme reactors prepared by LbL assembly (A) reactor on quartz plate for color-indication of glucose (B) multi-enzyme reactor for starch digestion on ultrafilter. Adapted from [26], M. Onda etal, Biotechnol. Bioeng. 1996, 57, 163 and [27], M. Onda et al.,J. Ferment. Bioeng. 1996, 82, 502.

R. Kindervater, W. Kiinnecke, and R.D. Schimid, Exchangeable immobilized enzyme reactor for enzyme inhibition tests in flow-injection analysis using a magnetic device. Determination of pesticides in drinking water. Anal. Chim. Acta 234, 113-117 (1990). 

R.Q. Thompson, H. Kim, and C.E. Miller, Comparison of immobilized enzyme reactors for flow-injection... 

L. Korecka, Z. Bilkova, M. Holeapek, J. Kralovsky, M. Benes, J. Lenfeld, N. Mine, R. Cecal, J.-L. Viovy, and M. Przybylski, Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments. Journal of Chromatography, B Analytical Technologies in the Biomedical and Life Sciences 808, 15-24 (2004). 

L Gorton, G Marko-Varga. In S Lam, G Malikin, eds. Analytical Applications of Immobilized Enzyme Reactors. London Blackie Academic Professional, Chapman Hall, 1994, pp 1-21. 

Figure 3 CL detection systems in combination with HPLC. P, pump I, injector C, column M, mixing tee D, detector RC, reaction coil MC, mixing coil RE, recorder E, eluent R, reagent W, waste IMER, immobilized enzyme reactor.

System C is used when an immobilized enzyme reactor (IMER) is introduced into system B. The analyte(s) separated by HPLC is converted to a suitable species for CL detection with an IMER, and then mixed with the CL reagent. In this system, a buffer solution as a mobile phase and an ion-exchange-type column are preferable for an enzyme reaction. 

PVA/chitosan blend membranes can be applied for the synthesis of monoglyceride, when used as a membrane enzyme reactor [277]. 

So far, all applications of the chromatographic enzyme reactor were limited to conversions of small molecules serving mainly as case studies . In the near future these studies will hopefully be extended to larger substrate molecules and... 

A horseradish peroxidase-osmium redox polymer-modified glassy carbon electrode (HRP-GCE) has also been applied to this analysis to improve sensitivity and reduce problems with faradic interference. Kato and colleagues (1996) employed this electrode in measurement of basal ACh in microdialysates using a precolumn enzyme reactor. This system was three to five times more sensitive than a conventional Pt electrode. ACh in rat hippocampus dialysate was quantitated at 9 5 fmol/15 pi (n = 8). ACh was analyzed in PC12 cells in a similar assay by Kim and colleagues (2004). No precolumn enzyme reactor was employed. 

Kato T, Liu JK, Yamamoto K, Osborne PG, Niwa O. 1996. Detection of basal acetylcholine release in the microdialysis of rat frontal cortex by high- performance liquid chromatography using a horseradish peroxidase-osmium redox polymer electrode with pre-enzyme reactor. J Chromatogr B 682 162-166. 

Osborne PC, Yamamoto K. 1998. Disposable, enzymatically modified printed film carbon electrodes for use in the high-performance liquid chromatographic-electrochemical detection of glucose or hydrogen peroxide from immobilized enzyme reactors. J Chrom B 707 3-8. 

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