Enzymes column reactor

The modeling of real immobilized-enzyme column reactors, mainly the fluidized-bed type, has been described (Emeiy and Cardoso, 1978 Allen, Charles and Coughlin, 1979 Kobayashi and Moo-Young, 1971) by mathematical models based on the dispersion concept (Levenspiel, 1972), by incorporation of an additional term to account for back-mixing in the ideal plug-flow reactor. This term describes the non-ideal effects in terms of a dispersion coefficient. 

Ellman s reagent, 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), produces 2-nitro-5-sulfhydrylbenzoic acid by the reaction with thiols, which shows visible absorption at 412nm. Acetyl-CoA thioester is separated by ion-pair LC followed by conversion to thio-CoA with a postcolumn immobilized enzyme column reactor of phosphotransacetylase. Thio-CoA thus liberated can be determined spectrophotometri-cally after reaction with Ellman s reagent with the detection limit of 0.05 nmol. 

A significant advantage of immobilized enzymes is the total absence of catalytic activity in the product. Moreover, the degree of substrate-to-product conversion can be controlled during processing, eg, by adjusting the flow rate through a packed-bed column reactor of immobilized enzyme. 

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. 

Many interesting biocatalytic reactions involve organic components that are poorly water-soluble. When using organic-aqueous biphasic bioreactor, availability of poorly water-soluble reactants to cells and enzymes is improved, and product extraction can be coupled to the bioreaction. Many applications in two-phase media can use the existing standard-type bioreactors, such as stirred-tank, fluidized-bed, and column reactors with minor adjustments. 

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. 

Caussette, M. Gaunnand, A. Planche, H. Colombie, S. Monsan, P. Lindet, B. Lysozyme Inactivation by Inert Gas Bubbling Kinetics in a Bubble Column Reactor. Enzyme Microb. Technol. 1999, 24, 412 -18. 

Mayer determined acetylcholine and choline by enzyme-mediated liquid chromatography with electrochemical detection [195]. The two compounds were separated by passing the eluted fractions through a post-column reactor containing immobilized Acetylcholineesterase and choline oxidase. In the presence of either compound, the dissolved oxygen was converted into hydrogen peroxide, which was detected amperometrically at a platinum electrode. This method was used to determine choline in rat brain homogenates. 

Figure 8.6 Schematic difference between an enzyme-immobilized column reactor and a forced-flow membrane bioreactor (Nakajima et al., 1988]...

The immobilized enzyme was removed from the tube and rinsed with D.I. water, followed sequentially by 2N urea solution, 2N NaCl solution and tris buffer, pH 7.5, containing 0.2% sodium azide and 20 mM CaClg, then stored at 4 C. About 0.18-0.2 g of immobilized enzyme beads were used to slurry pack stainless steel 10 cm x 2.1 mm column reactors. Complete details of enzyme preparation and assay for activity are described elsewhere [12],... 

Stefuca et al. (1990) proposed an ET method offering a rapid, convenient, and general approach to determine kinetic constants of immobilized biocatalysts. Here, a differential reactor (DR) was used for the measurement of the initial reaction rate of sucrose hydrolysis (Vallat et al. 1986). The enzyme column of the ET has been considered as a differential packed-bed reactor, and with a mathematical model, intrinsic kinetic constants of immobilized invertase were calculated from experimental DR and ET data. 

Enzyme-antibody complex formation represents the simplest among the immunoaffinity immobilization procedures and the immunocomplexes can be readily formed simply by mixing of the enzyme solution with the antibody or even antiserum. Interestingly neither pure enzyme nor pure antibody may be required for the formation of immunocomplexes. Several early [14,36,39] and some recent studies [22,24,28] indicate high retention of catalytic activities by various enzymes in the immunocomplexes and marked stability enhancement against various forms of inactivation. The small particle dimensions of the enzyme-antibody complexes may however lead to their compact packing and consequently to slow flow-rates in the column reactors. Their usefulness can be however remarkably enhanced by entrapping the complexes in a polymeric matrix [60,61]. 

The possibility of separation and analysis of enzymes by CZE has been explored. Banke, et al. separated alkahne proteases from crude fermentation broth, and collected fractions from CZE for enzyme analysis. CZE was also used to monitor the progress of an enzyme reaction [23]. Konse, et al. reported modification of a microtitre plate assembly which was used to coUect fractions on polyvinylidene difluoride (PVDF) membranes. Fractions blotted onto the PVDF membranes were then subsequently analyzed by a sequencer [24]. Emmer and Roeraade described an on-hne micro-post column reactor which they used in conjunction with on-column detection. By using the two detector system, they were able to rapidly monitor enzyme activity in samples. Through careful optimization of conditions in the reactor, the loss of efficiency at the point of detection through the reactor capiUary was minimal [25]. 

The catalytic behavior of enzymes in immobilized form may dramatically differ from that of soluble homogeneous enzymes. In particular, mass transport effects (the transport of a substrate to the catalyst and diffusion of reaction products away from the catalyst matrix) may result in the reduction of the overall activity. Mass transport effects are usually divided into two categories - external and internal. External effects stem from the fact that substrates must be transported from the bulk solution to the surface of an immobilized enzyme. Internal diffusional limitations occur when a substrate penetrates inside the immobilized enzyme particle, such as porous carriers, polymeric microspheres, membranes, etc. The classical treatment of mass transfer in heterogeneous catalysis has been successfully applied to immobilized enzymes I27l There are several simple experimental criteria or tests that allow one to determine whether a reaction is limited by external diffusion. For example, if a reaction is completely limited by external diffusion, the rate of the process should not depend on pH or enzyme concentration. At the same time the rate of reaction will depend on the stirring in the batch reactor or on the flow rate of a substrate in the column reactor. 

A plug flow reactor may be realized using immobilized enzymes within a column reactor or using soluble enzymes within a cascade of membrane reactors. A batch or a repetitive batch process with soluble enzymes (see below) has the same productivity as the plug flow reactor. 

Biocatalyst consumption per unit weight of product was found to be about 6000 U/ kg at a conversion of 78 %. For production purposes, enzyme membrane reactors with a working volume up to 500 mL were employed for the synthesis of approximately 2 kg of N-acetylneuraminic acid and other derivatives such as keto-desoxynonulosonic acid (KDN)[129, 132L Downstream processing was achieved mainly by anion exchange chromatography on a 30 L column followed by reverse osmosis to concentrate solutions before lyophilization. 

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